# Patent

## Title
Vaccines for control of epidemic Aeromonas hydrophila generated by markerless gene deletion

## Abstract

Disclosed are attenuated bacteria, compositions comprising attenuated bacteria, and vectors and methods for preparing attenuated bacteria. The attenuated bacteria may include attenuated Aeromonas hydrophila for use in vaccinating aquatic animals such as channel catfish against Motile Aeromonas Septicemia (MAS).

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Applications No. 62/003,953, filed on May 28, 2014, the content of which is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with U.S. government support under grant nos. 2012-38500-19665, 2013-67015-21313, and 2014-38500-22308 awarded by the U.S. Department of Agriculture. The U.S. government has certain rights in the invention.


BACKGROUND
The field of the present invention relates to compositions and methods for treating or preventing disease in aquatic animals such as farmed fish (e.g. catfish or tilapia) and crustaceans (e.g., shrimp). In particular, the present invention relates to compositions and methods comprising or utilizing attenuated strains of Aeromonas hydrophila for treating or preventing diseases such as Motile Aeromonas Septicemia (MAS) in aquatic animals such as farmed fish.
Aquaculture is one of the fastest growing industries in the world. It is developing, expanding and intensifying in almost all regions of the world due to the vast global population demand of aquatic food products and the leveling-off of the capture fisheries. According to FAO, in 2010 alone, the World aquaculture production reached 60 million tons (excluding aquatic plants and non-food products), with an estimated, total value of US $119 billion. In 2010, 181 countries and territories had aquaculture production, with Asia accounting for 89 percent of world aquaculture production by volume, in which 61.4 percent was from China. During the last thirty years, the production of global food fish protein has expanded by almost 12 times, at a rate of 8.8 percent more per year (FAO 2012) According to FAO, global aquaculture production will need to achieve 80 million tons by 2050 just to maintain the current level of per capita consumption. It is not possible for the capture fisheries industry itself to meet this big global aquatic food need. Even with the fast growing rate and the significant contribution of the aquaculture industry, it is still a big challenge for the aquaculture to achieve this goal. (FAO 2005).
In United States, the aquaculture industry is dominated by finfish production (FAO 2012). Being the largest sector of the aquaculture industry, channel catfish farming produced more than 400 million dollars which accounts for approximately half of the total aquaculture production in U.S. in 2010, in the top 10 fish and seafood that consumed among Americans rank, Catfish raised by farm was sixth, about 0.8 pound per person per year. (Hanson & Sites, 2012). Most catfish are produced in the south of the United States and Mississippi, Alabama, Arkansas, and Texas are the top four States for catfish in the United States, which accounts for 94 percent of total sales (USDA, 2014).
Even though the U.S. catfish industry is the dominant aquaculture practiced in the United States, catfish production is vulnerable to adverse impacts of disease and environmental conditions. Before the 1990's, the strategy of management practices was ‘low-density’, which resulted in good pond water quality, lower overall stress on fish populations and less efficient pathogen transmission. However, due to the great competition from the Asian countries especially China, the producers applied much more intense production strategies, such as multiple batch cropping systems, higher stocking density, more feed put into the culture systems. All of these practices lead to the emergence of the infectious diseases which now becomes the primary limiting factor in catfish production. Disease outbreaks in recent years are very common even on efficient and well-built catfish farming facilities. According to MSU (Mississippi State University) reports, infectious diseases have caused approximately 45 percent of inventory losses on catfish fingerling farms, and about 60 percent of the overall catfish losses are attributed to single or mixed bacterial infections, 30 percent due to parasitic infection, 9 percent from fungal infection, and 1 percent result from viral etiology. Economic losses resulting from infectious diseases are believed to cost producers millions of dollars in direct fish losses each year. Furthermore, infectious diseases can also impact the profitability by increasing treatment costs, reducing food consumption by fish due to the flavor change and appearance, increasing feed conversion ratios, and causing harvesting delays.
The major bacterial diseases in catfish that affect the catfish industry are: Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri (Hawke, 1979); Motile Aeromonas septicemia (MAS), which is caused by Aeromonas species (Austin & Adams, 1996) and Columnaris (also referred to as cottonmouth) which is caused by Flavobacterium columnare (Wagner, 2002). The economic losses due to the ESC, according to USDA were about 30 to 50 million dollars each year (Shoemaker et al., 2007; USDA, 2010a, 2010b). The yearly losses caused by the Columnaris are estimated to be 30 million dollars (Declercq, 2013). MAS also causes huge amount of economic losses which are not limited to channel catfish but also including tilapia, catfish, goldfish, common carp, and eel (Pridgeon et al., 2011).
Prior to 2009, MAS in channel catfish caused by A. hydrophila was not a significant concern because the catfish aquaculture operations in the southeastern United States had not experienced a major outbreak (Hemstreet, 2010). However In 2009, catfish farmers in west Alabama reported severe disease outbreaks which were then proved to be caused by a highly virulent strain of A. hydrophila, ML09-119, to channel catfish (Ictalurus punctatus). From 2009-2011, Alabama catfish famers lost more than 7.5 million pounds of catfish that were market-size and estimated to be $3 million due to this epidemic strain of A. hydrophila (Pridgeon, 2011; Liles, 2011). It is reported that A. hydrophila epidemic strain, ML09-119, is highly virulent to channel catfish, causing severe mortality within 24 h post exposure with certain amount of dose (Pridgeon, 2011). The epidemic MAS outbreaks caused by Aeromonas hydrophila are so devastating that it is highly essential to investigate the virulence nature of this pathogen, identify the virulence related genes and create live avirulent bacterial mutants that are vaccine candidates for this bacterial disease. So far there are only a few factors identified for the A. hydrophila epidemic strain, and no commercial vaccine or treatment for the epidemic MAS are available right now. Three attenuated A. hydrophila vaccines were reported to offered 86-100% protection against their virulent parents at 14 days post vaccination (dpv), when the channel catfish were vaccinated with the mutants at dosage of 4×105 CFU/fish. These mutants were developed from the virulent 2009 West Alabama isolates through selection for resistance to both novobiocin and rifampicin (Julia and Klesius, 2011). But these antibiotic resistant mutants are spontaneous mutants that could more readily revert to a virulent strain compared to targeted, stable genetic deletions in gene(s) responsible for virulence.
Therefore, there is a need for a better understanding of the virulence of A. hydrophila in order for vaccine development to progress. Here, the inventors disclose methods for identifying virulence factors of A. hydrophila and producing attenuated strains of A. hydrophila that have been made deficient in one or more virulence factors.
SUMMARY
Disclosed are attenuated bacteria, compositions comprising attenuated bacteria, and vectors and methods for preparing attenuated bacteria. The attenuated bacteria may include attenuated Aeromonas hydrophila for use in vaccinating aquatic animals such as channel catfish against Motile Aeromonas Septicemia (MAS).
The attenuated bacteria may be attenuated by making the bacteria deficient in one or more target genes that are associated with pathogenicity. Suitable genes may include but are not limited to the genes associated with the pathway for O-antigen and/or O-antigen capsule synthesis and secretion and myo-inositol catabolism and regulation. Genes associated with the pathway for O-antigen and/or O-antigen capsule synthesis and secretion may include, but are not limited too ymcA, ymcB, ymcC, waaL, wzy, polysaccharide export protein, and wzz. (See FIG. 19). Genes associated with myo-inositol catabolism and regulation may include, but are not limited to iolA, iolB, iolC, iolD, iolE, iolG1, and iolG2 (See FIG. 8).
The bacteria may be made deficient of the one or more target genes (e.g., target genes associated with pathogenicity) by a method that includes deleting at least a portion of the target gene by recombination and insertion of a selectable marker in place of the deleted portion of the target gene. Subsequently, the selectable marker may be deleted in order to prepare a markerless bacterium that is deficient in the target gene.
Suitable methods for preparing the markerless bacteria that are deficient in the one or more target genes may include recombineering systems. The recombineering systems may include: (a) a mobilizable recombineering vector that expresses protein components for facilitating homologous recombination; and (b) a linear DNA molecule that is configured for recombining at a target gene and replacing at least a portion of the target gene with a selectable marker that is flanked by recombinase recognition target sequences. After the linear DNA molecule is recombined at the target sequence, a recombinase that recognizes the recombinase recognition target sequences may be expressed in order to recombine the target sequences and remove the selectable marker that is flanked by recombinase recognition target sequences.
Also disclosed are vaccine compositions comprising the attenuated bacteria disclosed herein, preferably together with a suitable carrier. The vaccine compositions may include live attenuated bacteria or attenuated bacteria that have been killed, for example by chemical treatment or heat treatment. Optionally, the vaccines may include an adjuvant.
Preferably, the vaccine compositions comprise an effective concentration of the bacteria for treating and/or preventing disease in an aquatic animal after the vaccine compositions are administered to the aquatic animal. Accordingly, also contemplated herein are methods of vaccinating an aquatic animal against infection by the bacteria that include administering the vaccine composition to the aquatic animal.
Also disclosed herein are vectors and kits comprising one or more vectors for preparing the bacteria disclosed herein. Contemplated vectors include recombineering vectors as disclosed herein, and contemplated kits may include one or more recombineering vectors.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (A)-(D) Schematic map of conjugally transferable recombinogenic and flp recombinase plasmids constructed in this study. The plasmid map was generated by CLC Genomics Workbench (Version 4.7.1).
FIG. 2. Effect of primer modifications on the mutant frequency in A. hydrophila. Modified primers were generated by adding four consecutive phosphorothioate bonds at the 5′ end of the primers. To generate type a PCR substrate, leading strand specific modified plasmid was used; type b, lagging strand specific primers were modified, type c and d both or none primers were modified, respectively. PCR products (type c) generated by modified forward and reverse primers provided significantly more mutants than other types of PCR substrates used for recombineering in this study (p-value=0.0026). Average number of mutants represents in the graph, derived from three independent recombineering experiments.
FIG. 3. Schematic representation of strategy use for PCR free cloning of large genetic insert using recombineering.
FIG. 4. Plasmid pYmcABC constructed by PCR-free cloning using recombineering.
FIG. 5. Myo-inositol assay. Growth of bacterial strains in M9 medium containing myo-inositol as the sole carbon source for 144 hours. All the ΔiolA deletion mutants, both created by traditional SOE technique and the recombineering technique, were incapable of growth using myo-inositol as a sole carbon source. Wild type A. hydrophila ML09-119 reached stationary phase after 48 hours of incubation.
FIG. 6. Cumulative survival rate of the ΔiolAtra and ΔiolARec1 mutants compared to wild type A. hydrophila ML09-119. Channel catfish that were i.p injected with 106 CFU/fish of the ΔiolAtra mutant had 100±0% survival rate, while 2.5%±0.08 survival rate was observed in the positive control treatment group in which channel catfish were i.p injected with 106 CFU/fish wild type ML09-119 (P<0.0001). The iolARec1 treatment group had a 2.5%±0.08 survival rate which was the same as the wild type positive control group (P>0.05).
FIG. 7. Sub-challenge of the survivors in the ΔiolAtra group with wild type. 21 days post challenge, all survivors of ΔiolAtra group were I.P injected with 1×106 CFU/fish ML09-119. Naive channel catfish were I.P injected with the same dosage with ML09-119 as a positive control. Significant differences were observed between the ML09-119 group and ΔiolAtra mutant group (P<0.05).
FIG. 8. The relative positions of the iolA and iolR gene
FIG. 9. Pre-trial challenge of the iolA mutants compared with wild type A. hydrophila ML09-119. A 0% survival rate was observed in the channel catfish i.p injected with the ΔiolARec mutant, as did the ΔiolARec2, ΔiolARec5, ΔiolARec7, and ΔiolARec8 mutants. In contrast, the ΔiolAtra mutant had an 83% survival rate, and there was 25%, 33.3%, and 16.7% survival rates observed for the ΔiolARec3, ΔiolARec4, and ΔiolARec6 treatment groups, respectively.
FIG. 10. Cumulative survival rate of the catfish challenged with the ΔiolA mutants. The channel catfish in the ΔiolAtra treatment group had a 83.3±11.5% survival rate, while a 0±0% survival rate was observed in the ML09-119 treatment group (P<0.0001). Channel catfish challenged with the ΔiolARec3 or ΔiolARec4 mutants had a 12.1±9.1% and 17.3±17.2% survival rates, respectively.
FIG. 11. SAS Duncan comparisons. Significant differences were observed between iolAtra and ML09-119 treatment groups (P=5.23E−06), indicating that the ΔiolAtra is an attenuated mutant of ML09-119. No significant differences were observed between the iolARec3 or iolARec4 treatment groups and the wild type (P=0.09).
FIG. 12. Cumulative survival rate of the channel catfish survivors sub-challenged with ML09-119. Sub-challenge of the channel catfish survivors in the ΔiolAtra treatment group with the wild type ML09-119 showed a 71.4±14.3% survival rate, compared to challenge of naïve fish that resulted in a 0±0% survival rate (P=0.00098).
FIG. 13. Titers of ΔiolAtra antibody against ML09-119 by ELISA. All the replicates of the ΔiolAtra mutant induced strong antibody reaction to ML09-119. The levels of antibody to ML09-119 were highest in the serum from ΔiolAtra mutant immunized channel catfish.
FIG. 14. Challenge with O-antigen mutants created by traditional Allelic exchange technique. The channel catfish in the Δwaaltra and Δwzytra treatment groups had 100±0% survival rate, while a 5±0.08% survival rate was observed in the positive control treatment ML09-119 group. A significant difference was observed between Δwaaltra or Δwzytra and ML09-119 treatment groups, P<0.0001.
FIG. 15. Sub-challenge of the survivors of each treatment after 21 days with wild type ML09-119. The exact concentration of ML09-119 used in this experiment was not determined. However, significant differences were observed between the sham negative control group and the treatment groups (P=0.0044<0.05). No significant differences were observed between the treatment groups and the positive control group (PΔwaaltra>0.05 and PΔwaaltra=0.97). A 35±0.18% survival rate was observed in the Δwaaltra group, and a 27±0.3% survival rate was observed in the Δwzytra group.
FIG. 16. Cumulative survival rate of the catfish challenged with the ΔymcA mutant. The channel catfish in the ΔymcA treatment group had a 68.1±16.8% survival rate, while a 0±0% survival rate was observed for wild-type ML09-119. Note that a 83.3±11.6% survival rate was observed in the negative control treatment group, with some deaths attributable to non-bacterial causes. A significant difference was observed between the ΔymcA and ML09-119 treatment groups, P=0.000186<0.05.
FIG. 17. Cumulative survival rate of the channel catfish survivors sub-challenged with ML09-119. The ΔymcA treatment group surviving fish that were challenged with the wild type ML09-119 showed a 90.5±8.3% survival rate, in contrast to the 0±0% survival rate observed in the naive channel catfish challenged with ML09-119. Significant differences were observed between the ML09-119 group and the ΔymcA mutant group, P=4.52E−0.5<0.05.
FIG. 18. Antibody titers offish exposed to ΔymcA against ML09-119 by ELISA. All of the replicates of the ΔymcA mutant-challenged fish induced a strong antibody reaction to ML09-119. The levels of antibody to ML09-119 were highest in the serum from ΔymcA mutant immunized channel catfish.
FIG. 19. Organization of O-antigen Biosynthesis Gene Cluster
FIG. 20. Aquaria virulence studies of vAh strain ML09-119, the ΔymcA mutant (containing the chloramphenicol resistance cassette), and the markerless mutants of the O-antigen capsular genes ymcA and ymcC in channel catfish. For each treatment, 3 replicate groups of 10 fish each were injected intraperitoneally with 200 μl of bacterial cells adjusted to a fixed concentration ( ̃6.09×106 CFU/fish).
FIG. 21. Protective effect of ΔymcA and ΔymcC markerless mutants. Fingerling catfish that had previously been IP injected with 107 CFU/fish for each respective mutant, as well as naïve fish, were challenged with ML09-119 at the dosage of 4×106 CFU/fish 21 days post-injection. Survival rate is recorded after 24 hours. The average survival rate of naïve fish was 25±35.4%, while the ΔymcA markerless treatment group's survival rate was 88.9±16.7%, and the ΔymcC markerless treatment group's survival rate was 75±20.4%, suggesting that both mutants can provide protection against epidemic vAh strain ML09-119.



DETAILED DESCRIPTION
Disclosed herein are microbiocidal compositions. The disclosed microbiocidal compositions may be described using several definitions as discussed below.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” In addition, singular nouns such as “a bacterium,” “a carrier,” and “a vector” should be interpreted to mean “bacteria,” “carriers,” and “vectors,” unless otherwise specified or indicated by context.
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≦10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
As used herein, a “subject” or an “individual” and means an animal in need of treatment or prevention. Animals may include aquatic animals such as farmed fish (e.g. catfish or tilapia) and crustaceans (e.g., shrimp) having or at risk for developing an infection by a pathogenic microorganism such as Aeromonas hydrophila, Edwardsiella ictaluri, Edwardsiella tarda, Flavobacterium columnare, Streptococcus iniae, Yersinia ruckeri, Vibrio species and/or the oomycete fungus Saprolegnia. An animal may include a catfish at risk for developing Motile Aeromonas Septicemia (MAS).
Disclosed herein is a bacterium that has been made deficient in one or more genes associated with virulence. As used herein, “deficient” means that the bacterium does not express a functional form of a protein encoded by the gene. As such, deficiencies may include deletions, insertion, premature stop codons and the like, but preferably the bacteria disclosed herein have been made deficient in one or more genes associated with virulence via deletion of at least a portion of the gene, and preferably the entirety of the gene. For example, bacteria contemplated herein may be made deficient in one or more genes associated with the pathway for O-antigen and/or O-antigen capsule synthesis and secretion and myo-inositol catabolism and regulation.
Genes associated with the pathway for O-antigen and/or O-antigen capsule synthesis and secretion may include, but are not limited to ymcA, ymcB, ymcC, waaL (O-antigen ligase), wzy (O-antigen length determinant protein), polysaccharide export protein, and wzz (O-antigen length determinant protein). (See FIG. 19). Genes associated with myo-inositol catabolism and regulation may include, but are not limited to iolA (methylmalonate-semialdehyde dehydrogenase), iolB (5-deoxy-glucuronate isomerase), iolC (5-dehydro-2-deoxygluconokinase), iolD (COG3962 acetolactate synthase), iolE (myo-inosose-2-dehdratase), iolG1 (myo-inositol 2-dehdrogenase), and iolG2 (myo-inositol 2-dehdrogenase). (See FIG. 8). As such, bacteria contemplated herein may be made deficient in a gene encoding any of the polypeptides of SEQ ID NOs:11 (YmcA), 15 (unknown), 17 (YmcB), 19 (YmcC), 21 (O-antigen ligase), 23 (O-antigen length determinant protein Wzy), 25 (Polysaccharide Export Protein), 27 (O-antigen length determinant protein Wzz), 29 (methylmalonate-semialdehyde dehydrogenase IolA), 31 (COG3962 acetolactate synthase IolD), 33 (myo-inositol 2-dehydrogenase IolG1), 35 (myo-inositol 2-dehydrogenase IolG1), 37 (5-dehydro-2-deoxygluconokinase IolC), 39 (myo-inosose 2-dehdratase IolE), or 41 (5-deoxy-glucuronate isomerase IolB) or encoding a variant polypeptide having at least about 95% sequence identity to the polypeptide of any of SEQ ID NOs:11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41, wherein the variant polypeptide maintains or lacks the biological activity associated with any of 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41. Accordingly, bacteria contemplated herein, may be made deficient in a gene (or one or more genes) comprising a polynucleotide sequence of any of SEQ ID NOs:12 (ymcA), 16 (unknown), 18 (ymcB), 20 (ymcC), 22 (waaL), 24 (wzy), 26 (polysaccharide export protein), 28 (wzz), 30 (iolA), 32 (iolD), 34 (iolG2), 36 (ioG1), 38 (iolC), 40 (iolE), or 42 (iolB), or a gene comprising a polnucleotide sequence having at least 95% sequence identity to any of SEQ ID NOs:12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42.
Reference may be made herein to polypeptides and proteins, which terms may used interchangeably herein. For example, polypeptides contemplated herein may comprise the amino acid sequences of any of SEQ ID NOs:2, 3, 4, 5, 8, 11 or 13, or may comprise an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:2, 3, 4, 5, 8, 11 or 13. Mutant polypeptides or variant polypeptides may include one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to the wild-type polypeptide, where optionally the mutant polypeptides or variant polypeptide may exhibit the biological activity of the wild-type polypeptide or alternatively may lack the biological activity of the wild-type polypeptide.
Reference also is made herein to polynucleotides and nucleotide sequences, which terms may be used interchangeably herein. For example, polynucleotides that encode the polypeptides disclosed herein are contemplated (e.g., polynucleotides that encode the polypeptide of any of SEQ ID NOs:2, 3, 4, 5, 8, 11 or 13 or mutants or variants thereof). For example, contemplated herein are polynucleotides (e.g., DNA or RNA) comprising the nucleotide sequence of any of SEQ ID NOs:1, 6, 7, 12, or 14, or mutants or variants thereof, for example polynucleotides having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:1, 6, 7, 12, or 14.
Also contemplated are bacterial expression vectors that express the disclosed polypeptides or variants or mutants thereof. Vectors may include plasmids or other related vectors that may be used to transform appropriate host cells (e.g., E. coli and/or A. hydrophila), and the terms “vector” and “plasmid” may be used interchangeably in some embodiments disclosed herein. The transformed host cell may be cultured such that the polypeptide is expressed constitutively or after adding a reagent that induces expression (e.g., via an inducible promoter). Expression vectors as contemplated herein may include control sequences that modulate expression of the encoded polypeptide. Expression control sequences may include constitutive or inducible promoters (e.g., T3, T7, Lac, trp, or phoA, arabinose-inducible promoters, rhamnose-inducible promoters), ribosome binding sites, or transcription terminators.
The vectors disclosed herein may be utilized to transform host cells. Suitable host cells include bacterial. Suitable bacteria include, but are not limited to: Gram-negative bacteria such as Escherichia species (e.g., E. coli) and Aeromonas species (e.g. Aeromonas hydrophila), and other Gram-negative bacteria, (e.g., Edwardsiella species such as Edwardsiella ictaluri)
The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).
The terms “amino acid” and “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence (which terms may be used interchangeably), or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid, sequence associated with the recited protein molecule.
The amino acid sequences contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative polypeptide may include conservative amino acid substitutions relative to a reference polypeptide. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Table 1 provides a list of exemplary conservative amino acid substitutions.










TABLE 1







Original
Conservative



Residue
Substitution









Ala
Gly, Ser



Arg
His, Lys



Asn
Asp, Gln, His



Asp
Asn, Glu



Cys
Ala, Ser



Gln
Asn, Glu, His



Gly
Ala



His
Asn, Arg, Gln, Glu



Ile
Leu, Val



Leu
Ile, Val



Lys
Arg, Gln, Glu



Met
Leu, Ile



Phe
His, Met, Leu, Trp, Tyr



Ser
Cys, Thr



Thr
Ser, Val



Trp
Phe, Tyr



Tyr
His, Phe, Trp



Val
Ile, Leu, Thr










Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation of a reference polypeptide or a 5′-terminal or 3′-terminal truncation of a reference polynucleotide).
A “fragment” is a portion of an amino acid sequence or a polynucleotide which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptides respectively. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.
A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
“Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.
The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences, “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
Percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See. e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence listing, may be used to describe a length over which percentage identity may be measured.
A “variant,” “mutant,” or “derivative” of a particular polypeptide sequence is defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of polypeptides may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides. A “variant” or a “derivative” may have substantially the same functional activity as a reference polypeptide. For example, a variant or derivative of a cysteine protease may have cysteine protease activity (e.g., autoproteolytic cysteine protease activity).
The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.
“Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial, combination is often, accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. I 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
“Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
“Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A “composition comprising a given amino acid sequence” and a “composition comprising a given polynucleotide sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. The compositions may be stored in any suitable form including, but not limited to, freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. The compositions may be aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, and the like).
The presently disclosed composition and methods may include or utilize Gram negative bacteria such as Aeromonas spp, and Edwarsiella spp. Aeromonas spp. may include but are not limited to Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii. (See also Aeromonas spp, disclosed in Martino et al., “Determination of Microbial Diversity of Aeromonas Strains on the Basis of Multilocus Sequence Typing, Phenotype, and Presence of Putative Virulence Genes,” Applied Environmental Microbiology. 2011 July; 77)14): 4986-5000). The complete genome of Aeromonas hydrophila ML09-110 (5,024,500 bp) has been sequenced and is deposited in GenBank under accession number CP005966.1. Edwarsiella spp. may include but are not limited to Edwardsiella ictaluri. The complete genome of Edwardsiella ictaluri 93-146 (3,812,301 bp) has been sequenced and is deposited in the National Center for Biotechnology Information (NCBI) database under accession number NC_012779.2.
The disclosed compositions and methods may include or utilize Aeromonas hydrophila or an Aeromonas species that is closely related to Aeromonas spp. an Aeromonas species that is closely related to Aeromonas hydrophila may be defined as a species comprising a 16S rDNA sequence comprising SEQ ID NO:9 or comprising a 16S rDNA sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.
The disclosed compositions and methods may include or utilize Edwarsiella ictaluri or an Edwardsiella species that is closely related to Edwarsiella ictaluri. An Edwardsiella species that is closely related to Edwarsiella ictaluri may be defined as a species comprising a 16S rDNA sequence comprising SEQ ID NO: 10 or comprising a 16s rDNA sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:10.
“Percentage sequence identity” between two polynucleotide sequences may be determined by aligning two sequences using the Basic Local Alignment Search Tool (BLAST) available at the National Center for Biotechnology Information (NCBI) website (i.e., “bl2seq” as described in Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety). For example, percentage sequence identity between nucleotide sequences disclosed herein may be determined by aligning these two sequences using the online BLAST software provided at the NCBI website.
“Percentage sequence identity” between two deoxyribonucleotide sequences may also be determined using the Kimura 2-parameter distance model which corrects for multiple hits, taking into account transitional and transversional substitution rates, while assuming that the four nucleotide frequencies are the same and that rates of substitution do not vary among sites (Nei and Kumar, 2000) as implemented in the MEGA 4 (Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599), preferably version 4.0.2 or later. The gap opening and extension penalties are set to 15 and 6.66 respectively. Terminal gaps are not penalized. The delay divergent sequences switch is set to 30. The transition weight score is 35 set to 0.5, as a balance between a complete mismatch and a matched pair score. The DMA weight matrix used is the IUB scoring matrix where x's and n's are matches to any IUB ambiguity symbol, and all matches score 1.9, and all mismatched score O.
The presently disclosed vaccine composition may be administered to treat or prevent infection by bacterial pathogens of aquatic animals such as farmed fish (e.g. catfish or tilapia) and crustaceans (e.g., shrimp). In particular, the methods may be utilized to control or prevent the infection or colonization of catfish (e.g., Ictaluri punctatus Rafinesque) by pathogenic bacteria or fungi or colonization of environments in which catfish live or are raised (e.g., aquaculture ponds).
The term “sample” is used herein in its broadest sense. A sample may comprise a biological sample from an animal (e.g., a biological sample obtained from aquatic animals such as farmed fish (e.g. catfish or tilapia) and crustaceans (e.g., shrimp)) or a sample taken from an environment, (e.g., a water sample from a pond or a swabbed surface sample taken from a container or instrument).
Aeromonas hydrophila, a free-living, Gram-negative bacterium, is one of the most common bacteria in freshwater habitats worldwide. A. hydrophila infection results in hemorrhagic septicemia and heavy mortalities in cultured and wild fish. Antibiotics and chemotherapeutic drugs have been used for disease management in feed additives and in direct administration into fish pond water; however, there has been an increase in drug resistant strains (Son et al. 1997. Letters in Appl. Microbiol. 24: 479-482; (Harikrishnan and Balasundaram. 2005. Reviews in Fisheries Science 13: 281-320). Extensive research efforts and strategies have not yet resulted in the development of a safe and effective vaccine. There is still no product that has been licensed for use against the motile aeromonads within the United States (Cipriano, R. C. 2001. Revision of Fish Disease Leaflet 68, U.S. Dept. Interior, Fish and Wildlife Service Div. of Fishery Res., Washington, D.C.). Thus, there is a need, particularly in the aquaculture industry, for an efficacious and safe vaccine.
The disclosed vaccine compositions may be administered to aquatic animals by any suitable method. Suitable administration methods may include injection (e.g., intraperitoneal injection), oral administration, or by administering the compositions to an aqueous environment in which the aquatic animal resides (e.g., bath immersion).
The disclosed vaccines preferably may be administered to protect an aquatic animal from infection by a homologous strain of bacteria that was used to prepare the vaccine composition and/or to protect the aquatic animal from infection by a heterologous strain of bacteria (i.e., strains which are different from those used in the preparation of the vaccine composition). Moreover, the vaccine compositions may include live attenuated bacteria, or killed attenuated bacteria where the killed bacteria has been inactivated by chemical treatment (e.g., treatment with formalin, phenol, or beta-propiolactone) or by physical treatment (e.g., treatment with heat and/or pressure). Preferably, the disclosed vaccine compositions induce both antibody and cellular immune responses and can provide years of protection after the vaccine compositions are administered to an aquatic animal.
The vaccine compositions preferably control infection by A. hydrophila in a variety of aquatic animals when administered thereto, including channel catfish (Ictaluri punctutus). In addition, the vaccine compositions preferably control infection by A. hydrophila in a variety of other aquatic animals, including but not limited to tilapia (Oreochromis sp.), American, European, and Japanese eels (Anguilla sp), salmonids (Oncorhynchus sp. and Salmo sp.), striped bass and hybrid-striped bass (Morone chrysops×M. saxatilis), flounders (Seriola sp.), seabream (Sparus sp.), sea perch (Lates calcarifer), and the estuarine grouper (Epinephelus tawine), walleye (Zander vitreum), centrachids (such as large-mouth bass, Micropterus salmoides), brown bullheads (Nebulosus sp.) bait minnows (Pimephales promelas), golden shiners (Netemigonus crysoleucas), goldfish (Carassius auratus), carp (Cyprinus carpio) and aquarium fish species such as black mollies (Poecilia sphenops) and platies (Xiphophorus maculates).
“Vaccine” is defined herein in its broad sense to refer to any type of biological agent in an administrable form capable of stimulating a protective immune response in an animal inoculated with the vaccine. For purposes of this invention, the vaccine may comprise one or more live attenuated mutants of A. hydrophila or killed or inactivated mutants of A. hydrophila. 
Vaccination may be accomplished by administering a vaccine composition by injection or through oral ingestion or by means of aqueous immersion. The bacterial agent is prepared for administration by formulation in an effective immunization dosage with an acceptable carrier, diluent, or excipient. The expressions “effective immunization dosage” and “immunologically effective amount or dosage” may be defined as being that amount which will induce complete or partial immunity in a treated fish against subsequent challenge by a virulent strain of A. hydrophila. Immunity is considered as having been induced in a population of fish when the level of protection for the population (evidenced by a decrease in the number of infected fish or a decrease in the severity of infection) is significantly higher than that of an unvaccinated control group (measured at a confidence level of at least 80%, preferably measured at a confidence level of 95%). One measure of protection following experimental challenge is relative percent survival (RPS) as described by Amend (1981, Dev. Bio. Stand. 49: 447-454) herein incorporated by reference. RPS is calculated according to the following formula:




  RPS
  =
  
    1
    -
    
      
        
          %
          ⁢
          
            
          
          ⁢
          vaccinate
          ⁢
          
            
          
          ⁢
          mortality
        
        
          %
          ⁢
          
            
          
          ⁢
          control
          ⁢
          
            
          
          ⁢
          mortality
        
      
      ×
      100
    
  




In some embodiments, a positive vaccine effect is indicated by a RPS equal to or greater than about 50%, 60%, 70%, 80%, 90%, or higher. The vaccine may be administered to 7-10 day old aquatic animals such as fish by bath immersion, injection, and/or any oral delivery or immersion device. For example, fish may be vaccinated by immersion in water containing about 1×104 to about 1×108 colony forming units (CFU)/mL of attenuated A. hydrophila for 10 min at a density of about 50/L and a temperature of about 25° C. However, these parameters may be varied such that a sufficient level of vaccination is obtained without inducing stressful conditions or loss of fish. Suitable concentrations of A. hydrophila may range from about 1×104 CFU/ml, 1×105 CFU/ml, 1×106 CFU/ml, or 1×107 CFU/ml, to about 1×108 CFU/ml of immersion medium. Suitable vaccination times may range from about 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 40 min, or 50 min to about 60 min, preferably from about 2 min to about 15 min. Temperature of the inoculation media may range within the physiologically acceptable limits of the fish involved, channel catfish at tilapia preferably from about 18° C. to about 32° C., most preferably from about 20° C. to about 30° C. Concentrations of fish treated in the inoculation medium typically range from about 10, 20, 30, 40, 50, 60, 70, 80, or 90, to about 100 fish/L, but, in the alternative, may be determined on a weight basis and range from about 0.1, 0.2, 0.5, 1.0, 1.5, or 2.0 to about 2.5 kg/L.
The vaccine compositions also may be administered to aquatic animals such as fish by intraperitoneal injection using about 1×104 CFU, 1×105 CFU, 1×106 CFU or 1×107 CFU to about 1×108 CFU per fish. The vaccine can be effectively administered any time after the fish attains immunocompetence, which for channel catfish, after 7-10 days post-hatch and for tilapia is at about two to fourteen days post-hatch. Other species of fish susceptible to A. hydrophila can be immunized after 21-30 days post-hatch or when they become immunocompetent to modified live vaccine administered by immersion.
To produce large amounts of the recombinant A. hydrophila bacteria for preparation of a vaccine composition, the bacteria may be cultivated under any conventional conditions and on media which promote growth of A. hydrophila. Without being limited thereto, the recombinant A hydrophila may be grown on a variety of solid or liquid media types, including but not limited to tryptic soy agar or Helellea agar. In the alternative to growth on solid media, it is also envisioned that the strain may be grown in liquid culture. Without being limited thereto, conventional tryptic soy broth is preferred.
Following propagation, the recombinant A hydrophila may be recovered for use as a vaccine composition. Cells, particularly those produced by liquid culture, may be optionally concentrated, for example, by centrifugation or filtration. Live cells of the A. hydrophila strain are prepared for administration by formulation in an immunologically effective amount or dosage to the fish. The dose may further include pharmaceutically acceptable carriers and adjuvants known in the art such as water, physiological saline, mineral, oil, vegetable oils, aqueous sodium carboxymethyl cellulose, or aqueous polyvinylpyrrolidone. The vaccine formulations may also contain optional adjuvants, antibacterial agents or other pharmaceutically active agents as are conventional in the art. Without being limited thereto, suitable adjuvants include but are not limited to mineral oil, vegetable oils, alum, and Freund's incomplete adjuvant. Still other preferred adjuvants include microparticles or beads of biocompatible matrix materials. The microparticles may be composed of any biocompatible matrix materials as are conventional in the art, including but not limited to, agar and polyacrylate. The practitioner skilled in the art will recognize that other carriers or adjuvants may be used as well. For example, other adjuvants which may be used are described by Webb and Winkelstein (In: Basic & Clinical Immunology, 1984. Stites et al. (Eds.), Fifth Edition, Lange Medical Publications, Los Altos, Calif., pages 282-285), the contents of which are incorporated by reference herein.
ILLUSTRATIVE EMBODIMENTS
The following embodiments are illustrative and are not intended to limit the claimed subject matter.
Embodiment 1
An attenuated Aeromonas spp. bacterium that has been genetically modified by recombination to be deficient of gene encoding the polypeptide of any of SEQ ID NOs:11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41 or to be deficient of a gene encoding a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of any of SEQ ID NOs:11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41, optionally where the gene encoding the polypeptide is a gene comprising the polynucleotide of any of SEQ ID NOs: 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42 or a polynucleotide having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide of any of SEQ ID NOs:12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42.
Embodiment 2
The attenuated Aeromonas spp. of embodiment 1, wherein the bacterium is selected from the group consisting of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii. 
Embodiment 3
The attenuated Aeromonas spp. bacterium of embodiment 1 or 2, wherein the bacterium has been genetically modified by a method that includes (a) deleting at least a portion of the gene ymcA by recombination and inserting a selectable marker in place of the deleted portion of the ymcA gene, and (b) subsequently deleting the selectable marker to create a markerless bacterium deficient of gene ymcA, wherein optionally the selectable marker is a gene expressing a protein for antibiotic resistance (e.g., chloramphenicol resistance gene (SEQ ID NO:7) expressing the chloramphenicol resistance protein (SEQ ID NO:8).
Embodiment 4
The attenuated Aeromonas spp. bacterium of embodiment 3, wherein the bacterium has been genetically modified by (a) transferring a recombineering system into the bacterium, wherein the recombineering system deletes at least a portion of the ymcA gene (e.g. SEQ ID NO:12) and replaces the portion with the selectable marker flanked by two recombinase recognition target sites (e.g., SEQ ID NO:6 or SEQ ID NO:14); (b) selecting the bacterium for expression of the selectable marker; (c) curing the selected bacterium of the recombineering system; (d) transferring a vector that expresses a recombinase into the selected bacterium (e.g., flp recombinase (SEQ ID NO:5) or cre recombinase (SEQ ID NO:13)), wherein the recombinase recognizes the two recombinase recognition target sites (e.g., SEQ ID NO:6 or SEQ ID NO:14); (e) selecting the bacterium for lack of expression of the selectable marker; and (f) curing the selected bacterium of the vector that expresses the recombinase.
Embodiment 5
The attenuated Aeromonas spp. bacterium of embodiment 4, wherein the recombineering system comprises: a mobilizable recombineering vector; and a linear DNA molecule comprising the following contiguous sequences in 5′ to 3′ order: (i) a first nucleotide sequence of at least 10 nucleotides (or at least 20, 30, 40, 50 or more nucleotides) having sequence identity with the gene ymcA (SEQ ID NO:2), (ii) a second nucleotide sequence comprising the first of the recombinase recognition target sites (e.g., SEQ ID NO:6or SEQ ID NO:14), (iii) a third nucleotide sequence that expresses a selectable marker (e.g., chloramphenicol resistance gene (e.g., (SEQ ID NO:7) expressing the chloramphenicol resistance protein (SEQ ID NO:8)), (iv) a fourth nucleotide sequence comprising the second of the recombinase recognition target sites (e.g., SEQ ID NO:6 or SEQ ID NO:14), and (v), a fifth nucleotide sequence of at least 10 nucleotides (or at least 20, 30, 40, 50 or more nucleotides) having sequence identity with the gene ymcA (SEQ ID NO:2) that is different than the first nucleotide sequence of (i) and is upstream or downstream of the first nucleotide sequence of (i), wherein after a recombinase is expressed in the bacteria (e.g., flp recombinase (SEQ ID NO:5) or cre recombinase (SEQ ID NO:13)), the recombinase recombines the recombinases recognition target sites (e.g., SEQ ID NO:6 or SEQ ID NO:14) to remove the selectable marker (e.g., (SEQ ID NO:7) expressing the chloramphenicol resistance protein (SEQ ID NO:8)) and the portion of the ymcA gene (e.g., SEQ ID NO:2) that is deleted is replaced with one recombinase recognition target site (e.g., SEQ ID NO:6 or SEQ ID NO:14).
Embodiment 6
A vaccine composition comprising the attenuated Aeromonas spp. bacterium of any of embodiments 1-5 and a suitable carrier, diluent, or excipient, and optionally an adjuvant.
Embodiment 7
The vaccine composition of embodiment 6, wherein the attenuated Aeromonas spp. bacterium has been inactivated by chemical treatment, such as formalin treatment, phenol treatment, or beta-propriolactone treatment, and/or by physical treatment such as heat and/or pressure.
Embodiment 8
The vaccine composition of embodiment 7, wherein the attenuated Aeromonas spp. bacterium has been inactivated by formalin treatment.
Embodiment 9
A method for vaccinating an aquatic animal against infection by an Aeromonas spp. bacterium, the method comprising administering the attenuated Aeromonas spp. of any of claims 1-5 to the animal or administering a vaccine composition of any of claims 6-8 to the aquatic animal.
Embodiment 10
The method of embodiment 9, wherein the aquatic animal is a channel catfish (Ictaluri punctata).
Embodiment 11
The method of embodiment 9 or 10, wherein the aquatic animal is administered the vaccine composition by intraperitoneal injection.
Embodiment 12
The method of any of embodiments 9-11, wherein the aquatic animal is administered the vaccine composition at a dose that delivers 104-108 CFU of attenuated Aeromonas spp. bacteria per aquatic animal.
Embodiment 13
The method of embodiment 9 or 10, wherein the aquatic animal is administered the vaccine composition by immersing the aquatic animal in an aqueous medium comprising the vaccine composition.
Embodiment 14
The method of embodiment 13, wherein the aqueous medium has a concentration of 104-108 CFU/ml of attenuated Aeromonas spp. bacteria.
Embodiment 15
A mobilizable recombineering vector: (a) that comprises a polynucleotide sequence comprising oriT (SEQ ID NO:1) or a polynucleotide sequence having at least about 95% sequence identity with oriT (SEQ ID NO:1) wherein the polynucleotide sequence functions as an origin of transfer; and (b) a polynucleotide sequence that expresses lambda Gam polypeptide (SEQ ID NO:2) or a polypeptide having at least 95% sequence identity to lambda Gam polypeptide (SEQ ID NO:2), wherein the polypeptide functions as an inhibitor of E. coli RecBCD exonuclease; (c) a polynucleotide sequence that expresses lambda Exo polypeptide (SEQ ID NO:3) or a polypeptide having at least 95% sequence identity to lambda Exo polypeptide (SEQ ID NO:3), wherein the polypeptide functions as a 5′→3′ double-stranded DNA specific nuclease; and (d) a polynucleotide sequence that expresses lambda Beta polypeptide (SEQ ID NO:4) or a polypeptide having at least 95% sequence identity to lambda Beta polypeptide (SEQ ID NO:4), wherein the polypeptide functions as a ssDNA annealing protein.
Embodiment 16
The vector of embodiment 13, wherein the aqueous medium has a concentration of 104-108 CFU/ml of attenuated Aero 15, wherein the lambda Gam polypeptide (SEQ ID NO:2), the lambda Exo polypeptide (SEQ ID NO:3), and the lambda Beta polypeptide (SEQ ID NO:4) are inducibly expressed from the vector.
Embodiment 17
The vector of embodiment 15 or 16, wherein the vector further expresses a recombinase selected from bacteriophage P1 cre recombinase (SEQ ID NO:13) or a recombinase having at least about 95% sequence identity to bacteriophage P1 cre recombinase (SEQ ID NO:13), and Saccharomyces cerevisiae flp recombinase (SEQ ID NO:5) or a recombinase having at least about 95% sequence identify to Saccharomyces cerevisiae flp recombinase (SEQ ID NO:5).
Embodiment 18
The vector of embodiment 17, wherein the recombinase is inducibly expressed.
Embodiment 19
A method for genetically modifying an Aeromonas spp. bacterium to obtain a recombinant bacterium, the method comprising one or more of the following steps: (a) transferring a recombineering system into the bacterium, wherein the recombineering system deletes at least a portion of a target sequence and replaces the portion with a selectable marker flanked by two recombinase recognition target sites; (b) selecting the bacterium for expression of the selectable marker; (c) curing the selected bacterium of the recombineering system; (d) transferring a vector that expresses a recombinase into the selected bacterium, wherein the recombinase recognizes the two recombinase recognition target sites; (e) selecting the bacterium for lack of expression of the selectable marker; and (f) curing the selected bacterium of the vector that expresses the recombinase.
Embodiment 20
The method of embodiment 19, wherein deletion of the target sequence results in attenuating the bacterium.
EXAMPLES
The following examples are illustrative and are not intended to limit the claimed subject matter.
Example 1
Conjugal Transfer of a Recombineering System to Generate and Complement Markerless Genome Modifications\
Abstract
The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. In this study, we were unable to use an available recombineering system to construct genetic mutants in the fish pathogens Edwardsiella ictaluri and Aeromonas hydrophila due to an inability to introduce plasmids into these disease isolates via electroporation. Herein we describe the development of a modified PCR-based λ Red-mediated recombineering system for efficient deletion of genes in gram-negative bacteria, which we have used in different E. ictaluria and A. hydrophila strains. A series of conjugally transferrable plasmids were constructed by cloning oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we knocked out a total of 16 different genes from the genomes of three different strains of E. ictaluri and A. hydrophila. To generate a markerless mutant, we engineered the λ Red cassette and flp recombinase under the control of arabinose- and rhamnose-inducible promoters, respectively, and introduced this construct onto a conjugally transferrable and temperature sensitive plasmid. Using this system, we generated markerless gene deletion mutants in A. hydrophila including a mutant in a genetic operon. In order to formally demonstrate the contribution of this specific operon to virulence, we needed to complement this entire operon. To accomplish this we developed a highly efficient and novel PCR-free cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferable plasmid. This system should be applicable in diverse Gram-negative bacteria for modification and complementation of genomic elements including larger elements such as operons, genomic islands, and prophages in bacterial isolates that cannot be manipulated using currently available genetic tools.
Introduction
Genetic manipulation of bacterial strains provides critical information on the contributions of specific loci to virulence or other cellular functions, and many systems have been developed to achieve genetic knockouts and modifications (1-3). The modification of bacterial genomes using counter-selectable plasmid-based double-crossover methods are labor intensive and sometimes very difficult to achieve due to the low frequency of the recombination events (4-6). In contrast, the λ Red recombineering system (7,8) has many advantages as a fast, efficient and reliable means of generating targeted genetic modifications in prokaryotes (9,10) and eukaryotes (11). The λ Red system expresses Exo, Beta and Gam proteins that work coordinately to recombine single and double stranded DNA (9, 10, 12, 13), and has been exploited for genome modifications in Escherichia coli and other Gram-negative bacteria (9, 10, 12). Exo has a 5′ to 3′ double stranded DNA (dsDNA)-dependent exonuclease activity for generating 3′ single stranded DNA (ssDNA) overhangs (14-16) which then serve as a substrate for ssDNA-binding protein. Beta to anneal complementary DNA strands for recombination (17-19). Gam, an inhibitor of host exonuclease activity due to RecBCD (20), helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA. Unlike recA-dependent homologous recombination which requires longer regions of sequence homology with the targeted genetic region (21), the λ Red apparatus can efficiently recombine DNA with homologous regions as short as 30 to 50 bp which can directly be incorporated into oligonucleotide primers in a PCR (9,10). The recombineering technique is widely used to generate precise deletions (10), substitutions (22), insertions (23) or tagging (24) of targeted genes. One of the biggest advantages of the recombineering method is that modifying DNA can precisely eliminate the antibiotic selection markers for subsequent modification of the targeted DNA (9, 10, 25).
While this recombineering system works well in a model bacterium such as E. coli (7,8) bacteria often express restriction endonucleases that make them recalcitrant to foreign DNA even among naturally competent strains (26,27). In fact, it was through experimental infections of E. coli strains with bacteriophage λ that led to the discovery of restriction-modification (RM) systems (28). Overcoming host RM systems can be accomplished via the passage of plasmids through a methylation-minus E. coli strain (29), but in highly methylated bacterial strains it may be necessary to use an in vitro or in vivo methylation strategy to achieve more efficient electroporation (30-32). However, modulating the plasmid DNA methylation status is inefficient and labor-intensive compared to using conjugal transfer to introduce foreign DNA into a bacterial strain using a broad host range plasmid like IncP when electroporation is problematic (33-35).
Our need to generate target genetic deletions in gram-negative bacterial pathogens of farmed catfish led to the development of recombinogenic plasmids that could be introduced via conjugation. Our studies focused on two bacterial pathogens, including Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), which is responsible for significant economic loss to the channel catfish industry in the Southeastern United States (36). Fish diseases caused by strains of E. ictaluri are also frequently reported in catfish farming in Asia (37). In addition to E. ictaluri, we also had an interest in studying the pathogenesis of Aeromonas hydrophila, because beginning in 2009 US catfish farmers experienced epidemic disease outbreaks of motile Aeromonas septicemia (MAS) caused by a highly virulent Aeromonas hydrophila strain (38). This newly emergent and virulent A. hydrophila strain, which has been implicated to have an Asian origin (39), is responsible for killing millions of pounds of food-sized channel catfish in the US (39). Though, both E. ictaluri and A. hydrophila pose serious threats to the US catfish industry (36, 40, 41) as well as global fish farming (37,42), highly efficient genome modification techniques have not been developed yet to study the virulence mechanisms and permit generation of markerless vaccines for these two pathogens.
Though recombineering techniques are widely being used for genome modification of domesticated laboratory isolates, the implementation of these techniques for primary pathogenic isolates is quite challenging. In this study, we modified the available λ Red recombination tools (13,43) to generate markerless mutants of E. ictaluri and A. hydrophila. A novel dual inducible Redαβλγ and Flp recombinase plasmid was constructed to facilitate the removal of antibiotic resistance marker followed by recombineering. In addition, we also developed a novel in vivo error-free cloning system that can be used to clone large fragments of genomic DNA without PCR amplification of the inserts and used to complement larger genomic regions.
Materials and Methods
Bacterial Strains and Plasmids.
The list of bacterial strains and plasmids used in this study is presented in Table 2. E. ictaluri and A. hydrophila strains were routinely grown on Trypticase Soy Broth or Agar (TSB/TSA) medium at 28° C. and 30° C., respectively. E. coli SM10λpir (44) was routinely used for the conjugal transfer of mobilizable plasmids to strains of E. ictaluri and A. hydrophila. E. coli BW25141 and BT340 (10) were received from the Yale University Genetic Stock Center. When antibiotic selection was required, bacterial growth media were supplemented with chloramphenicol (15.0 and 25.0 μg/ml for strains of E. ictaluri and A. hydrophila, respectively), tetracycline (10.0 μg/ml) and/or colistin (10.0 μg/ml).
Recombinant DNA Techniques, and Conjugal Transfer of Recombinogenic Plasmids.
The list of primers used in this study are presented in Table 3. All primers were purchased from Eurofins MWG Operon (Huntsville, Ala.). For cloning purposes, we routinely used electrocompetent E. coli (“E. cloni 10G”, Lucigen Corp., Middleton, Wis.). PCR amplifications were carried out using EconoTaq DNA polymerase (Lucigen Corp.), Pfu DNA polymerase (Life Technologies, Grand Island, N.Y.) and TaKaRa Ex Taq (Clontech, Mountain View, Calif.) as appropriate. Genomic DNAs and plasmids were extracted using E.Z.N.A. DNA Isolation Kit (Omega Biotek, Atlanta, Ga.) and FastPlasmid Mini Kit (5 Prime, Gaithersburg, Md.), respectively. Restriction enzymes and T4 DNA Ligase (Quick ligase) used for restriction digestion of DNAs, and cloning, respectively were purchased from New England Biolabs (Ipswich, Me.). Restriction digested DNAs with sticky ends were blunt-ended using a DNA Terminator kit (Lucigen Corp.). Digested DNAs and ligation mix were purified using DNA Clean and Concentrator-5 (Zymo Research, Irvine, Calif.). DNA samples were quantitated using a Qubit 2.0 Fluorometer (Life Technologies). The mobilizable recombinogenic plasmids pMJH46 and pMJH65, and dual expression plasmid pMJH95 bearing the λ-Red cassette were introduced into E. coli SM10λpir by electroporation according to a previous published method (45). Plasmids were conjugally transferred into E. ictaluri and A. hydrophila by filter mating experiments according to the methods described previously (Maurer et al., 2001). E. ictaluri and A. hydrophila transconjugants were selected of LB plates supplemented with chloramphenicol and colistin, or tetracycline and colistin, respectively. The introduction of plasmids into E. ictaluri or A. hydrophila was confirmed by their growth in the presence of appropriate antibiotics and by conducting PCR with a plasmid-specific printer set.
Construction of Broad Host Range Recombinogenic Plasmids.
A list of primers used in this study is presented in Table 3. The mobilizable plasmid pMJH46 was constructed by introducing the oriT sequence and chloramphenicol acetyltransferase (cat) into the recombinogenic plasmid pKD46 (46) which contains an arabinose-inducible λ-Red cassette (exo, bet and gam genes) required for recombineering (FIG. 1). The oriT sequence and cat gene were PCR amplified from pGNS-BAC (47) using primers MobF and MobR, and CatF and CatR, respectively. Amplicons for the oriT sequence and cat gene were fused by splicing by overlap extension (SOE) PCR (48) using primers Mob-intF (forward) and Cat-intR (reverse). The oriT-cat cassette and pKD46 plasmid were digested with EcoRV and NcoI, respectively. NcoI digested pKD46 plasmid was blunt-ended and ligated to oriT-cat cassette using a DNA Terminator kit (Lucigen Corp., Middleton, Wis.) and T4 DNA ligase (Promega, Wis.), respectively. The ligation mixture was then transformed into electrocompetent E. coli (E. cloni 10G, Lucigen, Corp.). Transformants were selected on 2×YT medium supplemented with ampicillin and chloramphenicol after incubation overnight at 30° C. The introduction of the oriT-cat cassette into pKD46, resulting in pMJH46, was confirmed by PCR and sequencing. To construct the recombinogenic plasmid pMJH65, plasmid pMJH46 was digested with BstZ17I and SfiI, and blunt-ended using the DNA Terminator kit. A tetracycline resistance gene (tetA) cassette was PCR amplified from pACYC184 using primers TetAF and TetAR and ligated to blunt-ended pMJH46 using T4 DNA ligase. The ligation mixture was then transformed into electrocompetent E. coli (E. cloni 10G, Lucigen Corp.), Transformants were selected on 2×YT medium supplemented with tetracycline after overnight incubation at 30° C. The construction of recombinogenic plasmid pMJH65 was confirmed by PCR and sequencing.
Construction of Dual Inducible Plasmid for Red and flp/FRT Recombination.
To construct a dual expression plasmid with arabinose-inducible Red cassette (exo, bet and gam) and rhamnose-inducible flippage (flp) gene under araPBAD and rhaPBAD promoters, respectively, we modified our recombinogenic plasmid pMJH46 by replacing the beta-lactamase (bla) and cat genes with rhaSRT and flp-tetA cassettes (FIGS. 1A, B, and C). The rhaSRT cassette amplified from genomic DNA of E. coli BL21. (DE3) using primers rhaSRT-BsaIF and rhaSRT-ApaLIR was cloned into a Bsal- and ApaLI-digested pMJH46 that resulted in plasmid pMJH52. To introduce rhamnose-inducible flp gene in pMJH52, the flp gene was amplified from pCP20 using primers Flp-pRhamF and Flp-pRhamR and was cloned into the pRham N-His vector under control of the rhaPBAD promoter that resulted in plasmid pPham-flp. This plasmid was then digested with XbaI and blunt-ended in order to insert a tetracycline resistance gene (tetA) that was PCR amplified from pMJH65 using primer tetAF and tetAR. After cloning this tetA cassette into the pRham-flp plasmid, resulting in plasmid pRham-flp-tetA, the flp-tetA cassette was digested with AlwNI and BsaAI, and blunt-ended for cloning into pMJH52. This flp-tetA cassette was then cloned into a BstZ17I and SfiI-digested and blunt-ended plasmid pMJH52. The resulting dual expression plasmid pMJH95 that contained arabinose-inducible λ-Red and rhamnose-inducible flip was verified by digestion with BsaI and ApaLI and agarose gel electrophoresis. The plasmid was introduced into A. hydrophila mutants to determine their efficiency in the excision of an antibiotic cassette flanked by FRT sequences.
Preparation of Linear Double Stranded DNA (dsDNA) Substrate for Recombineering.
The linear dsDNA fragments used for deletion of the ompLC gene from E. ictaluri with recombineering were generated by PCR amplification of the kanamycin resistance gene (kanR) cassette with its flanking FRT sequences using plasmid pKD4 as a template (10). All other linear dsDNA used for deletion of E. ictaluri genes eihA, dtrA and ptrA were PCR amplified from a kanR cassette located within the genome of this E. ictaluri Alg-08-183 ΔompLC mutant generated by recombineering. The linear dsDNA substrate used for recombineering in A. hydrophila were generated by PCR amplification of the cat gene or cat gene flanking with FRT sequences integrated within the genome of A. hydrophila ML09-119. Recombineering primers contained 50-60 bp of homology to the targeted genes at their 5′ ends and 20-22 bp of homology to the cat cassette at their 3′ ends. Primers were modified with four consecutive 5′ phosphorothioates bonds to reduce the chance of degradation by exonucleases during recombination. PCR amplification of the respective antibiotic resistance gene cassette using these gene-targeted primers was performed using high fidelity Takara Ex Taq Polymerase (Clontech) and EconoTaq PLUS GREEN (Lucigen Corp.). At least 10 positive PCRs of 50 μl volume were pooled together and purified by phenol-chloroform extraction followed by ethanol precipitation (45) or using the Wizard DNA Clean-Up System (Promega, Madison, Wis.). Purified PCR products were resuspended in nuclease-free water and used for transformation into electrocompetent E. ictaluri and A. hydrophila strains harboring recombinogenic plasmids pMJH46 and pMJH65, respectively.
Deletion of E. ictaluri and A. hydrophila Genes by Recombineering.
Electrocompetent E. ictaluri and A. hydrophila harboring recombinogenic plasmids pMJH46 and pMJH65, respectively, were prepared as described follows. E. ictaluri and A. hydrophila strains were grown and selected for mutants at 28° C. or 30° C. in the presence of chloramphenicol and tetracycline, respectively. Overnight grown cultures were diluted 1:70 in 40 ml of Super Optimal broth (SOB) medium supplemented with appropriate antibiotics and 10 mM of L-arabinose and grown with vigorous shaking until the OD600 attained a value of 0.45 or 0.6 for E. ictaluri and A. hydrophila, respectively. Cells were harvested by centrifugation at 5000×g for 8 min at 4° C., washed three times with ice-cold 10% glycerol and finally cells were concentrated 400-fold by resuspending with 100 μl of ice-cold GYT (10% glycerol, 0.125% yeast extract and 0.25% tryptone) medium or 10% glycerol. Freshly prepared electrocompetent cells were immediately used for electroporation. For deletion of targeted genes from E. ictaluri using recombineering, dsDNA substrate of appropriate concentrations were mixed with 50-55 μl of electrocompetent cells in a pre-chilled electroporation cuvette (0.1-cm gap), and pulsed at 1.8 kV, 25 μF and 200Ω using an Eppendorf Electroporator 2510 (Hamburg, Germany). For A. hydrophila, the same electroporation procedures were followed with the exception that cells were pulsed at 1.2 kV. Immediately after electroporation, 950 μl of SOC supplemented with 10 mM of L-arabinose (for catabolite repression) was added and incubated at an appropriate temperature with vigorous shaking for at least 4 hrs. Cells were then spread onto 2×YT agar plates supplemented with kanamycin and chloramphenicol for E. ictaluri and A. hydrophila, respectively, and incubated at an appropriate temperature to obtain mutants with the targeted deletions. The correct deletions of the targeted genes were confirmed by PCR and sequencing as previously described (10). To determine the effect of 1) phosphorothioate-modified primers, 2) the size of the gene-specific region of homology and 3) the concentration of the dsDNA substrates on recombination frequencies, each experiment was repeated independently at least three times.
Flp-Mediated Excision of Antibiotic Resistance Gene Cassettes to Generate Unmarked Mutants.
Before removal of the antibiotic resistance gene cassettes using Flp/FRT mediated recombination, recombinogenic plasmids were cured from the mutants of E. ictaluri and A. hydrophila. Plasmid pMJH46 was cured from E. ictaluri mutants by growing cells on TSB medium at 28° C. until the OD600 attained a value of 1.0 and then heat-inducing cells by incubation at 43° C. for 1 hr with shaking at 250 rpm. Heat-induced cultures were serially diluted in sterile water and spread for isolated colonies onto BHI Blood Agar plates that were then incubated at 28° C. for 36 hours. To cure plasmid pMJH65 from A. hydrophila mutants, cultures were grown in TSB broth at 37° C. overnight and streaked onto TSA plates for isolated colonies. The loss of plasmid pMJH46 and pMJH65 from E. ictaluri and A. hydrophila mutants were confirmed by determining the lack of ability of individual colonies to grow on TSA plates supplemented with chloramphenicol and tetracycline, respectively. Plasmid pCP20 that contains the Flp recombinase (49) required for FRT sequence-specific recombination was electroporated into E. ictaluri mutants according to the methods described above. E. ictaluri mutants harboring pCP20 were selected on 2×YT agar plates supplemented with chloramphenicol. These E. ictaluri mutants were grown in TSB at 28° C. until OD600 of 1.0 and temperature was shifted by incubating at 37° C. for 1 hr with shaking at 250 rpm to induce the removal of kanamycin resistance gene cassette by FLP recombinase. To obtain isolated colonies diluted cultures were plated onto BHI Blood Agar plates and incubated at 28° C. for up to 36 hours. Dual expression plasmid pMJH95 (pCMT-flp) constructed in this study was conjugally transferred to A. hydrophila mutants as described above and induced for the removal of chloramphenicol resistance gene cassette by incubating at 37° C. Colonies grown on non-selective plates that subsequently failed to grow on antibiotic selective plates were tested by PCR and sequencing to confirm the Flp-mediated excision of antibiotic resistance gene cassettes introduced by recombineering.
Cloning Large Genomic Inserts without PCR Amplification.
To construct a small, conjugally transferrable, and low copy-number plasmid backbone, the cat gene and p15A origin of replication (oriR) were PCR amplified from the genome of A. hydrophila ML09-119Δvgr3 (generated in this study) and pACYC184, respectively. The reverse primer used for amplification of the cat gene contains the 87 bp oriT sequence (Table 3) to facilitate the conjugal transfer of large insert clones to Gram-negative bacteria. The cat-oriT cassette and oriR sequence were fused together to construct a 2003 bp plasmid backbone cat-oriT-oriR (pMJH97) using SOE PCR with outermost primers. To clone the ymcABC operon of A. hydrophila ML09-119, the pMJH97 plasmid backbone was PCR amplified using primers that contain 60 and 63 bp, respectively, of homologous sequence specific to the upstream region of the ymcABC operon. To facilitate the restriction digestion of the regions flanking ymcABC and its contiguous cat-oriT-oriR cassette integrated within the genome, an Acc65I restriction site (GGTACC) was introduced between the 60 bp homologous sequence and cat gene priming site of the forward primer. Purified PCR products were introduced into A. hydrophila ML09-119 harboring plasmid pMJH65 by electroporation for genomic integration by recombineering. Colonies selected on 2×YT plates containing chloramphenicol were subjected to PCR to confirm the correct integration of the pMJH97 backbone plasmid into the genome, and amplicons of the expected size were selected for sequencing. Once the correct integration was confirmed, genomic DNA was extracted from ML09-119::cat-oriT-oriR that was restriction digested with Acc65I. Blunt-ended and purified genomic DNA fragments were self-ligated, electroporated and selected on 2×YT plates with chloramphenicol for cloning into E. coli (E. cloni 10G, Lucigen Corp.). The cloned plasmid pYmcABC was verified by PCR and sequencing for the presence of the complete ymcABC operon as an insert. Once the complete ymcABC cloning was confirmed, the pYmcABC was introduced into E. coli SM10λpir electroporation and conjugally transferred to A. hydrophila as described above.
Nucleotide Sequence Accession Numbers.
The sequences of pMJH46 and pMJH65 were deposited to the NCBI GenBank sequence database under accession numbers JQ070344 and KF195927, respectively.
Results
Construction of Conjugally Transferable Recombinogenic Plasmids.
The expression of exo, bet and gam within bacterial cells substantially improves their recombination frequencies that can be exploited to modify bacterial genomes by recombineering (13). Though published reports indicate that some E. ictaluri strains are capable of accepting foreign DNA of up to 45 kb by electroporation (51), our repeated attempts failed to introduce the recombinogenic plasmid pKD46 (13) into primary disease isolates of E. ictaluri or A. hydrophila. To introduce the recombinogenic λ-Red cassette into E. ictaluri, a mobilizable plasmid was constructed by introducing the ‘mob cassette’ (oriT region, traJ and traK) along with a chloramphenicol resistance (cat) gene into pKD46, resulting in plasmid pMJH46 (FIG. 1A, accession no. JQ070344). The cat gene introduction broadens the applicability of this plasmid since some E. ictaluri strains are resistance to ampicillin (52); therefore, the original plasmid pKD46 expressing the bla gene is incompatible for these E. ictaluri isolates. We successfully transferred this recombinogenic plasmid pMJH46 into different E. ictaluri strains by conjugation with E. coli SM10λpir. In subsequent studies, the pMJH46 plasmid was modified by replacing the cat gene with tetA to make it compatible for the genomic modification of primary disease isolates of A. hydrophila. The resulting plasmid pMJH65 (FIG. 1B, accession number KF195927) was successfully introduced into highly virulent catfish isolate A. hydrophila ML09-119 (53) for genomic modifications through recombineering.
Deletion of E. ictaluri and A. hydrophila Genes by Recombineering.
To determine the feasibility of using this recombineering system in E. ictaluri, we deleted the ompLC gene that is required for phage ΦeiAU-183 attachment to E. ictaluri strain Alg-08-183 (54). The PCR screening of colonies grown on antibiotic selection plates showed that approximately 1% colonies were true mutants (data not shown). Unfortunately, a large number of colonies grown on 2×YT plates supplemented with kanamycin were determined to be false positive even though the suicide plasmid pKD4 (13) used as template was treated with Dpnl before electroporation into E. ictaluri. To avoid the occurrence of background colonies, we subsequently used the genomic DNA of E. ictaluri Alg-08-183 ompLC::kanR mutant as the PCR template for amplification of the kanamycin resistance gene cassette. Using this chromosomal template to prepare amplicons we obtained 20 to 25 colonies per experiment on average, of which  ̃90% of them were true mutants. We deleted three additional genes including dtrA and ptrA of E. ictaluri Alg-08-183 (Hossain et al., 2012), and eihA of E. ictaluri R4383 (55) (Table 2). Using this recombineering approach, we also deleted 12 different genes from the primary disease isolate A. hydrophila ML09-119 (Table 2). PCR and sequencing confirmed that all genes that were targeted for deletion from E. ictaluri and A. hydrophila strains were successfully deleted by recombineering.
Effects of Primer Modification, Length of Homology and dsDNA Substrate Concentration in Recombination Frequency.
To determine the effect of primer modifications on recombination frequencies in A. hydrophila, four different combinations of primers were used for the preparation of dsDNA substrates to delete the waaL gene of A. hydrophila ML09-119. In one combination, both the leading and lagging strand-specific primers were modified with four consecutive 5′ phosphothioate bonds, whereas in another combination both the strands specific primers were unmodified. In two other combinations the leading strand and lagging strand were modified with for consecutive 5′ phosphothioates bonds, vice versa. We found that dsDNA substrate prepared with both of the modified primers provided significantly more mutants compared to other combinations (FIG. 2). Once we determined and modified primers provided significantly more mutants, all of our subsequent primers used for recombineering in A. hydrophila were modified on both strands.
To determine the effect of the length of the gene-specific homologous arms on recombination efficiency, three different dsDNA substrates that Included approximately 50 bp, 250 bp ord 500 bp of homologous sequence were used for deletion of the waaL gene. We found that the recombination frequencies were not significantly different due to the varying length of homologous arms flanking to the targeted gene (data not shown).
To determine the effect of dsDNA concentration on recombination frequencies in A. hydrophila, we used four different concentrations that included 0.75, 1.5, 3.0 and 5.0 μg of PCR products as a substrate for recombineering. Our findings demonstrated that gradual increment of the dsDNA substrate concentrations did not change the recombination frequency significantly (data not shown). The number of mutants we routinely obtained in this experiment was within the range of approximately 30-200.
Removal of Antibiotic Resistance Cassette by Flp Recombinase.
Temperature induction of E. ictaluri Alg-08-183ompLC::kanR, dtrA::kanR and E. ictaluri R4383 eihA::kanR mutant at 43° C. for 1 hr followed by plating on BHI blood agar plates resulted in the curing of the recombinogenic plasmid pMJH46 (data not shown). We found that only highly rich BHI medium supplemented with 5% Sheep Blood, unlike TSA, supported the growth of the high temperature-induced E. ictaluri strains. The introduction of plasmid pCP20, that contains the Flp recombinase (49) followed by their growth at 37° C. resulted in removal of the antibiotic marker from the E. ictaluri ompLC mutant PCR amplification of the targeted genes with their flanking primers indicated a 100% frequency for removal of the antibiotic selection marker. The antibiotic resistance markers from the E. ictaluri dtrA and eihA mutants were also removed using the Flp recombinase. We found that, in addition to the removal of the antibiotic resistance marker, heat induction efficiently cured the plasmid pCP20 from all mutant colonies tested. Cured mutants lacking the antibiotic resistance cassette could be subsequently targeted for deletion of additional genes. Since genes from A. hydrophila were replaced using the cat gene cassette, plasmid pCP20 containing the cat gene was not compatible for conducting Flp/FRT mediated recombination in A. hydrophila mutants. Therefore, we constructed a new flp recombinase plasmid pCMT-flp (FIG. 1D) that contained a tetA selectable marker that was conjugally transferred into A. hydrophila mutants for markerless mutant construction.
Dual Inducible Expression Plasmid for Red and Flp Recombination.
The construction of markerless mutants involves several steps including the introduction of λ-Red recombinogenic plasmid, curing of the plasmid after recombineering, and introduction of flp recombinase plasmid to remove the antibiotic resistance marker by flp/FRT recombination. If a double mutation is desired, then it would be necessary to cure the flp recombinase plasmid followed by re-introduction of the λ-Red recombinogenic plasmid. In this study, for faster and streamlined markerless mutant generation, we constructed a conjugally transferable, dual expression plasmid pMJH95 (FIG. 1C) that contains an arabinose-inducible red recombination cassette and a rhamnose-inducible Flp recombinase protein. Since the induction of genes under the transcriptional control of the rhaBAD promoter requires the RhaS and RhaR, and RhaT proteins for promoter activation and transport of rhamnose into the cells, respectively (56,57), we cloned, the rhaSRT genetic cluster into the plasmid pMJH95 to facilitate rhamnose transport to enable induction of the rhaBAD promoter by rhaS. This plasmid was conjugally transferred to the A. hydrophila ML09-119hlyA mutant and after induction, flp recombinase removed the cat marker by FRT/flp recombination (data not shown). After introducing into A. hydrophila by conjugation, we constructed an A. hydrophila double mutant ΔaeroA-hlyA using this dual inducible plasmid.
Cloning without PCR Amplification of Large Inserts.
Since cloning of large inserts using traditional cloning techniques are challenging and PCR amplification of the targeted inserts can introduced unwanted mutations, we developed a novel technique to clone large genomic inserts of A. hydrophila that does not require any PCR amplifications of the targeted inserts (FIG. 3). As a proof of concept of this technique, we targeted for cloning the 3.6 kb ymcABC operon of A. hydrophila strain ML09-119. For this purpose, we constructed a small conjugally transferrable low copy-number plasmid backbone (pMJH97) and integrated it contiguously to the ymcABC operon of A. hydrophila ML09-119 by recombineering (data not shown). We confirmed the correct integration of the plasmid backbone upstream of the ymcABC operon by PCR and sequencing. We successfully cloned the complete ymcABC operon (FIG. 3) and conjugally transferred the plasmid construct into A. hydrophila. 
Discussion
The genetic manipulation of primary pathogenic isolates, compare to domesticated laboratory isolates, can be challenging due to many factors including antibiotic resistance (58) (59), poor recombination efficiency and wide-spread occurrence of restriction-modification systems (43,60). Our attempts to genetically modify the fish pathogens E. ictaluri and A. hydrophila were inhibited due to our inability to introduce the λ Red recombineering system into these bacterial isolates. Similar difficulties were observed by several other researchers who reported reduced transformation efficiency of pKD46 in E. coli by electroporation (61), demonstrating the need for an alternative route to introduce the recombineering system, i.e., via conjugation. In this study we describe the development of a fast, efficient, and reliable technique for genetic modification of E. ictaluri and A. hydrophila (and presumably other Gram-negative bacteria) using a recombineering system that is readily transferrable by conjugation. The introduction of a mob cassette to pKD46 (13) permitted the resulting plasmid pMJH46 to transfer into different E. ictaluri strains by conjugation. Additional modified recombinogenic plasmids were constructed to make it compatible for knocking out genes from the emerging catfish pathogen A. hydrophila. Furthermore, we demonstrated the applicability of this method by creating multiple mutants in E. ictaluri and A. hydrophila. 
Our first experiments using recombineering in E. ictaluri unfortunately were plagued by a large number of background colonies on the antibiotic selection plates that were not successful recombinants. These results were obtained even though we used suicide plasmid pKD4 as a template for PCR amplification of antibiotic cassette and treated the DNA with DpnI treatment, as has been shown to reduce the number of background colonies (62). The solution to reducing the high background of antibiotic resistant colonies was to use genomic DNA isolated from a successful genomic integrant (E. ictaluri Alg-08-183ompLC::kanR) constructed in this study as a template for PCR of the recombineering construct. Therefore, all of our subsequent recombineering experiments for gene deletion in E. ictaluri and A. hydrophila used genomic DNA as template for PCR amplification of antibiotic resistance gene cassettes.
We were able to use the Flp recombinase encoded on the temperature-sensitive plasmid pCP20 (49) to successfully remove a FRT-flanked antibiotic resistance cassette used for genome modification in E. ictaluri. Before introducing pCP20 into E. ictaluri mutants, pMJH46 was cured by heat induction since both plasmids contain the cat gene. Unlike E. coli (13), E. ictaluri mutants required a highly rich medium (BHI supplemented with 5% sheep blood) to recover after heat-induction at 43° C., which may be due to the mesophilic growth temperature (28° C.) of E. ictaluri. Because of antibiotic resistance marker incompatibility, a new conjugally transferable flp recombinase plasmid, pCMT-flp, was constructed that can efficiently remove FRT-flanked antibiotic resistance gene cassettes. To avoid repeated curing of recombinogenic and flp recombinase plasmids to generate markerless mutants with multiple mutations, we constructed a dual expression plasmid pMJH95 with the Red cassette and flp gene under the control of arabinose- and rhamnose-inducible promoters, respectively. This plasmid can be used to delete genes by recombineering after arabinose-induction and then the FRT-flanked antibiotic resistance cassette can readily removed by rhamnose-induction of the flp recombinase.
In addition to developing techniques for genetic modification in E. ictaluri and A. hydrophila, we devised a novel technique for cloning large fragments of bacterial genomes without PCR amplification of the targeted region. This cloning system would be advantageous to clone larger fragments of genomic DNA without the need for PCR amplification, given the difficulties in producing larger amplicons and the potential for incorporating PCR-mediated errors. This method was validated by the cloning of a genetic operon from A. hydrophila, as an example of this method that can overcome the shortcomings of PCR-based methods for the cloning and conjugal transfer of genetic elements such as genomic islands, prophages, and other genetic clusters.
We have described a highly efficient and rapid procedure for the generation of markerless mutants in E. ictaluri and A. hydrophila by recombineering. The newly constructed conjugally transferable recombinogenic plasmids pMJH46, pMJH66 and pMJH95, and recombinase plasmid pCMT-flp can presumably be used for other Gram-negative bacteria for generating markerless mutants, especially for bacterial isolates that are recalcitrant to electroporation. Finally, the development of a PCR-free system for cloning and transfer will facilitate complementation of much larger genetic elements.







TABLE 2







List of bacterial strains and plasmids used in this study.









Bacterial strains or




plasmid
Features
References





E. coli




SM10λpir
thi-1thr leutonAlacYsupE recA::RP4-2-TcT::Mu Kmr
(46)



λpir


BW25113/pKD46
F−, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ−, rph-1,
(13)



Δ(rhaD-rhaB)568, hsdR514, pKD46


BT340
F−, Δ(argF-lac)169, φ80dlacZ58(M15), glnV44(AS),
(13)



λ−, rfbC1, gyrA96(NalR), recA1 endA1, spoT1, thiE1,



hsdR17, pCP20


BW25141/pKD4
F−, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), Δ(phoB-
(13)



phoR)580, λ−, galU95, ΔuidA3::pir+, recA1,



endA9(del-ins)::FRT, rph-1, Δ(rhaD-rhaB)568,



hsdR514, pKD4


E. ictaluri


Alg-08-183
Pathogenic isolates from diseased catfish
(53)


R4383
Highly hemolytic E. ictaluri strain from diseased
(65)



catfish


Alg-08-183
Replacement of hemolysin ompLC gene with kanR
This Study


ompLC::kanR
gene


Alg-08-183
Replacement of hemolysin dtrA gene with kanR gene
This Study


drtA::kanR


Alg-08-183
Replacement of hemolysin ptrA gene with kanR gene
This Study


prtA::kanR


R4383 eihA::KanR
Replacement of hemolysin eihA gene with kanR gene
This Study


R4383 ΔeihA
In-frame deletion of hemolysin gene eihA
This Study


Plasmids


pACYC184
Include
reference


pKD46
Temperature-sensitive recombinogenic plasmid
(13)


pKD4
Template for recombineering substrate
(13)


pMJH46
Conjugally transferrable recombinogenic plasmid
This Study


pMJH65
Conjugally transferrable recombinogenic plasmid
This Study


pMJH95
Conjugally transferrable recombinogenic and
This Study



recombinase plasmid


pCMT-flp
Temperature-sensitive Flp recombinase plasmid
This Study


pMJH97
cat-oriT-oriR backbone plasmid for PCR-free cloning
This Study


pCP20
Temperature-sensitive Flp recombinase plasmid
(51)


pGNS-BAC
Conjugally transferable BAC vector
(49)
















TABLE 3







List of primers used in this study.








Primer Name
Sequence in 5′-3′ direction





pKD4-ompLCf
AACTGGTAGATCATACCAACGCCAACGATGTTGTCGG



TGCTGATACCGGCGTGTAGGCTGGAGCTGCTTC



(SEQ ID NO: 43)


 


pKD4-ompLCr
GTTCAAAAAATTCCCGATGGAATCAAATTAGGCAGTG



GCAGGTGTCAAAACATATGAATATCCTCCTTAGT



(SEQ ID NO: 44)


 


ML44-RedF
ATGCTTACAACAAAAAATATGCCAGCCAATGCTGGGC



TGGCAGCGTTTTCTGGTGTAGGCTGGAGCTGCTTC



(SEQ ID NO: 45)


 


ML44-RedR
TTAGCAAGGGGGAAGATGCTCTGGTGGTGATGGTCTG



TTTTTCTGATGATAGCATATGAATATCCTCCTTAGT



(SEQ ID NO: 46)


 


Hemo-redF
TTCCTTTTAACTCTGCTTTGGCGCCCATGGGCGCTGA



TATGAGGCAATCTCTGTGTAGGCTGGAGCTGCTTC



(SEQ ID NO: 47)


 


Hemo-redR
ACGGCGGCCCGCAGGCCGCCGTTGAGGATGGATAACG



TCGCCACTATCCGGTCATATGAATATCCTCCTTAGT



(SEQ ID NO: 48)


 


ML82-RedF
GTGATACGTAGACAAGGTGCGACCATCGTACTGTGGA



TATTACTGCTGTTTTGTGTAGGCTGGAGCTGCTTC



(SEQ ID NO: 49)


 


ML82-RedR
TCAGCGGGTCACCTCTATCTTCAGCGTCTTTTGCAGC



GCACTGACGTCCGGGTCATATGAATATCCTCCTTAGT



(SEQ ID NO: 50)


 


RedtrackF
GATGTCTATCTGTTCAGCTC



(SEQ ID NO: 51)


 


RedtrackR
GTACGCAATACCAATAGTG



(SEQ ID NO: 52)


 


RE33-165F
TATGCAAGCTTGTAGTTCTTGCTGGTCTC



(SEQ ID NO: 53)


 


RE33-165R
TATGCAAGCTTGTAACGCAACATTCTAAC



(SEQ ID NO: 54)


 


k1
CAGTCATAGCCGAATAGCCT



(SEQ ID NO: 55)


 


k2
CGGTGCCCTGAATGAACTGC



(SEQ ID NO: 56)


 


Kt
CGGCCACAGTCGATGAATCC



(SEQ ID NO: 57)


 


MobF
ATGCAGATATCGGATCCTTTTTGTCCG



(SEQ ID NO: 58)


 


MobR
ACGCAGCAGTCAGTCACGATACAGCCGACCAGGCT



(SEQ ID NO: 59)


 


CatF
TATCGTGACTGACTGCTGCGTGTAGACTTCCGTTGA



ACT



(SEQ ID NO: 60)


 


CatR
ATGCAGATATCGCCTAATGAGTGAGCTAA



(SEQ ID NO: 61)


 


MobicatF
AGAGTGCTGACAGATGAG



(SEQ ID NO: 62)


 


MobicatR
ACGCAGCAGTCAGTCACGATAATGATGTGGTCTGTC



CT



(SEQ ID NO: 63)


 


MCF-int
CATGCGATATCACCGCTAACCTGTCTT



(SEQ ID NO: 64)


 


CatR-int
CATGCGATATCTAATGAATCGGCCAAC



(SEQ ID NO: 65)









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Example 2
Mutagenesis of Virulence Genes in an Epidemic Strain of Aeromonas hydrophila ML09-119 for Development of an Attenuated Vaccine Strain
An epidemic strain of A. hydrophila (ML09-119) caused a devastating outbreak of Motile Aeromonad Septicemia of catfish (MAS) on the fish farms of Southeastern United States in 2009. A. hydrophila ML09-119 was reported to cause severe mortality in commercial catfish farms. Research has been done on the virulence of this epidemic strain on channel catfish including molecular identification of the specific strain and unique DNA sequences.
Previously our lab sequenced 11 A. hydrophila isolates, 6 of which are epidemic stains, while the others were historical A. hydrophila isolates not affiliated with an epidemic outbreak of disease that we describe as “reference” strains. A comparative genomic analysis indicated that 53 epidemic-associated genetic regions with 313 predicted genes were uniquely present in the epidemic isolates but absent from the reference isolates. Thirty four genes from this region were predicted to be related to the virulence of the epidemic strains. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was identified and demonstrated to be functional based on the ability of epidemic A. hydrophila isolate ML09-119 to use myo-inositol as a sole carbon source while the reference strain AL06-06 cannot. A novel O-antigen cluster was found in all the epidemic isolates and one reference isolates.
In this study, the gene iolA coding for the enzyme aldehyde dehydrogenase for myo-inositol catabolism was inactivated by traditional allelic exchange to generate the A. hydrophila ΔiolAtra mutant. The iolA-iolR genetic region was also mutagenized using a recombineering technique to obtain ΔiolArec mutants. An in vivo challenge in channel catfish showed that there was no mortality in the channel catfish that were challenged with ΔiolAtra mutant, but there was mortality in the channel catfish challenged with ΔiolArec mutants similar to wild type ML09-119.
Eight mutants were created by knocking out an upstream portion of the iolA gene in the iolA-iolR promoter region. Results of the in vivo challenge in channel catfish showed that ΔiolArec3, ΔiolArec4 exhibited some decrease in mortality, but there were no significant difference in the mortality between the channel catfish challenged with ΔiolArec3, ΔiolArec4 and the channel catfish challenged with the wild type ML09-119. ELISA titer of the survivors of the ΔiolAtra after 21 days showed that ΔiolAtra can induce strong antibody response against the wild type A. hydrophila ML09-119, indicating that this mutant can serve as a promising vaccine candidate against the epidemic A. hydrophila. 
Lipid A-Core ligase (waaL) and O-antigen polymerase (wzy) mutants were created by both traditional splicing PCR and conjugation technique and recombineering technique respectively, and termed ΔwaaLtra or ΔwaaLRec, Δwzytra or ΔwzyRec. An in vivo channel catfish challenge study was performed on channel catfish to study the role of O-antigen in the virulence of the epidemic strain of A. hydrophila. The results show that the channel catfish that were challenged with ΔwaaLtra, Δwzytra had 100% survival rate, but 0% survival rate was observed in the channel catfish that were challenged with ΔwaaLRec, ΔwzyRec.
A ΔymcA mutant was created by knocking out the ymcA gene by a recombineering technique to study the role ymcA gene of the O-antigen in the virulence of A. hydrophila. A 68.13±16.75% survival rate was observed in the channel catfish that were challenged with ΔymcA mutant. Sub-challenge of the survivors of ΔymcA treatment group 21 days post first challenge showed that a 90.48±8.25% survival rate was observed. A significant difference was observed between the ΔymcA treatment group and the positive control group which were naive channel catfish challenged with wild type. ELISA titer of the survivors of the ΔymcA treatment group 21 days post first challenge showed that ΔymcA induced strong antibody response against the wild type A. hydrophila ML09-119 indicating that ΔymcA mutant can serve as a promising vaccine candidate.
A. DETERMINING THE ROLE OF THE MYO-INOSITOL PATHWAY IN A. hydrophila ML09-119 VIRULENCE
Abstract
In this study, the gene iolA coding for the enzyme aldehyde dehydrogenase for myo-inositol catabolism was inactivated by traditional allelic exchange to generate the A. hydrophila ΔiolAtra mutant. The iolA-iolR genetic region was also mutagenized using a recombineering technique to generate ΔiolArec mutants. An in vivo challenge in channel catfish showed that there was no mortality in the channel catfish that were challenged with ΔiolAtra mutant, but there was mortality in the channel catfish challenged with ΔiolArec mutants similar to wild type ML09-119.
Because of this observation, we hypothesized that the avirulent phenotype of the ΔiolAtra mutant was due to a polar effect on the upstream and divergently transcribed iolR gene, which is known to be a negative transcriptional regulator in other bacteria. Eight mutants were created by knocking out the upstream of the iolA gene in the iolA-iolR promoter region. Results of the in vivo challenge in channel catfish showed that ΔiolArec3, ΔiolArec4 exhibited some decrease in mortality, but there were no significant difference in the mortality between the channel catfish challenged with ΔiolArec3, ΔiolArec4 and the channel catfish challenged with the wild type ML09-119. ELISA titer of the survivors of the ΔiolAtra after 21 days showed that ΔiolAtra can induce strong antibody response against the wild type A. hydrophila ML09-119, indicating that this mutant can serve as a promising vaccine candidate against the epidemic A. hydrophila. 
Materials and Methods
Bacterial, Isolates and Plasmids.
The A. hydrophila ML09-119 and reference strain AL06-06 used in this study were picked out from single colony on the plate that were streaked using the −80° C. stock. The epidemic strain was from the west Alabama MAS outbreak in 2009, while the reference strain. The bacteria were routinely grown on fresh Trypticase Soy Broth (TSB) medium overnight before use. The A. hydrophila ML09-119 used for experiment was from the bacterial stocks of the fish disease lab in Auburn University. This epidemic strain was originally isolated from the kidneys of channel catfish naturally infected with A. hydrophila. The pure culture of epidemic strain was used first in a small test infection of 10 catfish. Moribund catfish that showed clinical signs of A. hydrophila ML09-119 was collected for necropsy. A. hydrophila was re-isolated from the fresh dying fish by poking a sterile plastic bacteriology loop into the kidney and inoculating a BHI plate. By doing this, it is expected than the virulence of the epidemic strain stock can be recovered. ML09-119 was then confirmed by biochemistry and selective media following the established identification procedures with modifications (Furuwatari, et al., 1994; Holt, et al., 1994), Briefly, the identification biochemical tests included Gram stain, cytochrome oxidase, glucose utilization, 0/129, sucrose, esculin hydrolysis, V-P, DL-lactate utilization and urocanic acid utilization, and then test on selective media M9 minimum media with inositol added. E. coli SM10-λ-pir (Simon et al., 1983) was used for the conjugal transfer of mobilizable plasmid to A. hydrophila ML09-119. The list of bacterial strains used in this study is presented in Table 4.
Construction of Defined A. hydrophila ΔiolAtra Mutant by Traditional Splicing PCR and Conjugation Technique.
To investigate the role of myo-inositol utilization pathway in the virulence of epidemic A. hydrophila ML09-119 in channel catfish, a iolA knockout mutants, ΔiolAtra were constructed using plasmid pDMS197, a sacB containing suicide plasmid (Edwards, Keller et al. 1998). The primers needed for this study were listed in the Table 5.
The two pairs of primers, AupF/AupR and AdnF/AdnR, were used to amplify approximately 350 bp upstream and downstream sequences of iolA gene, respectively using PCR kit (TaKara Ex Taq) to construct the ΔiolA mutant. The template used in this PCR was the genomic DNA of A. hydrophila ML09-119 which was extracted using a E.Z.N.A.® Bacterial DNA Kit (Omega Bio-Tek, USA). The chloramphenicol acetyltranferase gene (cat) was amplified from pMHH46 plasmid (Hossain et al 2013) using primers catF and catR. The primers AupR and AdnF, used for the amplification of upstream and downstream sequences of iolA gene contained the reverse complemented sequences of catF and catR primers which were added respectively at their 5′ ends when the primers were designed. The Cat-cassette which was the chloramphenicol resistance gene (CmR) with two arms of the upstream and downstream homologous of iolA gene was created by fusing the two arms and the CmR gene by splicing through overlap extension PCR (SOB) (Morton, Hunt et al. 1989). The primers for this PCR were Aup-intF and And-intR. The PCR products were purified by agarose gel purification.
The suicide plasmid pDMS197 was digested by restricted digestion enzyme XbaI (New England Biolabs, NEB, USA) following the protocol provided by the manufacturer. A 50 ul reaction was used for the digestion, including 25 ul of the suicide plasmid pDMS197 DNA, 3 ul of the XbaI restricted digestion enzyme, 5 ul of the 10×CutSmartTM Buffer, 1×BSA and 16 ul RNase free H2O. The reaction system was incubated at 37° C. for one hour. The reaction system was then incubated at 65° C. for 20 min to stop the reaction. The digested product was purified by DNA Clean & ConcentratorTM (Zymo research), and the concentration was measured by Qubit® dsDNA BR Assay Kit (Life technologies). The product was blunted using end-repair kit DNA terminator (Lucigen, USA) following the producer's instruction. The product was purified by DNA Clean & ConcentratorTM (Zymo research) again before ligation.
The purified restriction enzyme XbaI digested and blunted suicide plasmid pDMS197 was ligated with the Gel purified Cat-cassette using Quick Ligase (NEB, USA) under the room temperature for 30 minutes. Briefly, 50 ng of blunted suicide plasmid pDMS197 and around 3-fold molar excess of the Cat-cassette insert was mixed together and the volume was adjusted to 10 ul with RNase free H2O. 10 ul of the 2× Quick Ligation Buffer and 1 μl of Quick T4 DNA Ligase were added into the mixture. The mixture was centrifuged briefly and incubated at room temperature (25° C.) for 30 minutes before it was chilled on ice. A SB gel electrophoresis was done to confirm the ligation product (data not shown).
The making of the electrocompetent cells of E. coli SM10-λ-pir was following a published protocol (Inoue, et al., 1990) with minor changes. A 0.5 ml of the overnight culture of E. coli SM10-λ-pir bacteria was inoculated into 200 ml of Hanahan's Broth (SOB Medium) with 10 mM MgCl2. The culture was incubated in the 37° C. water bathe incubator with shaking bed at 200 rpm for around 2.5 hours and the OD600=0.4. The culture was chilled in ice for 10 min before loaded into 200 ml centrifuge tubes. The culture was centrifuged at 6000 rpm for 8 min at 4° C., the supernatant was discarded and the pellet was washed by resuspended with 10% glycerol and centrifuge again at 6000 rpm for 8 min. The wash step was repeated for 3 times before the pellet was gently resuspended in 200 ul GYT medium. The whole procedure was performed on ice.
The ligation product was then used in the electroporation (Chassy, et al., 1988; Dower et al, 1988) to create the plasmid pDMS197iolA, which contains a deletion of the entire iolA gene. 50 ul of the premade electrocompetent cells of the E. coli SM10-λ-pir was mixed gently with 2.5 ul of the ligation product and chilled on ice for 5 min. The mixture was transferred into ice cold cuvettes (Bulldog bio) before the cuvettes were loaded onto the Eppendorf® Eporator® (Eppendorf). Voltage was set up at 1800V. The mixture was mixed with recovery medium (SOC medium) right after the electronic pulse shock. The culture was transferred to a 2 ml test tube and incubated at 37° C. with shaking bed at 200 rpm for 2 hrs. The successful electroporated E. coli SM10-λ-pir with the plasmid pDMS197iolA was selected on 2XYT agar medium plate with 25 ul/ml chloramphenicol, 5 ul/ml tetracycline.
The suicide plasmids pDMS197iolA were independently introduced into A. hydrophila ML09-119 by conjugation with E. coli SM10-λ-pir bearing plasmid pDMS197iolA. A single colony was selected on the selective medium plate for SM10-λ-pir bearing plasmid pDMS197iolA for inoculation of 5 ml LB broth medium. The culture was incubated at 37° C. with shaking at 200 rpm until the OD600 was above 1. A single colony of A. hydrophila ML09-119 was picked to inoculate 5 ml TSB broth medium. The culture was incubated at 30° C. with shaking at 200 rpm until the OD600 was above 1. A 4 ml ML09-119 culture and 1 ml SM10-λ-pir bearing plasmid were mixed together. The 5 ml culture mixture was filtered through a MicroFunnel 300 SP (MicroFunnelTM) by vacuum pressure. 5 ml fresh LB broth medium was used for washing the cells onto the membrane. The membrane was transferred to the sheep blood agar medium after 2× wash step. The sheep blood agar medium was incubated at 30° C. overnight.
The membrane with the cell culture mixture was vortexed with 3 ml fresh TSB broth medium for selection. Single cross-over mutants were selected on TSA plate supplemented with chloramphenicol, tetracycline and colistin. Double-cross over mutants were obtained by plating onto LB (without NaCl) plates supplemented with 15% sucrose and 12.5 μg/ml chloramphenicol. Mutants grown on this selective plate were subjected to phenotypic and genotypic characterizations. The complete deletion of the iolA genes were confirmed by PCR followed by sequencing.
Construction of Defined A. hydrophila ΔiolArec Mutants by Recombineering.
A recombineering technique was used to create a precise deletion of the iolA gene and generate the ΔiolARec mutant, in order to compare with the ΔiolAtra created by the traditional technique by splicing through overlap extension PCR (SOE) (Horton, Hunt et al. 1989), as well as to better determine the role of myo-inositol utilization pathway in the virulence of epidemic A. hydrophila ML09-119 in channel catfish.
The chloramphenicol acetyltranferase (cat) gene was amplified from pMHH46 plasmid (Hossain et al 2013) using primers iolA5RecF and iolA5RecR to generate the cat-cassette with 50 bp of the upstream and downstream of the targeted iolA gene. The primers iolA5RecF contained 50 bp of the upstream of the targeted iolA gene and iolA5RecR contained the reverse complemented sequences of 50 bp of the upstream of the targeted iolA gene which were added respectively at the 5′ ends of each respective primer. The PCR product was validated using gel electrophoresis before another 24×PCR was done using this PCR product to generate more cat-cassette insertion.
The PCR product was purified and concentrated using Wizard® DNA Clean-Up system (Promega, USA) following the protocol provided by the manufacturer. Briefly, the 24 different PCRs were pooled together in a 15 ml conical tube, and a Wizard® DNA Clean-Up kit (Promega, Madison, Wis.) was used to purify the PCR products according to the manufacturer's protocol. The concentration of the final concentrated PCR product was measured using Qubit® dsDNA BR Assay Kit (Life Technologies).
A. hydrophila ML09-119 containing the plasmid pMJH65, which was constructed for the purposes of introducing a recombineering cassette into gram-negative bacteria (Hossain et al, manuscript in preparation), was prepared for electroporation using a standard protocol (Inoue, et al., 1990) with minor changes. 0.5 ml of the overnight culture of ML09-119 bacteria was inoculated into 150 ml of Hanahan's Broth (SOB Medium) with 1.5 ml 1M Arabinose, 300 ul 25 mg/ml Tetracycline and 600 2M MgCl2. The culture was incubated in the 30° C. water bath incubator with shaking at 200 rpm for around 4 hours and the OD600=0.5. The culture was chilled on ice for 10 min before loaded into 200 ml centrifuge tubes. The culture was centrifuged at 6000 rpm for 8 min at 4° C. The supernatant was discarded and the pellet was washed by re-suspending with 10% glycerol and centrifuged again at 6000 rpm for 8 min. The wash step was repeated 4 times before the pellet was gently resuspended in 200 ul 10% glycerol. The whole procedure was performed on ice.
The concentrated and purified PCR product was then used in the electroporation (Chassy, et al., 1988; Dower et al, 1988) to create the precise iolA gene deletion mutant ΔiolARec. 50 ul of the premade electrocompetent cells of A. hydrophila ML09-119 (pMJH65) was mixed gently with 3 ug of the concentrated PCR product and chilled on ice for 5 min. The mixture was transferred into ice cold cuvettes (BulldogBio) before the cuvettes were loaded onto the Eppendorf® Eporator® (Eppendorf) with a voltage setting of 1200 V. The mixture was mixed with recovery medium (SOC medium) right after the pulse shock. The culture was transferred to a 2 ml test tube and incubated at 30° C. with shaking at 200 rpm overnight.
The successfully electroporated A. hydrophila ML09119 iolA deletion mutant was selected on a TSA agar medium plate with 25 ul/ml chloramphenicol. A similar strategy was followed for the construction of ΔiolARec2 through ΔiolARec8 which represent progressively larger deletions of the iolA-iolR promoter region, with each successive mutant having a deletion of the iolA gene and an additional 50 bp upstream of the iolA-iolR promoter region, respectively (FIG. 7).
Evaluating the Growth Response of A. hydrophila Mutants Using Myo-Inositol as a Sole Carbon Source.
A 2 ml TSB culture of the A. hydrophila isolate was started by inoculating the medium using a single colony of the bacteria. The culture was grown at 30° C. overnight with shaking at 200 rpm. The cell culture next day was centrifuged at 10,000×g for 10 min. The supernatant was poured out, and the pellet was resuspended in M9 minimal medium supplemented with 5.5 mM of myo-inositol (M9I). The centrifugation and re-suspension in M9I was repeated twice to remove any TSB residue. At last, the re-suspension of the bacteria, cells in M9I was adjusted to an OD600 of 0.5. A 1:100 dilution of the suspension was achieved by 10 fold serial dilution from the original M9I suspension. A 100 ul of the dilution was used to inoculate 1.9 ml of M9I. The bacterial cultures were then incubated at 30° C. with shaking at 200 rpm for 144 hours and the OD600 was recorded at 24 hrs intervals to record the growth condition of the bacteria strains in M9I. The results were used to evaluate the ability of each strain to use myo-inositol as a sole carbon source. A. hydrophila isolates ML09-119 and AL06-06 were used as positive and negative control, respectively, for the myo-inositol utilization assay.
Virulence Study of A. hydrophila Mutants in Channel Catfish.
All experiments conducted with vertebrate animals (catfish) were approved by the Institutional Animal Care and Use Committee (IACUC) review board at Auburn University in accordance with the animal welfare guidelines specified in the United States.
All the channel catfish (I. punctatus, Kansas Random Strain), used in this study were spawned at the hatchery of the Auburn University Fish Genetics Research Unit artificially prior to transferring to troughs or glass aquaria at the Auburn University Fish Pathology wet lab S6. Fish were maintained at recirculation systems (temperature around 25° C. and pH 7.5) using well water sources with constant aeration. Fish were fed daily with commercial feed. Water quality factors including temperature, pH, salt level, total ammonia level, total nitrite level were tested on daily basis to ensure that catfish fingerlings remained unstressed and naive to A. hydrophila. Catfish fingerlings were grown out in this system until their body weight (BW) reached 20±5 g.
A bacterial suspension of exponential phase growth was prepared by overnight culture of in 5 ml TSB broth medium with shaking at 200 rpm at 30° C. The next day 1 ml of the overnight bacterial culture was used to inoculate 100 ml fresh TSB broth culture which was incubated with shaking at 200 rpm at 30° C. for 4 hours. The bacterial culture was centrifuged at 6000 rpm for 10 min. The supernatant was discarded and the bacterial pellet was resuspended in fresh TSB media. The optical density of the bacterial culture was measured by the thermospectronic spectrophotometer (Thermo Spectronic, Rochester, N.Y., USA) at 600 nm and adjusted to an OD=1, which was expected to be 1×109 CFU/ml. After adjusting the bacterial suspension to an appropriate OD, a 1:100 dilution was performed using fresh TSB broth to get the desired concentration (around 1×107 CFU/ml) of A. hydrophila. Another 1:2 dilution was done with fresh TSB. This culture was put on ice and used for challenge within 3 hours. A plate count assay was conducted right after the fish challenge to calculate the accurate CFU/ml concentration used in this study. The bacterial culture used in the fish challenge were serial diluted and 100 ul of each dilution was spread on the TSA plates, with 3 replicates were done for each strain of bacteria.
Channel catfish in Auburn University Fish Pathology wet lab S-6 were randomly distributed into glass aquarium tanks. MS-222 (30 mg/l) was used during the handling of fish to calm the fish down to decrease the stress. Each tank contained 10 fish. A recirculating system was applied during the acclimation period, which was lasted for 10 days. Water temperature was originally 25° C. and salt level was kept around 1.8 ppt to decrease the stress caused by environmental changes as well as eliminating the chance of F. columnare infection. Water temperature was gradually brought up to 30±1° C., and salt was gradually brought down to 0.8 during the first 3 clay of the acclimation time. Every environmental factor was kept stable prior to the challenge. Fish were fed with commercial catfish fed once a day at 4% of their body weight. Water was changed once per day for the recirculating system with constant aeration. At the time of challenge, recirculating system was changed into flow through system, with the temperature at 30±1° C. Fish of each treatment tank were euthanized by immersing in a bucket with MS222 (30 mg/l), before 200 ul of ML09-119 bacterial culture was injected intraperitoneally into each fish. Fish were then put back to their cohabitation tanks. Fish of control groups were injected with pure TSB broth medium. Challenged fish were kept the same way as they were during the latter acclimating time. Mortalities were recorded daily for 14 days post challenge. Any moribund or dead fish were removed from the system daily for bacteriological identification and tissue sampling. Prior to sampling, fresh dying or dead fish were inspected externally and internally for any clinical signs. The identification of A. hydrophila isolated from anterior kidney of the fresh dying or dead fish was performed by the biochemistry and selective medium method described previously. Survivors of the challenge were kept for 28 days, before they are challenged again with the wild type ML09-119 to test if any protection effect was provided. The procedure of the re-challenge was similar to the previous challenge. At seven days post re-challenge, blood samples were then drawn from the survivors for the ELISA titer in the later experiment.
Immunogenicity of the Mutants and the Enzyme-Linked Immunosorbent Assay (ELISA).
Blood samples collected after the fish challenge were put in the room temperature for 2 hrs then 4° C. overnight allowing to clot completely. Serum of each blood sample was collected followed by centrifuging at 5000 rpm for 10 min. The supernatant of each sample was collected for Enzyme-linked Immunosorbent assay (ELISA) analysis.
Antibody responses of channel catfish to A. hydrophila were quantified by evaluating the presence of specific immunoglobulin to A. hydrophila wild type ML09-119 using indirect ELISA. Protein DetectorTM ELISA kit was use to conduct the ELISA experiment.
The protocol followed was similar to the product introduction with minor changes. 96-well plastic plates were coated with 100 ul of a solution of 10 ug/ml (107 CFU/ml) A. hydrophila epidemic strain. A. hydrophila were suspended in carbonate-bicarbonate coating solution. The coating solution was prepared by diluting one time coating buffer tablet in 10 times of sterile reagent quality water. The plates with coating buffer and antigen were placed in 4° C. pH 9.6 overnight. The plates were washed 4 times with washing buffer provided by the kit the next day, followed by adding 1×BSA blocking buffer to block for 15 min at room temperature. After another wash step, the plates were used to do ELISA analysis. 100 ul 1% BSA blocking buffer was added into each well on the A. hydrophila ML09-119 coated plate. 200 ul of the 1/10 fish blood serum sample diluted with 1% BSA blocking buffer was added to the column A2-A11, A1 and A12 were served as positive and negative control. 100 ul of the solution from A1-A12 was transferred to B1-B12 and mixed carefully by pipetting 3-5 times, and this step was repeated across the plate until E1-E12. The final 100 ul from the wells in the row E after mixing was discarded. The plate was then incubated at room temperature for 1 hour. The plate was emptied, and residual liquid was tapped out. Plate was washed out by the washing buffer that came with the kit for 5 times. 100 ul of Rat Anti-catfish monoclonal antibody (Mab) was diluted 32 times and added into each well that contained the primary antibody, after which the plate was incubated at room temperature for 1 hour. After incubation the plated was emptied, and residual liquid was tapped out and the plate was washed out five times using the washing buffer that came with the kit 50 ul of tertiary antibody (goat anti-rat antibody conjugated with horseradish peroxidase) (0.1 ug/ml) was added into each wall that contained the secondary antibody. The plate was incubated at room temperature for 1 hour, after which the plate was washed as above. 50 ul of the substrate solution that came with the kit was added into each well that contained the tertiary antibody. The plated was incubated at room temperature for 5-15 min before the reaction as stopped by adding 50 ul of stop solution into each well for full color development and the plate was then read at OD405. A reaction was defined as positive if its OD450 value was at least two times the negative control. Ending points were the highest dilution with a positive reaction.
A criss-cross serial dilation analysis was done prior to the ELISA analysis of the samples to optimize the reagent concentration in the immunoassay procedure. 100 ul of 1% BSA blocking buffer was added into each well of the A. hydrophila ML09-119 coated plate. 200 ul of the 1/10 ML09-119 infected survivor fish blood serum sample diluted with 1% BSA blocking buffer was added to the respective columns and serially diluted across the plate to identify the best concentration range for the sample. Prior to adding the Mab, 100 ul of 1% BSA blocking buffer was added into each well, followed by 200 ul of the secondary Rat anti-channel catfish Mab. This Mab solution was serially diluted across the plate to identify the optimum concentration for the Mab.
Statistical Analysis.
Mortality data of this study was presented as mean±standard error (SE) and analyzed by one-way analysis of variance and Tukey's multiple range comparison using SAS software (SAS 9.2, SAS Institute Inc., Cary, N.C.). Significant level was set at 5% (p<0.05). Variances were considered significant when probability (P) values<0.05 were calculated.
Results
Evaluating the Growth Response of A. hydrophila Mutants Using Myo-Inositol as a Sole Carbon Source.
The iolA gene encodes aldehyde dehydrogenase and terminally located in the inositol catabolic (iol) gene cluster of epidemic A. hydrophila isolates. It has been demonstrated that the iolA gene is required for the conversion of malonate semialdehyde to acetyl-CoA (Hossain et al., 2012). It was predicted that the iolA deletion mutants that were created in this study, which were created by replacing the iolA gene and 50 bp of upstream of the iolA gene with cat gene using both traditional technique and recombineering technique, would be unable to utilize myo-inositol as a sole carbon source. The growth assay was carried out with M9I for 144 hours, and it was determined that all of the iolA mutants were unable to utilize myo-inositol as a sole carbon source (FIG. 4), whereas wild type A. hydrophila ML09-119 reached stationary phase after 48 hours of incubation. The results of this myo-inositol assay were comparable to the results in previously published research (Hossain et al., 2012). Like A. hydrophila ML09-119 iolA mutants, wild type A. hydrophila AL06-06 that does not carry the iol cluster did not show any growth after 144 hours of incubation. The lack of the ability of the iolA mutant to utilize myo-inositol as a sole carbon source clearly demonstrated that an iolA mutation had been constructed and that IolA function is required for A. hydrophila utilization of myo-inositol as a sole carbon source.
Cumulative Survival Rate of the Channel Catfish Challenged with the iolA Mutants.
For better understanding of the virulence factors of the A. hydrophila epidemic strain and to identify possible live vaccine candidates, the iolA gene was knocked out by a traditional allelic exchange technique. It has been observed that all of the A. hydrophila epidemic strains A. hydrophila can utilize myo-inositol as a sole carbon source (Hossain, et al, 2013). Since iolA gene is required for the conversion of malonate semialdehyde to acetyl-CoA (Kohler, et al., 2011), the hypothesis is that the iolA gene can be the key virulence factor and by knocking out the iolA gene, the ML09-119 strain may be attenuated and serve as a vaccine candidate.
The results of the in vivo channel catfish i.p challenge with ΔiolAtra showed that this mutant is avirulent. The channel catfish in the ΔiolAtra treatment group had a 100±0% survival rate, while the wild-type strain-injected group had a 2.5%±0.08 survival rate. The percentage survival rates were transformed by arcsine square root transformation and then analyzed by SAS 9.2, and significant differences were observed between iolA and ML09-119 treatment groups (P<0.0001). This indicates that the ΔiolAtra was an attenuated strain of ML09-119 (FIG. 5). However, the channel catfish i.p challenged with the ΔiolARec1 mutant, which was created by precisely knocking out the iolA gene using the recombineering method, was still fully virulent with a 2.5%±0.08 survival rate that was the same as the wild type positive control group (P>0.05). This indicated that the ΔiolARec1 was not an attenuated strain of ML09-119 (FIG. 5).
Sub-challenge of the channel catfish survivors in the ΔiolAtra treatment group with wild type ML09-119 showed a 56.9%±0.154 survival rate observed in the ΔiolAtra group (FIG. 6). In contrast, there was a 4.4%±0.141 survival rate in the positive control group. All survivors of the ΔiolAtra group were I.P injected with 1×106 CFU/fish ML09-119 again to determine if any immonogenicity developed. Naive channel catfish were I.P injected the same dosage with ML09-119 as a positive control. Significant differences were observed between the ML09-119 group and iolA mutant group (P<0.05) suggesting that a certain extent of immunogenicity to ML09-119 was developed by exposing catfish to the ΔiolAtra mutant.
Investigation of the Virulence of the Different iolA Mutants.
The vast difference of the virulence between ΔiolAtra and iolARec1 mutants prompted us to remake the ΔiolAtra mutant using the recombineering method to identify if any secondary mutation was introduce while the ΔiolAtra mutant was constructed. The hypothesis for the difference between the ΔiolAtra and iolARecI mutants is that there might be a second site mutation that happened during the construction of the ΔiolAtra mutant that resulted in an attenuated strain.
Furthermore, it was noticed that during the construction of the ΔiolAtra mutant that a part of the promoter region between the iolA and iolR genes was deleted as well (FIG. 7). This promoter region is hypothesized to contain the binding region for the transcriptional regulator protein IolR to regulate its own transcription, as it is expected to do for other genes in the IolR regulon (Kohler., et al. 2011). This prompted us to create the ΔiolARec2 through ΔiolARec8 mutants to determine if the loss of the IolR binding region (as yet undefined) will affect the expression of the iolR gene as result in an attenuated strain. The hypothesis is that by deleting the binding region for the transcriptional repressor IolR that this will increase the transcription of iolR, resulting in suppression of other possible virulence factor genes such as aerolysin (Zhang D, et al, 2013) that could possibly be co-regulated by IolR and be in the “IolR regulon”. Thus the mutant will be attenuated.
A pretrial was carried out to determine the virulence of each mutant as well as to help select specific mutants for vaccine and immunogenicity studies. The result of this pretrial showed that the remake of the ΔiolAtra mutant using the recombineering method, ΔiolArec, did not lose its virulence with a 0% survival rate, as did the ΔiolARec2, ΔiolARec5, ΔiolARec7, and ΔiolARec8 mutants. In contrast the ΔiolAtra had a 83% survival rate, and the ΔiolARec3, ΔiolARec4, and ΔiolARec6 treatment groups had 25%, 33%, and 17% survival rates, respectively. (FIG. 8). These results indicate that there may be some variability in the virulence among these mutants and that there may be some contribution of the IolR regulon to A. hydrophila virulence. Even though this pretrial did not give a valid statistical analysis, it did provide a preliminary determination of the virulence of these iolA mutants. The ΔiolARec3 and ΔiolARec4 mutants were picked for the formal vaccine candidate and immunogenicity challenge study.
Vaccine Candidate and Immunogenicity Challenge Study.
To determine the virulence of the iolA mutants and to evaluate their efficacy as a live vaccine against A. hydrophila ML09-119, an in vivo channel catfish challenge study was conducted. The results of the in vivo channel catfish i.p challenge with ΔiolAtra again showed that this mutant is attenuated in catfish with a 83.3±11.5% survival rate, while 0±0% survival rate was observed for the fish in the positive control treatment group (P<0.0001) (FIG. 9). However, channel catfish i.p challenged with ΔiolARec3 or ΔiolARec4 mutants, that have deletions of the iolA gene and 100 bp or 500 bp upstream of iolA gene, respectively, had 12.1±9.1% survival and 17.3±17.2% survival rates, respectively (FIG. 10). No significant difference was observed between the iolARec3 or iolARec4 and the wild type treatment group (P>0.05) (FIG. 10).
A sub-challenge of the channel catfish survivors was carried out 21 days post challenge. The ΔiolAtra treatment group surviving fish that were challenged with the wild type ML09-119 showed a 71.4±14.3% survival rate, in contrast to the 0±0% survival rate observed in the naïve fish challenged with ML09-119 (P<0.05) (FIG. 11). This suggests that a certain extent of immunity against ML09-119 was developed by exposing catfish to the ΔiolAtra mutant.
Enzyme-Linked Immunosorbent Assay (ELISA).
The Enzyme-linked Immunosorbent Assay (ELISA) was carried out to determine the efficacy of protective immunity induced by the ΔiolAtra mutant immunized channel catfish were i.p injected with 1×106 CFU/fish of the mutant. We hypothesized that the ΔiolAtra mutant expressed epitopes that would retain a similar immunogenicity as the wild type. Thus, a positive reaction should be observed in the titer of the ELISA assay. All of the replicates of the ΔiolAtra mutant induced a strong antibody reaction to ML09-119 (FIG. 13). The levels of antibody to ML09-119 were highest in serum from the ΔiolAtra mutant immunized channel catfish, indicating strong antibody induction by the ΔiolAtra mutant.
Discussion
This study provided valuable insight into role of the myo-inositol pathway in the virulence of A. hydrophila ML09-119. One of the iolA gene deletion mutants created proved to be attenuated and can provide protection against A. hydrophila ML09-119 in an in vivo channel catfish challenge study. This mutant may be a promising live vaccine candidate against epidemic A. hydrophila. 
The recent epidemic outbreak of the MAS, which caused by highly virulent A. hydrophila has drawn a lot attention since the catfish farming operations in the southeastern United States have not experienced a large-scale outbreak of MAS before (Hemstreet, 2010). In 2009 and in all subsequent years, catfish farmers in west Alabama have reported severe disease outbreaks which were then proved to be caused by a highly virulent strain of A. hydrophila, represented by strain ML09-119, to channel catfish (I. punctatus). From 2009-2011, Alabama catfish farmers lost more than 10 million pounds of catfish that were market-size and estimated to be more than $3 million due to this epidemic strain of A. hydrophila (Pridgeon et al., 2011; Liles et al., 2011). It is reported that A. hydrophila epidemic strain, ML09-119, is highly virulent to channel catfish, causing severe mortality within 24 h post exposure with certain amount of dose. Also, this epidemic A. hydrophila has expanded its geographic territory and caused frequent outbreaks in the summer months, resulting in millions of pounds of losses in Alabama, Mississippi and Arkansas. (Pridgeon and Klesius, 2011). Due to its highly virulent nature and huge economic loss so far, it is essential that the virulent factors be studied and an effective vaccine be developed.
A previous study showed that epidemic strains can utilize myo-inositol as a sole carbon source. All of the epidemic strains encode the myo-inositol catabolic pathway (Hossain et al, 2013). This prompted us to investigate the role of the myo-inositol pathway in the virulence of A. hydrophila ML09-119.
The ΔiolAtra mutant was created using a traditional allelic exchange technique, and the in vivo channel catfish challenge study showed that this mutant is attenuated compared to its wild-type parent strain ML09-119. However, when we created a precise iolA gene deletion mutant ΔiolARec using a more efficient and accurate recombineering technique, we observed that this mutant was still virulent in channel catfish. There are two hypotheses that could explain this difference in virulence between these two iolA mutants: 1) the truncation of the IolR binding region causes the over expression of iolR gene, repressing other virulence factors such as aerolysin, and/or 2) the ΔiolAtra mutant has a secondary mutation responsible for some degree of virulence attenuation.
One difference between the ΔiolAtra and the ΔiolArec mutants is that when the ΔiolAtra was constructed part of the promoter region between the iolA and iolR genes was deleted. IolR is a transcriptional repressor for multiple genes in the myo-inositol pathway, including iolR (Kohler, et al. 2011). It is possible that when the ΔiolAtra was constructed, the deleted promoter region contained a binding region for IolR (Kohler, et al. 2011). Without the binding region for the IolR repressor, the transcription of the iolR gene may be increased and the synthesis of more IolR might repress other genes that are related to the virulence of A. hydrophila in the IolR regulon such as aerolysin (Zhang et al., 2013; Cordero-Alba et al., 2012). We hypothesize that by deleting the region between the iolA and iolR gene that the expression of the iolR gene might change along with the virulence of the mutants. The results of RT-PCR using iolR-specific primers showed that there might be differences between the ΔiolARec4 and other ΔiolARec mutants (data not shown); however, no quantification of these data has been performed to date. Our in vivo channel catfish challenge study showed that there is some attenuation within the ΔiolARec3 and ΔiolARec4 mutants; however, the statistical analysis did not support a difference at P<0.05, and additional experiments with more animals and groups may be needed in order to observe a statistically significant difference between the ΔiolARec3 or ΔiolARec4 mutants and wild type ML09-119.
Even though the reason for the attenuation of the ΔiolAtra has not been completely characterized, the immunogenicity study showed that this mutant can provide around 70% survival rates for channel catfish at doses that result in no survival for naïve fish. The ELISA assay evaluating the antibody induced by the ΔiolAtra mutant against A. hydrophila ML09-119 showed that the ΔiolAtra mutant could induce strong antibody reaction. This indicates that ΔiolA5tra mutant can serve as a promising live vaccine candidate against the recent MAS epidemic outbreak. This study also raised some interesting studies for the future research including: (1) The reason of the attenuation of the ΔiolAtra mutant; (2) The role of the iolR gene and what are the genes that are included in the IolR regulon; and (3) The delivery route for the live vaccine of the channel catfish against the A. hydrophila epidemic strain.







TABLE 4







Summary of bacterial strains and plasmids used in this study









Bacterial strains and




Plasmid
Relevant features
References





Bacterial strains




A. hydrophila

Hossain et al., 2013


ML09-119


E. coli SM10-λ-pir
thi-1thr leu tonA lacY supE
(Simon, Priefer



recA::RP4-2-TcT::Mu Kmr
et al. 1983)



λpir


Plasmids


pDMS197
Suicide vector, sacB, TetR
(Edwards, Keller




et al. 1998)


pDMS197iolA

This study
















TABLE 5







The primers used in this study









primer ID
Primer sequence
Primer Application





iolA5RecF
5′-T*G*A*A*ATTTAATTTTCAACAAATTCCGTGATCAT
Amplifying the cat-cassette



CAGCCAAGAGAGAGATCGTAGACTTCCGTTGAACT-3′
for creating the ΔiolARec



(SEQ ID NO: 66)



 


iolA5RecR
5′-C*T*G*G*AGAGCGGGATAACCGAGGTGAGTCTGGAC
Amplifying the cat-cassette



GTGGCGCCTTCAGGAGAGCCTAATGAGTGAGCTAA-3′
for creating the ΔiolARec and



(SEQ ID NO: 67)
serve as the reverse primer




for the ΔiolARec2-ΔiolARec8


 


iolARecF1
5′-A*A*A*T*TTAATTTTCAACAAATTCCGTGATCATCA
Amplifying the cat-cassette



GCCAAGAGAGAGATCGAGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec1 mutant



(SEQ ID NO: 68)



 


iolARecR1
5′-CGCCTTCAGGAGAGAGGGGCGACTGTCCCTCTCTTTT
Amplifying the cat-cassette



GTTACACTGTCCGGCCTAATGAGTGAGCTAA-3′
for c the ΔiolARec1 mutant



(SEQ ID NO: 69)



 


iolARecF2
5′-T*G*C*T*GGATTGATCACAAAAAGGAATTTTTGTTT
Amplifying the cat-cassette



CACATAAGATTTTTATGGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec2 mutant



(SEQ ID NO: 70)



 


iolARecF3
5′-T*G*G*G*GGTTAAACCAGAACCAGGCCAACATCTGC
Amplifying the cat-cassette



GGGTATGGTGTCAAGAAGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec3 mutant



(SEQ ID NO: 71)



 


iolARecF4
5′-A*C*C*A*TTCTTTCATATCATGAAAAACTGTAAACC
Amplifying the cat-cassette



ATTTCGTAACGACAGACGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec4 mutant



(SEQ ID NO: 72)



 


iolARecF5
5′-G*C*T*T*CATTAACTGCTACAAGATCTCGTTTCCTG
Amplifying the cat-cassette



TCTGCAGCAATGGAACAGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec5 mutant



(SEQ ID NO: 73)



 


iolARecF6
5′-A*A*T*G*CCAAATCAATTGATATTGTTCAGAAATCT
Amplifying the cat-cassette



CAACCACTAAACCCCCGGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec6 mutant



(SEQ ID NO: 74)



 


iolARecF7
5′-C*T*G*T*TGCAATCATGTTTACCGGGGCGCCAGCCA
Amplifying the cat-cassette



GTTACACTTCTTACTTCGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec7 mutant



(SEQ ID NO: 75)



 


iolARecF8
5′-T*C*T*G*CTAGATTCTTCGCCACTGTCATTATGCAC
Amplifying the cat-cassette



TCCAATCTGTTGCAATCGTAGACTTCCGTTGAACT-3′
for c the ΔiolARec8 mutant



(SEQ ID NO: 76)



 


Cat F
5′-GTAGACTTCCGTTGAACT-3′
Amplifying the cat gene



(SEQ ID NO: 77)



 


CatR
5′-GCCTAATGAGTGAGCTAA-3′
Amplifying the cat gene



(SEQ ID NO: 78)



 


iolA_upF8
5′-TTGCAGAATATGGTGAGGTT-3′
PCR and sequencing to



(SEQ ID NO: 79)
identify the mutant from




ΔiolARec6 to ΔiolARec8


 


iolA_dnR8
5′-ATATGCCATTCAGTATGCCA-3′
PCR and sequencing to



(SEQ ID NO: 80)
identify the mutant from




ΔiolARec1-ΔiolARec8


 


Aup_intF
5′-CCAGCCAGTTACACTTCTTA-3′
fusing the two iolA gene



(SEQ ID NO: 81)
arms and the catR gene by




splicing through overlap




extension PCR (SOE)


 


And_intR
5′-TTATCTTCAGGCTTGATGCT-3′
fusing the two iolA gene



(SEQ ID NO: 82)
arms and the catR gene by




splicing through overlap




extension PCR (SOE)


 


AupF
5′-CACTCCAATCTGTTGCAATC-3′
Amplification of upstream



(SEQ ID NO: 83)
sequences of iolA gene


 


AupR
5′-GATCTCTCTCTTGGCTGATGAGTTCAACGGAAGTCTA
Amplification of upstream



C-3′
sequences of iolA gene



(SEQ ID NO: 84)



 


AdnF
5′-TCTCCTGAAGGCGCCATTAGCTCACTCATTAGGC-3′
Amplification of downstream



(SEQ ID NO: 85)
sequences of iolA gene


 


AdnR
5′-GTAACACCACCGTGAGCAAG-3′
Amplification of downstream



(SEQ ID NO: 86)
sequences of iolA gene









B. DETERMINING THE ROLE OF THE O-ANTIGEN IN A. hydrophila ML09-119 VIRULENCE
One difference between the ΔiolAtra and the ΔiolARec mutants is that when the Δioltra was constructed part of the promoter region between the iolA and iolR genes was deleted. IolR is a transcriptional repressor for multiple genes in the myo-inositol pathway, including iolR (Kohler, et al. 2011). It is possible that when the ΔiolAtra was constructed, the deleted promoter region contained a binding region for IolR (Kohler, et al. 2011). Without the binding region for the IolR repressor, the transcription of the iolR gene may be increased and the synthesis of more IolR might repress other genes that are related to the virulence of A. hydrophila in the IolR regulon such as aerolysin (Zhang et al., 2013; Cordero-Alba et al., 2012). We hypothesize that by deleting the region between the iolA and iolR gene that the expression of the iolR gene might change along with the virulence of the mutants. The results of RT-PCR using iolR-specific primers showed that there might be differences between the ΔiolARec4 and other ΔiolARec mutants (data not shown); however, no quantification of these data has been performed to date. Our in vivo channel catfish challenge study showed that there is some attenuation within the ΔiolARec3 and ΔiolARec4 mutants; however, the statistical analysis did not support a difference at P<0.05, and additional experiments with more animals and groups may be needed in order to observe a statistically significant difference between the ΔiolARec3 or ΔiolARec4 mutants and wild type ML09-119.
Even though the reason for the attenuation of the ΔiolAtra has not been completely characterized, the immunogenicity study showed that this mutant can provide around 70% survival rates for channel catfish at doses that result in no survival for naïve fish. The ELISA assay evaluating the antibody induced by the ΔiolAtra mutant against A. hydrophila ML09-119 showed that the ΔiolAtra mutant could induce strong antibody reaction. This indicates that ΔiolA5tra mutant can serve as a promising live vaccine candidate against the recent MAS epidemic outbreak. This study also raised some interesting questions regarding whether additional genetic loci were contributing to virulence.
Introduction
Lipopolysaccharides (LPS) of Gram-negative bacteria are major virulent determinants and are composed of lipid A, an inner core oligosaccharide, and repeating O-antigen polysaccharides. The virulent nature of LPS is attributed due to the core oligosaccharide and O-antigen polysaccharides. LPS contributes significantly in bacterial pathogenesis by intestinal colonization (Nevola, Laux et al. 1987; West, Sansonetti et al. 2005), lessening macrophage activation (Lugo, Price et al, 2007), promoting intracellular growth (Nagy, Danino et al. 2006), and serum resistance (DeShazer, Brett et al. 1998). The truncation or deletion of the components of the LPS, particularly the O-antigen polysaccharide, diminishes the virulence properties of the bacterial pathogen and this attenuation is necessary for development of a live, attenuated vaccine strain.
In our previous study, through whole genome comparative genomic analysis, we determined the genetic basis of O-antigen biosynthesis from twelve different A. hydrophila isolates obtained from diseased fish (Hossain et al 2013), and observed a unique O-antigen biosynthetic pathway in ML09-119 and other epidemic strains and a total of 5 different O-antigen types among the sequenced strains.
Gene knockout and mutant generation is a tool developed from naturally existing mechanisms by which genetic material is exchanged between different bacteria and viruses (Rocha, et al. 2005). After the genes are transferred into the host bacteria, these genes are then incorporated onto the host genome by homologous gene recombination (Ishikawa, et al., 2013; Thomason, et al. 2007).
Recombineering is a precise technique for the manipulation of bacterial genes and other organisms (Yu et al., 2000). This technique is very accurate and fast in target gene deletion, insertion, or substitution events; thus, in a very short time mutants for the study of gene functions can be generated (Datsenko & Wanner, 2000; Datta et al., 2008; Rivero-Müller et al., 2007). A novel recombineering method was developed (Hossain et al., manuscript in preparation) in order to introduce a recombineering plasmid into epidemic A. hydrophila via conjugation and mutagenize genes to determine their respective roles in virulence.
In this study, the Lipid A-Core ligase gene (waaL) and O-antigen polymerase gene (wzy) knockout mutants, ΔwaaLtra or ΔwaaLRec, Δwzytra or ΔwzyRec were created by both traditional allelic exchange and recombineering techniques. An in vivo channel catfish challenge study was conducted to study the role of O-antigen in the virulence of the epidemic strain of A. hydrophila ML09-119. A ΔymcA mutant was also created by knocking out the ymcA gene using the recombineering method to study the role of YmcA in the virulence of A. hydrophila. 
Materials and Methods
Bacterial Isolates and Plasmids.
The A. hydrophila ML09-119 used in this study was picked out from single colony on a TSA plate that was inoculated from a −80° C. cryostock. The epidemic strain was from a west Alabama MAS disease outbreak in 2009. The bacteria were routinely grown on fresh TSB medium overnight before use. The A. hydrophila ML09-119 used for experiments was from the bacteria stocks of the fish disease lab in Auburn University. This epidemic strain was originally isolated from the kidneys of channel catfish naturally infected with A. hydrophila. The pure culture of the epidemic strain was used first in a small test infection of 10 catfish. Moribund catfish that showed clinical signs of A. hydrophila ML09-119 were collected for necropsy. A. hydrophila was re-isolated from a dying fish by poking a sterile plastic bacteriology loop into the kidney and inoculating a TSA plate. By doing this, it is expected than the virulence of the epidemic strain stock can be recovered. ML09-119 was then confirmed by biochemistry and selective media following the established identification procedures with modifications (Furuwatari, et al., 1994; Holt, et al., 1994). Briefly, the identification biochemical tests included Gram stain, cytochrome oxidase, glucose utilization, 0/129, sucrose, esculin hydrolysis, V-P, DL-lactate utilization and urocanic acid utilization, and then testing on the selective minimal medium M9 with myo-inositol added. E. coli SM10-λ-pir and E. coli CC118-λ-pir (Simon et al., 1983) were used for the conjugal transfer of the mobilizable mutagenesis plasmids to A. hydrophila ML09-119. The list of bacterial strains used in this study is presented in Table 6.
Construction of Defined A. hydrophila Lipid A-Core Ligase (waaL) and O-Antigen Polymerase (Wzy) Knockout Mutants, ΔwaaLtra & ΔWzytra, by Traditional Splicing PCR and Conjugation Technique.
Lipid A-Core ligase (waaL) and O-antigen polymerase (wzy) knockout mutants, ΔwaaLtra & Δwzytra were constructed using suicide plasmid pDMS197 (Edwards, Keller et al. 1998). The primers needed for this study were listed in the Table 7.
The two pairs of primers, Li-upF/Li-upR and Li-dnF/Li-dnR, were used to amplify approximately 400 bp upstream and downstream sequences of waaL gene, respectively using EconoTaq PLUS GREEN 2× Master PCR kit (Lucigen, USA) to construct the ΔwaaL mutant. The template used in this PCR was the genomic DNA of A. hydrophila ML09-119 which was extracted using a E.Z.N.A.® Bacterial DNA Kit (Omega Bio-Tek, USA). The chloramphenicol acetyltranferase gene (cat) was amplified from pMHH46 plasmid (Hossain et al 2013) using primers catF and catR. The primers Li-upR and Li-dnF were used for the amplification of upstream and downstream sequences of waaL gene, and contained the reverse complemented sequences of catF and catR primers which were added respectively at their 5′ ends. The CatR-cassette which was the chloramphenicol resistance gene (CMR) with two arms of the upstream and downstream homologous of waaL gene was created by fusing the two arms and the CMR gene by splicing through overlap extension PCR (SOE) (Horton, Hunt et al. 1989). The primers for this PCR were Liup-intF and Lidn-intR. The PCR products were purified by agarose gel purification.
The suicide plasmid pDMS197 was digested by restricted digestion enzyme XbaI (New England Biolabs, NEB) following the protocol provided by the manufacturer. A 50 ul reaction was used for the digestion, including 25 ul of the suicide plasmid pDMS197 DNA, 3 ul of the XbaI restricted digestion enzyme, 5 ul of the 10×CutSmartTM Buffer, 1×BSA and 16 ul RNase free H2O, the reaction system was incubated at 37° C. for one hour. The reaction system was then incubated at 65° C. for 20 min to stop the reaction. The digested product was purified by DNA Clean & ConcentratorTM (Zymo research), and the concentration was measured by Qubit® dsDNA BR Assay Kit (Life technologies). The product was blunted using end-repair kit DNA terminator (Lucigen, USA) following the producer's instruction. The product was purified by DNA Clean & ConcentratorTM (Zymo research) again before ligation.
The purified restriction enzyme XbaI digested and blunted suicide plasmid pDMS197 was ligated with the gel purified CatR-cassette using Quick Ligase (NEB, USA) incubated for 30 minutes. Briefly, 50 ng of blunted suicide plasmid pDMS197 and around 3-fold molar excess of the CatR-cassette insert was mixed together and the volume was adjusted to 10 ul with RNase free H2O. 10 ul of the 2× Quick Ligation Buffer and 1 μl of Quick T4 DNA Ligase were added into the mixture. The mixture was centrifuged briefly and incubated at room temperature (25° C.) for 30 minutes before it was chilled on ice. A SB gel electrophoresis was done to confirm the ligation product (data not shown).
The making of the electrocompetent cells of E. coli CC118-λ-pir and SM10-λ-pir was following a published protocol (Iuoue, et al., 1990) with minor changes. A 0.5 ml of the overnight culture of E. coli CC118λ-pir and SM10-λ-pir bacteria was inoculated into 200 ml of Hanahan's Broth (SOB Medium) respectively with 10 mM MgCl2. The culture was incubated in the 37° C. water bath incubator with shaking at 200 rpm for around 2.5 hours and the OD600=0.4. The culture was chilled in ice for 10 min before loaded into 200 ml centrifuge tubes. The culture was centrifuged at 6000 rpm for 8 min at 4° C., the supernatant was discarded and the pellet was washed by resuspended with 10% glycerol and centrifuged again at 6000 rpm for 8 min. The wash step was repeated for 3 times before the pellet was gently resuspended in 200 ul GYT medium. The whole procedure was performed on ice.
The ligation product was then used in the electroporation (Chassy, et al., 1988; Dower et al, 1988) to create the plasmid pDMS197waaL, which contains a deletion of the entire waaL gene. 50 ul of the premade electrocompetent cells of the E. coli CC11810-λ-pir was mixed gently with 2.5 ul of the ligation product and chilled on ice for 5 min. The mixture was transferred into ice cold cuvettes (Bulldog bio) before the cuvettes were loaded onto the Eppendorf® Eporator® (Eppendorf). Voltage was set at 1800V. The mixture was mixed with recovery medium (SOC medium) right after the pulse shock. The culture was transferred to a 2 ml test tube and incubated at 37° C. with shaking at 200 rpm for 2 hrs. The successfully electroporated E. coli CC118-λ-pir with the plasmid pDMS197waaL was selected on 2XYT agar medium plate with 25 ug/ml chloramphenicol and 5 ug/ml tetracycline. A similar strategy was followed for the construction of pDMS197wsy, which contains a deletion of the entire wzy gene. E. coli CC118-λ-pir with the plasmids were grown in fresh LB medium with 25 ug/ml chloramphenicol and 5 ug/ml tetracycline. The plasmid was extracted using E.Z.N.A.® Plasmid Midi Kit (Omega Bio-Tek, USA) respectively to get more pure suicide plasmids pDMS197waaL and pDMS197wzy.
The suicide plasmids pDMS197waaL and pDMS197wzy were independently introduced into A. hydrophila ML09-119 by conjugation with E. coli SM10-λ-pir bearing plasmid pDMS197waaL or pDMS197wzy, respectively. A single colony was selected on the selective medium plate for SM10-λ-pir bearing plasmid pDMS197waaL or pDMS197wzy, respectively, for inoculation of 5 ml LB broth, medium. The culture was incubated at 37° C. with shaking at 200 rpm until the OD600 was above 1. A single colony of A. hydrophila ML09-119 was picked to inoculate 5 ml TSB broth medium. The culture was incubated at 30° C. with shaking at 200 rpm until the OD600 was above 1. A 4 ml ML09-119 culture and 1 ml SM10-λ-pir bearing plasmid pDMS197waaL or pDMS197wzy were mixed together, respectively. The 5 ml culture mixture was filtered through a MicroFunnel 300 SP (MicroFunnelTM) by vacuum pressure and 5 ml of fresh LB broth medium was used for washing the cells onto the membrane. The membrane was transferred to the sheep blood agar medium after 2 washes. The sheep blood agar medium was incubated at 30° C. overnight.
The membrane with the cell culture mixture was vortexed with 3 ml fresh TSB broth medium for selection. Single cross-over mutants were selected oil TSA plate supplemented with chloramphenicol, tetracycline and colistin. Double-cross over mutants were obtained by plating onto LB (without NaCl) plates supplemented with 15% sucrose and 12.5 μg/ml chloramphenicol. Mutants grown on this selective plate were subjected to phenotypic and genotypic characterizations. The complete deletion of the waaL and wzy genes were confirmed by PCR followed by sequencing.
Construction of Defined A. hydrophila ΔymcA and ΔWzyRec Mutant by Recombineering.
A recombineering technique was used to create a precise deletion of the ymcA gene and wzy gene and generate the ΔymcA and Δwzyrec mutants in order to determine the role of O-antigen in the virulence of epidemic A. hydrophila ML09-119 in channel catfish.
The chloramphenicol acetyltranferase (cat) gene was amplified from pMHH46 plasmid (Hossain et al 2013) using primers ymcARecF and ymcARecR to generate the cat-cassette with 50 bp of the upstream and downstream of the targeted ymcA gene. The primer ymcARecF contained 50 bp of the upstream of the targeted ymcA gene and the primer ymcARecR contained the reverse complemented sequences of 50 bp of the downstream of the targeted ymcA gene which were added respectively at the 5′ ends of each respective primers. The PCR product was validated using agarose gel electrophoresis before another 24×PCR was done using this PCR product to generate more cat-cassette insertion.
The PCR product was purified and concentrated using Wizard® DNA Clean-Up system (Promega, USA) following the protocol provided by the manufacturer. Briefly, the 24 different PCRs were pooled together in a 15 ml conical tube, and a Wizard® DNA Clean-Up kit (Promega, Madison, Wis.) was used to purify the PCR products according to the manufacturer's protocol. The concentration of the final concentrated PCR product was measured using Qubit® dsDNA BR Assay Kit (Life Technologies).
A. hydrophila ML09-119 containing the plasmid pMJH65, which was constructed for the purposes of introducing a recombineering cassette into gram-negative bacteria (Hossain et al, manuscript in preparation), was prepared for electroporation using a standard protocol (Inoue, et al., 1990) with minor changes. 0.5 ml of the overnight culture of ML09-119 bacteria was inoculated into 150 ml of Hanahan's Broth (SOB Medium) with 1.5 ml 1M arabinose, 300 ul 25 mg/ml Tetracycline and 600 ul of 2M MgCl2. The culture was incubated in the 30° C. water bath incubator with shaking at 200 rpm for around 4 hours and the OD600=0.5. The culture was chilled on ice for 10 min before loaded into 200 ml centrifuge tubes. The culture was centrifuged at 6000 rpm for 8 min at 4° C. The supernatant was discarded and the pellet was washed by re-suspending with 10% glycerol and centrifuged again at 6000 rpm for 8 min. The wash step was repeated 4 times before the pellet was gently resuspended in 200 ul 10% glycerol. The whole procedure was performed on ice.
The concentrated and purified PCR product was then used in the electroporation (Chassy, et al., 1988; Dower et al, 1988) to create the precise ymcA gene deletion mutant ΔymcA. 50 ul of the premade electrocompetent cells of A. hydrophila strain ML09-119 (pMJH65) was mixed gently with 3 ug of the concentrated PCR product and chilled on ice for 5 min. The mixture was transferred into ice cold cuvettes (BulldogBio) before the cuvettes were loaded onto the Eppendorf® Eporator® (Eppendorf) with a voltage setting of 1200 V. The mixture was mixed with recovery medium (SOC medium) right after the pulse shock. The culture was transferred to a 2 ml test tube and incubated at 30° C. with shaking at 200 rpm overnight.
The successfully electroporated A. hydrophila ML09-119 ymcA gene deletion mutant was selected on a TSA agar medium plate supplied with 25 ug/ml chloramphenicol. A similar strategy was followed for the construction of ΔwaaLrec or ΔwzyRec, which contains a deletion of waaL or wzy genes, respectively.
Virulence Study of A. hydrophila Mutants in Channel Catfish.
All experiments conducted with vertebrate animals (catfish) were approved by the Institutional Animal Care and Use Committee (IACUC) review board at Auburn University in accordance with the animal welfare guidelines specified in the United States.
All the channel catfish (I. punctatus, Kansas Random Strain), used in this study were spawned at the hatchery of the Auburn University Fish Genetics Research Unit artificially, prior to transferring to troughs or glass aquaria at the Auburn University Fish Pathology wet lab S-6. Fish were maintained at recirculation systems (temperature around 25° C. and pH 7.5) using well water sources with constant aeration. Fish were fed daily with commercial feed. Water quality factors including temperature, pH, salt level, total ammonia level, total nitrite level were tested on daily basis to ensure that catfish fingerlings remained unstressed and naive to A. hydrophila. Catfish fingerlings were grown out in this system until their body weight (BW) reached 20±5 g.
A bacterial suspension of exponential phase growth was prepared by overnight culture in 5 ml TSB broth medium on 200 rpm shaking at 200 rpm at 30° C. The next day 1 ml of the overnight bacterial culture was used to inoculate 100 ml fresh TSB broth culture which was incubated with shaking at 200 rpm at 30° C. for 4 hours. The bacterial culture was centrifuged at 6000 rpm for 10 min. The supernatant was discarded and the bacterial pellet was resuspended in fresh TSB media. The optical density of the bacterial culture was measured by the thermospectronic spectrophotometer (Thermo Spectronic, Rochester, N.Y., USA) at 600 nm and adjusted to an OD600=1, which was expected to be 1×109 CFU/ml. After adjusting the bacterial suspension to an appropriate OD, A 1:100 dilution was performed using fresh TSB broth to get the desired concentration (around 1×107 CFU/ml) of A. hydrophila. Another 1:2 dilution was done with fresh TSB. This culture was put on ice and used for challenge within 3 hours. A plate count assay was conducted right after the fish challenge to calculate the accurate CFU/ml concentration used in this study. The bacterial cultures used in the fish challenge were serially diluted and 100 ul of each dilution was spread on the TSA plates with 3 replicates for each strain of bacteria.
Channel catfish in Auburn University Fish Pathology wet lab S-6 were randomly distributed into glass aquarium tanks. MS-222 (30 mg/l) was used during the handling of fish to calm the fish down to decrease the stress. Each tank contained 10 fish. A recirculating system was applied during the acclimation period, which was lasted for 10 days. Water temperature was originally 25° C. and salt level was kept around 1.8 ppt to decrease the stress caused by environmental changes as well as eliminating the chance of F. columnare infection. Water temperature was gradually brought up to 30±1° C., and salt was gradually brought down to 0.8 during the first 3 day of the acclimation time. Every environmental factor was kept stable prior to the challenge. Fish were fed with commercial catfish fed once a day at 4% of their body weight. Water was changed once per day for the recirculating system with constant aeration. At the time of challenge, recirculating system was changed into flow through system, with the temperature at 30±1° C. Fish of each treatment tank were euthanized by immersing in a bucket with MS-222 (30 mg/l), before 200 ul of ML09-119 bacterial culture was injected intraperitoneally into each fish. Fish were then put back to their cohabitation tanks. Fish of control groups were injected with pure TSB broth medium. Challenged fish were kept the same way as they were during the latter acclimating time. Mortalities were recorded daily for 14 days post challenge. Any moribund or dead fish were removed from the system daily for bacteriological identification and tissue sampling. Prior to sampling, fresh dying or dead fish were inspected externally and internally for any clinical signs. The identification of A. hydrophila isolated from anterior kidney of the fresh dying or dead fish was performed by the biochemistry and selective medium method described previously. Survivors of the challenge were kept for 28 days, before they are challenged again with the wild type ML09-119 to test if any protection effect was provided. The procedure of the re-challenge was similar to the preciously challenge. At seven days post re-challenge, blood samples were then drawn from the survivors for the ELISA titer in the later experiment.
Immunogenicity of the Mutants and Enzyme-Linked Immunosorbent Assay (ELISA).
Blood samples collected after the fish challenge were put in the room temperature for 2 hrs then 4° C. overnight allowing to clot completely. Serum of each blood sample was collected followed by centrifuging at 5000 rpm for 10 min. The supernatant of each sample was collected for Enzyme-linked Immunosorbent assay (ELISA) analysis. Antibody responses of channel catfish to A. hydrophila were quantified by evaluating the presence of specific immunoglobulin to A. hydrophila wild type ML09-119 using indirect ELISA. Protein DetectorTM ELISA kit was use to conduct the ELISA experiment.
The protocol followed was similar to the product instructions with minor changes. Ninety six-well plastic plates were coated with 100 ul of a solution of 10 ug/ml (107 CFU/ml)) A. hydrophila epidemic strain. A. hydrophila were suspended in carbonate-bicarbonate coating solution. The coating solution was prepared by diluting one time coating buffer tablet in 10 times of sterile reagent quality water. The plates with coating buffer and antigen were placed in 4° C. pH 9.6 overnight. The plates were washed 4 times with washing buffer provided by the kit the next day, followed by adding 1×BSA blocking buffer to block for 15 min at room temperature. After another wash step, the plates were used to do ELISA analysis. 100 ul of 1% BSA blocking buffer was added into each well on the A. hydrophila ML09-119 coated plate. 200 ul of the 1/10 fish blood serum sample diluted with 1% BSA blocking buffer was added to the column A2-A11, A1 and A12 were served as positive and negative control. 100 ul of the solution from A1-A12 was transferred to B1-B12 and mixed carefully by pipetting 3-5 times. This step was repeated across the plate until E1-E12. The final 100 ul from the wells in the row E after mixing was discarded. The plate was then incubated at room temperature for 1 hour. The plated was emptied, and residual liquid was tapped out. The plate was washed out by the washing buffer that came with the kit for 5 times. 100 ul of Rat Anti-catfish monoclonal antibody (Mab) was diluted 32 times and added into each well that contained the primary antibody, after which the plate was incubated at room temperature for 1 hour. After incubation the plate was emptied, and residual liquid was tapped out and the plate was washed out five times by the washing buffer that came with the kit. 50 ul of tertiary antibody (goat anti-rat antibody conjugated with horseradish peroxidase) (0.1 ug/ml) was added into each wall that contained the secondary antibody. The plate was incubated at room temperature for 1 hour, after which the plate was washed as above. 5 minutes soaking time was given to the last wash. 50 ul of the substrate solution that came with the kit was added into each well that contained the tertiary antibody. The plated was incubated at room temperature for 5-15 min before the reaction as stopped by adding 50 ul of stop solution into each well for full color development and the plate was then read at OD405. A reaction was defined as positive if its OD450 value was at least two times the negative control. Ending points were the highest dilution with a positive reaction.
A criss-cross serial dilution analysis was done prior to the ELISA analysis of the samples to optimize the reagent concentration in the immunoassay procedure. 100 ul of 1% BSA blocking buffer was added into each well of the A. hydrophila ML09-119 coated plate. 200 ul of the 1/10 ML09-119 infected survivor fish blood serum sample diluted with 1% BSA blocking buffer was added to the respective columns and serially diluted across the plate to identify the best concentration range for the sample. Prior to adding the Mab, 100 ul of 1% BSA blocking buffer was added into each well, followed by 200 ul of the secondary rat anti-channel catfish Mab. This Mab solution was serially diluted across the plate to identify the optimum concentration for the Mab.
Results
Cumulative Survival Rate of the Channel Catfish Challenged with Δwaaltra or Δwzytra and ΔwaalRec or ΔwzyRec.
For better understanding of the virulence factors of the A. hydrophila epidemic strain and to identify possible live vaccine candidates, the waal and wzy genes that are expected to be required for O-antigen synthesis and assembly were knocked out by a traditional allelic exchange technique. The LPS of Gram-negative bacteria are major virulent determinant and are composed of lipid A, an inner core oligosaccharide and repeating O-antigen polysaccharide. The role of LPS in virulence is due to the core oligosaccharide and O-antigen polysaccharide, by contributing to intestinal colonization (Nevola, Laux et al. 1987; West Sansonetti et al. 2005), lessening macrophage activation (Lugo, Price et al. 2007), promoting intracellular growth (Nagy, Danino et al. 2006), and serum resistance (DeShazer, Brett et al. 1998). Since O-antigen significantly contributes to the virulence of many gram negative bacteria, the hypothesis is that the waal and wsy genes are virulence factors and by constructing targeted deletions of each of these genes that the resulting mutants of ML09-119 will be attenuated and can serve as promising vaccine candidates.
The results of the in vivo channel catfish i.p challenge with Δwaaltra and Δwzytra showed that the Δwaaltra and Δwzytra are both avirulent. The channel catfish in the Δwaaltra and Δwsytra treatment groups had a 100±0% survival rate, while the wild-type strain-injected group had a 5±0.08% survival. The percentage survival rates were transformed by arcsine square root transformation and then analyzed by SAS 9.2, and significant differences were observed between Δwaaltra or Δwzytra and ML09-119 treatment groups (P<0.0001). This indicates that the Δwaaltra and Δwzytra are both attenuated strains of ML09-119 (FIG. 14). However, the channel catfish i.p challenged with the ΔwaalRec and ΔwzyRec mutants, which were created by precisely knocking out the waal and wsy gene using the recombineering method, were still fully virulent with a survival rate comparable to the wild type ML09-119 (data not shown).
Sub-Challenge of the Channel Catfish Survivors in the Δwaaltra and Δwzytra Mutants Treatment Groups with Wild Type ML09-119 was Conducted.
Unfortunately, due to mistakes in the plate count technique, the exact concentration of ML09-119 used in this experiment was not determined. However, significant differences were still observed between the sham negative control group and the treatment groups (P<0.05). No significant differences were observed between the treatment groups and the positive control group (PΔwzy>0.05 and PΔwaal>0.05). A 35±0.1% survival rate was observed in Δwaal group, and a 27±0.3% survival rate was observed in Δwzy group, suggesting no immunity developed in either of the O-antigen mutant treatment groups (FIG. 15).
The investigation of the virulence of the ΔymcA mutant and the vaccine candidate and immunogenicity challenge study. The vast difference in the virulence between the Δwaaltra or Δwzytra mutants and ΔwaalRec or ΔwzyRec prompted us to investigate the molecular difference(s) between the mutations generated in these two groups. It was discovered that when the Δwaaltra mutant was constructed, a part of the transcription termination site (TTS) of the ymcA gene, located downstream of the waal gene, was deleted. It is also possible that insertion of the CMR gene cassette has a polar effect on ymcA transcription. This prompted us to create the ΔymcA mutant to determine if the ymcA gene contributes to the virulence of A. hydrophila ML09-119. The hypothesis was that by interrupting the ymcA gene, the ΔymcA mutant will be attenuated; therefore, the ΔymcA mutant was created using the recombineering technique.
To determine the virulence of the ΔymcA mutant and to evaluate the efficacy of the ΔymcA mutant as a live vaccine against A. hydrophila ML09-119, an in vivo channel catfish challenge study was carried out. The results of the in vivo channel catfish i.p. challenged with ΔymcA mutant showed that the ΔymcA mutant was avirulent. The channel catfish in the ΔymcA treatment group had a 68.1±16.8% survival rate, while a 0±0% survival rate was observed in the positive control treatment group. Note that a 83.3±11.6% survival rate was observed in the negative control treatment group. A significant difference was observed between the ΔymcA treatment group and the A. hydrophila ML09-119 treatment group, PΔymcA=0.000186<0.05. This indicates that ΔymcA is an attenuated mutant of ML09-119 (FIG. 16).
A sub-challenge of the channel catfish survivors was carried out 21 days post challenge. The ΔymcA treatment group surviving fish that were challenged with wild type ML09-119 showed a 90.5±8.3% survival rate, in contrast to the 0±0% survival rate observed in the naive channel catfish challenged with ML09-119 (FIG. 17). Significant differences were observed between the ML09-119 group and ΔymcA mutant group, PΔymcA=4.52E−0.5<0.05. This suggested that protective immunity against ML09-119 was developed by exposing catfish to the ΔymcA mutant.
Enzyme-Linked Immunosorbent Assay (ELISA).
The Enzyme-linked Immunosorbent Assay (ELISA) was carried out to determine the efficacy of protective immunity induced by the ΔymcA mutant immunized channel catfish after being i.p injected with 1×106 CFU/fish of the mutant. Since LPS contributes significantly to bacterial pathogenesis via multiple mechanisms, and the ymcA gene has been reported to be required for the biosynthesis and assembly of the O-antigen (Peleg, et al., 2005), we hypothesized that the ymcA gene is a virulence factor in A. hydrophila ML09-119, the deletion of the ymcA gene, by removing the O-antigen, might result in an A. hydrophila strain that is sensitive to complement, is less invasive, and allows development of antibodies targeting A. hydrophila antigens that are present in more typical A. hydrophila strains that are opportunistic pathogens in different fish species. All of the replicates of the ΔymcA mutant induced a strong antibody reaction to ML09-119 (FIG. 18). The levels of antibody to ML09-119 were highest in serum from the catfish that were immunized with the ΔymcA mutant, indicating strong antibody induction by the ΔymcA mutant.
Discussion
This study provided valuable insight into role of the O-antigen in the virulence of A. hydrophila ML09-119. The ymcA gene deletion mutant was observed to be attenuated in its virulence and can provide protection against A. hydrophila ML09-119 in an in vivo channel catfish challenge study. This mutant may be a promising live vaccine candidate against epidemic A. hydrophila. 
The recent epidemic outbreak of the MAS caused by highly virulent A. hydrophila has drawn a lot of attention since the catfish farming operations in the southeastern United States have not experienced a large-scale outbreak of MAS previously (Hemstreet, 2010). In 2009 and in all subsequent years, catfish farmers in west Alabama have reported severe disease outbreaks which were demonstrated to be caused by a highly virulent strain of A. hydrophila, represented by strain ML09-119, in catfish (I. punctatus). From 2009 to the present, Alabama catfish farmers lost more than 10 million pounds of catfish that were market-size and estimated to be more than $3 million due to this epidemic strain of A. hydrophila (Pridgeon et al., 2011; Liles et al., 2011). The A. hydrophila epidemic strain ML09-119 that has been used in research studies is highly virulent to channel catfish, causing severe mortality within 24 h post exposure at a dose of >1×106 CPU by i.p injection. Also, this epidemic A. hydrophila strain has expanded its geographic territory and caused frequent outbreaks in the summer months, resulting in millions of pounds of losses in Alabama, Mississippi and Arkansas, (Pridgeon and Klesius, 2011). Due to its highly virulent nature and the resulting huge economic losses, it is essential that the virulent factors expressed by this epidemic A. hydrophila be studied and an effective vaccine be developed.
A previous study showed that epidemic strains possess an unique O-antigen cluster compared to reference strains (Hossain et al, 2013). This prompted us to investigate the role of the O-antigen in the virulence of the A. hydrophila ML09-119, since the O-antigen is known to contribute significantly in bacterial pathogenesis, such as intestinal colonization (Nevola, Laux et al. 1987; West, Sansonetti et al. 2005), lessening macrophage activation (Lugo, Price et al. 2007), promoting intracellular growth (Nagy, Danino et al. 2006), and serum resistance (DeShazer, Brett et al. 1998).
The Δwaaltra and Δwzytra mutants were created using a traditional allelic exchange technique, and the in vivo channel catfish challenge study showed that these mutants were attenuated compared to their wild type parent strain ML09-119 strain. However, when we created the precise waal and wzy gene deletion mutants ΔwaalRec and ΔwzyRec mutants using a more efficient and accurate recombineering technique, we observed that those mutants were still virulent in channel catfish. It was found that during the construction of the Δwaal mutant that a region of the transcription termination site (TTS) between the waal gene and the ymcA gene was deleted, and the insertion of the gene cassette in the mutant may have a polar effect on ymcA transcription. The ymcA gene is reported to be required for the biosynthesis and assembly of the O-antigen (Peleg, et al. 2005). We therefore hypothesized that a ymcA mutant would be attenuated in its virulence. The in vivo channel catfish challenge study showed that the ΔymcA mutant is significantly attenuated in its virulence.
The immunogenicity study showed that this mutant can provide 90.5±8.3% protection for channel catfish in the in vivo channel catfish study, and the ELISA assay demonstrated that the ΔymcA mutant induced a strong antibody reaction. This indicates that ΔymcA mutant can serve as a promising live vaccine candidate against epidemic A. hydrophila. The fact that the ELISA titer of ΔymcA mutant is even higher than the positive control is probably due to that the serum for the positive control was collected months ago and stayed in the −20° C. for months, thereby losing some efficacy. We were not able to use fresh ML09-119 serum, since ail of the channel catfish in the positive control group were dead and there were no survivors from which to collect blood samples.
Interestingly, there has not been any previous research investigating the contribution of YmcA to the virulence of any bacterial pathogens. There have been studies on the contribution of YmcA in B. subtilis on the formation of biofilms in multicellular bacterial assemblages (Branda, et al., 2004; Branda, et al., 2006; Kobayashi, 2007). However, in these studies the exact function of YmcA is not determined. One study on the human pathogen Shigella flexneri mentioned that the ymcA gene exists and speculates that it might encode a putative outer membrane lipoprotein that is highly conserved among Shigella and E. coli (Sun, et al., 2012). Since none of these studies have provided any conclusive evidence for the function of YmcA, this makes the finding in this study is even more valuable, given the evidence that the ymcA gene is required for virulence in epidemic A. hydrophila. 
This study also raised some interesting studies for the future research including: (1) The exact function of YmcA; (2) The complementation of the ΔymcA mutant and determining if the complemented mutant is restored in its virulence; (3) The delivery route for the live vaccine of the channel catfish against the A. hydrophila the epidemic strain; (4) The protective effect of the ΔymcA mutant against other non-epidemic strains of A. hydrophila.







TABLE 6







Summary of bacterial strains and plasmids used in this study









Bacterial strains and




Plasmid
Relevant features
References





Bacterial strains




A. hydrophila

Hossain et al., 2013


ML09-119


A. hydrophila

Hossain et al., 2013


AL06-06


E. coli SM10-λ-pir
thi-1thr leu tonA lacY supE
(Simon, Priefer



recA::RP4-2-TcT::Mu Kmr
et al. 1983)



λpir


E. coli CC118-λ-pir
Δ(ara-leu) araD ΔlacX74
(Herrero, de



galE galKphoA20 thi-1 rpsE
Lorenzo et al. 1990)



rpoB argE(Am) recA1 λpir


Plasmids


pDMS197
Suicide vector, sacB, TetR
(Edwards, Keller




et al. 1998)


pDMS197waaL

This study


pDMS197wzy

This Study
















TABLE 7







The primers used in this study









Primer ID
Primer sequence
Primer Application





ymcARecF
5′-T*A*G*A*GATATCAATATTCGTATTGCCAATCTCCT
Amplifying the cat-cassette for



TGCTAATCGAGTACCAGAGTAGACTTCCGTTGAACT-3′
creating the ΔymcA



(SEQ ID NO: 87)



 


ymcARedR
5′-C*A*A*C*TGCTCGCCCTTTTTGATGAAAAAAGATCG
Amplifying the cat-cassette for



GCTCTATGCAACTTTTGAGCCTAATGAGTGAGCTAA-3′
creating the ΔymcA



(SEQ ID NO: 88)



 


ymcA_upF
5′-CCGAATGGTAATCCACAGTT-3′
PCR and Sequencing of ΔymcA for



(SEQ ID NO: 89)
identification of the mutant


 


Ymca_dnR
5′-TAGAACAGCTGGTCACGAGA-3′
PCR and Sequencing of ΔymcA for



(SEQ ID NO: 90)
identification of the mutant


 


Cat F
5′-GTAGACTTCCGTTGAACT-3′
Amplifying the cat gene



(SEQ ID NO: 91)



 


CatR
5′-GCCTAATGAGTGAGCTAA-3′
Amplifying the cat gene



(SEQ ID NO: 92)



 


Li-upF
5′-ACTTAAGCTCGCCGAACTC
Amplification of upstream



(SEQ ID NO: 93)
sequences of WaaL gene


 


Li-upR
5′-GCTGTCGAGGCCATGTGAGTTCAACGGAAGTCTAC-3′
Amplification of upstream



(SEQ ID NO: 94)
sequences of WaaL gene


 


Li-dnF
5′-AAGATCGGCTCTATGCAACTTTAGCTCACTCATTAGG
Amplification of downstream



C-3′
sequences of WaaL gene



(SEQ ID NO: 95)



 


Li-dnR
5′-TGATTATGATGTAATGACTGG
Amplification of downstream



(SEQ ID NO: 96)
sequences of WaaL gene


 


Liup-intF
5′-AGAAGCGGTGCTGATAACG
fusing the two waal gene arms and



(SEQ ID NO: 97)
the CMR gene by splicing through




overlap extension PCR (SOE)


 


Lidn-intR
5′-GGCAGTTACCATTCATGAGT
fusing the two waal gene arms and



(SEQ ID NO: 98)
the CMR gene by splicing through




overlap extension PCR (SOE)


 


wzyupF
5′-CCGCGACAACAACTCCTT
Amplification of upstream



(SEQ ID NO: 99)
sequences of wzy gene


 


wzyupR
5′-GCACTTCCTGTATCAAGATTAGTTCAACGGAAGTCTA
Amplification of upstream



C-3′
sequences of wzy gene



(SEQ ID NO: 100)



 


wzydnF
5′-CTAGCTGTGGTGCCAGAATATTAGCTCACTCATTAGG
Amplification of downstream



C-3′
sequences of wzy gene



(SEQ ID NO: 101)



 


wzydnR
5′-CATTCAATATAGTGTCTGAA
Amplification of downpstream



(SEQ ID NO: 102)
sequences of wzy gene


 


wzyup-inF
5′-GTGACGCCACCGATGATA
fusing the two wzy gene arms and



(SEQ ID NO: 103)
the CMR gene by splicing through




overlap extension PCR (SOE)


 


wzydn-inR
5′-CTGATGTTATTATTGACCAAG
fusing the two wzy gene arms and



(SEQ ID NO: 104)
the CMR gene by splicing through




overlap extension PCR (SOE)









C. CONCLUSIONS
A. hydrophila ML09-119 has been reported to cause severe mortality in commercial catfish farms. In this study, the virulence factors of the myo-inositol pathway and of the O-antigen synthesis pathway were studied to determine their role in the virulence of A. hydrophila ML09-119. Mutants lacking factors in each of these pathways were created by both allelic exchange technique and recombineering technique. The efficacy of the mutant as a live vaccine candidate against A. hydrophila ML09-119 was evaluated by in vivo channel catfish challenge study.
In this study, the gene iolA coding for the enzyme aldehyde dehydrogenase for myo-inositol catabolism was inactivated by traditional allelic exchange to generate the A. hydrophila ΔiolAtra mutant. An in vivo challenge in channel catfish showed that there was no mortality in the channel catfish that were challenged with ΔiolAtra mutant, but there was mortality in the channel catfish challenged with ΔiolArec mutants similar to wild type ML09-119. Results of the in vivo challenge in channel catfish showed that ΔiolArec3, ΔiolArec4 exhibited some decrease in mortality, but there were no significant difference in the mortality between the channel catfish challenged with ΔiolArec3, ΔiolArec4 and the channel catfish challenged with the wild type ML09-119. ELISA titer of the survivor's of the ΔiolAtra after 21 days showed that ΔiolAtra can induce strong antibody response against the wild type A. hydrophila ML09-119, indicating that this mutant can serve as a promising vaccine candidate against the epidemic A. hydrophila. 
In this study, Lipid A-Core ligase (waaL) and O-antigen polymerase (wzy) knockout mutants, ΔwaaL, Δwzy were created by both traditional splicing PCR and conjugation technique and recombineering technique respectively, ΔwaaLtra or ΔwaaLRec, Δwzytra or Δwzyrec. An in vivo channel catfish challenge study was committed on channel catfish to study the role of O-antigen in the virulence of the epidemic strain of A. hydrophila. The results show that the channel catfish that were challenged with ΔwaaLtra, Δwzytra were avirulent, but ΔwaaLRec, ΔwzyRec were virulent.
In this study, a ΔymcA mutant was created by knocking out the ymcA gene by recombineering technique. The results showed that ΔymcA mutant was attenuated. Sub-challenge of the survivors of ΔymcA treatment group 21 days post first challenge and ELISA titer of the survivors of the ΔymcA treatment showed that this mutant can provide 90.5±8.3% protection against wild type A. hydrophila ML09-119 indicating that the ΔymcA mutant can serve as a promising vaccine candidate.
Markerless versions of the ΔymcA and ΔymcC mutants also were created using the flippase-mediated removal of the chloramphenicol resistance gene. These markerless mutants were injected IP into fingerling catfish and within 24 hours significant mortality was observed in the wild-type ML09-119-injected fish, but a significant reduction was observed in the mortality of fish injected with the CmR ΔymcA, markerless ΔymcA, or the markerless ΔymcC mutants compared to the wild-type control (P<0.05) (FIG. 20).
Fingerling catfish that had previously been IP injected with 107 CFU/fish for each respective mutant (i.e., the ΔymcA markerless mutant and the ΔymcC markerless mutant), as well as naïve fish, were challenged with ML09-119 at the dosage of 4×106 CFU/fish 21 days post-injection. Survival rate was recorded after 24 hours (FIG. 21). The average survival rate of naïve fish was 25±35.4%, while the ΔymcA markerless treatment group's survival rate was 88.9±16.7%, and the ΔymcC markerless treatment group's survival rate was 75±20.4%, suggesting that both mutants can provide protection against epidemic vAh strain ML09-119.
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Example 3
Methods for the Development of Markerless ML09-119ΔymcA Mutant
Conjugal transfer of recombinogenic plasmid pMJH65 into Aeromonas hydrophila strain ML09-119. The mobilizable recombinogenic plasmids pMJH65 bearing the λ-Red cassette were introduced into E. coli strain SM10λpir by electroporation according to a previously published method (1). Plasmid pMJH65 in E. coli strain SM10λpir was conjugally transferred into A. hydrophila strain ML09-119 by filter mating experiments according to the methods described previously (2). A. hydrophila transconjugants were selected on LB plates supplemented with tetracycline and colistin. The conjugal transfer of plasmid pMJH65 into A. hydrophila was confirmed by the positive growth of transconjugants in the presence of appropriate antibiotics and with PCR using primers p46-intF (5′-TGTTCCTTCTTCACTGTC-3′) (SEQ ID NO:105) and p46intR (5′-GATGTACTTCACCAGCTC-3′) (SEQ ID NO:106) (3).
Deletion of gene ymcA from A. hydrophila strain ML09-119 by recombineering. Electrocompetent A. hydrophila strain ML09-119 harboring recombinogenic plasmid pMJH65 was prepared as described follows. Overnight grown ML09-119 culture was diluted 1:70 in 40 ml of Super Optimal broth (SOB) medium supplemented with tetracycline (10 μg/ml) and 10 mM of L-arabinose, and grown with vigorous shaking until the OD600 attained a value of 0.6 for A. hydrophila. Cells were harvested by centrifugation at 5000×g for 8 min at 4° C., washed three times with ice-cold 10% glycerol and cells were finally concentrated to 400-fold by resuspending with 100 μl of ice-cold 10% glycerol. Freshly prepared electrocompetent ML09-119 cells were immediately used for electroporation. Double stranded DNA (dsDNA) substrate used for deletion of ymcA gene from strain ML09-119 was generated by PCR using genomic DNA of A. hydrophila ML09-119vgr3A mutant (3) as a template using primers Lipo-FRT-F (5′-C*A*A*C*TGCTCGCCCTTTTTGATGAAAAAAGATCGGCTCTATGCAACTTTTGA GTGTAGGCTGGAGCTGCTTC-3′) (SEQ ID NO:107) and Lipo-FRT-R (5′-T*A*G*A*GATATCAATATTCGATTGCCAATCTCCTTGCTAATCGAGTACCAGA CATATGAATATCCTCCTTAGT3′) (SEQ ID NO:108) (3). For deletion of ymcA gene from A. hydrophila strain ML09-119 using recombineering, 1.0 μg of dsDNA substrate was mixed with 55 μl of electrocompetent ML09-119 cells in a pre-chilled electroporation cuvette (0.1-cm gap), and pulsed at 1.2 kV with 25 μF and 200Ω using Eppendorf Electroporator 2510 (Hamburg, Germany). Immediately after electroporation, 950 μl of SOC supplemented with 10 mM of L-arabinose was added and culture was incubated at 30° C. for overnight. Cells were then spread onto 2×YT agar plates supplemented with chloramphenicol (25.0 μg/ml) and incubated at 37° C. to obtain A. hydrophila ML09-119ΔymcA mutant and to cure the recombinogenic plasmid pMJH65 from the mutant. The correct deletion of ymcA gene from A. hydrophila strain ML09-119 was confirmed by PCR and sequencing using primers Liop_upF (5′-CCG AAT GGT AAT CCA CAG TT-3′) (SEQ ID NO:109) and Liop_dnR (5′-TAG AAC AGC TGG TCA CGA GA-3′) (SEQ ID NO:110). The removal of the recombinogenic plasmid pMJH65 from A. hydrophila ML09-119ΔymcA mutant was confirmed by the absence of its growth in TSB broth supplemented with tetracycline (10 μg/ml).
Flp-mediated excision of antibiotic resistance gene cassettes to generate unmarked mutants. A. hydrophila ML09-119ΔymcA mutant devoid of recombinogenic plasmid pMJH65 was mated with E. coli SM10λpir bearing FLP/FRT plasmid pMJH95 (3) according to the methods as described previously (2). The introduction of the plasmid pMJH95 into A. hydrophila ML09-119ΔymcA mutant was confirmed by its growth in the presence of tetracycline. Once the presence of plasmid pMJH95 was confirmed within A. hydrophila ML09-119ΔymcA mutant, culture was grown at 30° C. for overnight and induced for the removal of chloramphenicol resistance gene cassette by incubating at 37° C. for 6 hours. Broth culture was then streaked onto the TSA plates and incubated at 37° C. for overnight to obtain isolated colonies. Colonies grown on non-selective plates that subsequently failed to grow on antibiotic selective plates were tested by PCR and sequencing using primers Liop_upF (5′-CCG AAT GGT AAT CCA CAG TT3′) (SEQ ID NO:111) and Liop_dnR (5′-TAG AAC AGC TGG TCA CGA GA-3′) (SEQ ID NO:112) to confirm the Flp-mediated excision of antibiotic resistance gene cassettes introduced by recombineering. Sequence analysis demonstrated the precise deletion of ymcA gene and confirmed the generation of markerless ymcA mutant in A. hydrophila ML09-119.
REFERENCES FOR EXAMPLE 3


    1. Sambrook J, Fritsch E F, Maniatis T. 1998. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    2. Maurer K J, Lawrence M L, Fernandez D H, Thune R L. 2001. Evaluation and Optimization of a DNA Transfer System for Edwardsiella ictaluri. Journal of Aquatic Animal Health 13: 163-167.
    3. Hossain M J, Thurlow C M, Sun D, Nasrin S, Liles M R. 2015. Conjugal Transfer of a Recombineering System to Generate and Complement Markerless Mutants. Manuscript in Preparation.


It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein, without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

## Claims

1. An attenuated Aeromonas spp. bacterium that has been genetically modified by recombination to be deficient of one or more genes associated with O-antigen capsule synthesis or secretion, the gene being selected from the group consisting of:
(a) a gene encoding YmcA polypeptide (SEQ ID NO:11) or a gene encoding a polypeptide having at least 95% sequence identity to the YmcA polypeptide (SEQ ID NO:11); and
(b) a gene encoding YmcC polypeptide (SEQ ID NO:19) or a gene encoding a polypeptide having at least 95% sequence identity to the YmcC polypeptide (SEQ ID NO:19); and
(c) both (a) and (b).

2. The attenuated Aeromonas spp. of claim 1, wherein the bacterium is selected from the group consisting of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii.

3. The attenuated Aeromonas spp. bacterium of claim 1, wherein the bacterium has been genetically modified by a method that includes (a) deleting at least a portion of the gene ymcA by recombination and inserting a selectable marker in place of the deleted portion of the ymcA gene, and (b) subsequently deleting the selectable marker to create a markerless bacterium deficient of gene ymcA.

4. The attenuated Aeromonas spp. bacterium of claim 3, wherein the bacterium has been genetically modified by (a) transferring a recombineering system into the bacterium, wherein the recombineering system deletes at least a portion of the ymcA gene and replaces the portion with the selectable marker flanked by two recombinase recognition target sites; (b) selecting the bacterium for expression of the selectable marker; (c) curing the selected bacterium of the recombineering system; (d) transferring a vector that expresses a recombinase into the selected bacterium, wherein the recombinase recognizes the two recombinase recognition target sites; (e) selecting the bacterium for lack of expression of the selectable marker; and (f) curing the selected bacterium of the vector that expresses the recombinase.

5. The attenuated Aeromonas spp. bacterium of claim 4, wherein the recombineering system comprises: a mobilizable recombineering vector; and a linear DNA molecule comprising the following contiguous sequences in 5′ to 3′ order: (i) a first nucleotide sequence of at least 10 nucleotides having sequence identity with the gene ymcA (SEQ ID NO:12), (ii) a second nucleotide sequence comprising the first of the recombinase recognition target sites, (iii) a third nucleotide sequence that expresses a selectable marker, (iv) a fourth nucleotide sequence comprising the second of the recombinase recognition target sites, and (v), a fifth nucleotide sequence of at least 10 nucleotides having sequence identity with the gene ymcA (SEQ ID NO:12) that is different than the first nucleotide sequence of (i), wherein after the recombinase is expressed, the recombinase recombines the recombinases recognition target sites to remove the selectable marker and the portion of the ymcA gene that is deleted is replaced with one recombinases recognition target site.

6. The attenuated Aeromonas spp. bacterium of claim 1, wherein the bacterium further has been genetically modified by recombination to be deficient of a gene encoding YmcB polypeptide (SEQ ID NO:17) or to be deficient of a gene encoding a polypeptide having at least 95% sequence identity to the YmcB polypeptide (SEQ ID NO:17).

7. A vaccine composition comprising the attenuated Aeromonas spp. bacterium of claim 1 and a carrier.

8. The vaccine composition of claim 7, wherein the attenuated Aeromonas spp. bacterium has been inactivated by chemical treatment or physical treatment.

9. A method for vaccinating an aquatic animal against infection by an Aeromonas spp. bacterium, the method comprising administering the vaccine composition of claim 7 to the aquatic animal.

10. The method of claim 9, wherein the aquatic animal is a channel catfish.

11. The method of claim 9, wherein the aquatic animal is administered the vaccine composition by intraperitoneal injection.

12. The method of claim 9, wherein the aquatic animal is administered the vaccine composition at a dose that delivers 104-108 CFU of attenuated Aeromonas spp. bacteria per aquatic animal.

13. The method of claim 9, wherein the aquatic animal is administered the vaccine composition by immersing the aquatic animal in an aqueous medium comprising the vaccine composition.

14. The method of claim 13, wherein the aqueous medium has a concentration of 104-108 CFU/ml of attenuated Aeromonas spp. bacteria.

15. An attenuated Aeromonas spp. bacterium that has been genetically modified by recombination to be deficient of:
(a) a gene encoding YmcA polypeptide (SEQ ID NO:11) or a gene encoding a polypeptide having at least 95% sequence identity to the YmcA polypeptide (SEQ ID NO:11); and
(b) a gene encoding YmcC polypeptide (SEQ ID NO:19) or a gene encoding a polypeptide having at least 95% sequence identity to the YmcC polypeptide (SEQ ID NO:19).

16. The attenuated Aeromonas spp. of claim 15, wherein the bacterium is selected from the group consisting of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii.

17. The attenuated Aeromonas spp. bacterium of claim 15, wherein the bacterium has been genetically modified by a method that includes (a) deleting at least a portion of the gene ymcA by recombination and inserting a selectable marker in place of the deleted portion of the ymcA gene, and (b) subsequently deleting the selectable marker to create a markerless bacterium deficient of gene ymcA.

18. The attenuated Aeromonas spp. bacterium of claim 15, wherein the bacterium further has been genetically modified by recombination to be deficient of a gene encoding YmcB polypeptide (SEQ ID NO:17) or to be deficient of a gene encoding a polypeptide having at least 95% sequence identity to the YmcB polypeptide (SEQ ID NO:17).

19. A vaccine composition comprising the attenuated Aeromonas spp. bacterium of claim 15 and a carrier.

20. The vaccine composition of claim 19, wherein the attenuated Aeromonas spp. bacterium has been inactivated by chemical treatment or physical treatment.