bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of carbohydrate-reactive outer membrane proteins of Aeromonas hydrophila
Authors: Quinn D M , Wong C Y , Atkinson H M , Flower R L
Published: January 2020

Outer membrane proteins of Aeromonas hydrophila A6 were isolated by affinity chromatography on the basis of their reactivity with trisaccharide structures analogous to the terminal trisaccharide of the H antigen of the human ABO(H) blood group system and were characterized by using antisera raised against the isolate. The outer membrane extract for affinity chromatography was prepared from pressure-disrupted outer membranes by differential centrifugation, followed by solubilization of outer membrane components in a nondenaturing, nonionic detergent. Carbohydrate-reactive outer membrane proteins (CROMPs) were then purified by affinity chromatography on two different affinity matrices composed of trisaccharides resembling the terminal trisaccharide of the H antigen, attached to inert silica beads. The relative efficiencies of H type 1 and 2 terminal trisaccharides as affinity adsorbents were established. Reactive proteins were eluted under alkaline conditions (pH 11.0) and in the presence of soluble H substance prepared from group O secretor saliva, but not by 60 mM alpha-L-fucose or under acid conditions (pH 3.0). The eluate contained at least three components (M(r)s, 43,000, 40,000, and < 14,000), as detected by immunoblot analysis with a polyvalent, polyspecific rabbit antiserum to A. hydrophila A6 (serum 3/83). A specific antiserum (serum 3/91) prepared in a rabbit by repeated immunizations with nitrocellulose containing the 43,000-Da band reacted with three bands (M(r)s, 43,000, 40,000, and < 14,000) in immunoblot analysis of solubilized outer membranes of A. hydrophila A6, suggesting that the 40,000- and < 14,000-Da elements are immunologically related to components of the 43,000-Da protein. Furthermore, pretreatment of A. hydrophila A6 with serum 3/91 reduced the strength of bacterial hemagglutination. The purified CROMPs did not agglutinate human group O erythrocytes. The reactivity of isolated CROMPs with a second CROMP-specific antibody (lipopolysaccharide-absorbed serum 3/83) was investigated. CROMPs, proteinase K-treated CROMPs, and bovine serum albumin were bound to latex beads and reacted with lipopolysaccharide-absorbed serum 3/83. Antibodies eluted from CROMP-latex inhibited hemagglutination of human erythrocytes by A. hydrophila A6 to a titer of 4. Antibody eluted from proteinase K-treated CROMP-latex beads showed hemagglutination inhibition activity only when undiluted. There was no hemagglutination inhibition antibody activity detectable in the eluate from bovine serum albumin-latex beads. These results show that antibodies which react with the isolated CROMPs also react with an H-antigen-reactive hemagglutinin of A. hydrophila A6. The possibility that CROMPs act as an adhesin, or adhesins, and contribute to the virulence of this organism is discussed.
Group B streptococci invade endothelial cells: type III capsular polysaccharide attenuates invasion
Authors: Gibson R L , Lee M K , Soderland C , Chi E Y , Rubens C E
Published: January 2020

Group B streptococci (GBS) are the most common cause of neonatal sepsis and pneumonia. The pathogenesis of GBS disease is not completely defined. GBS-induced endothelial cell injury is suggested by histological findings at autopsy and in animal studies. We hypothesized that (i) type III GBS (COH-1) invade and injure human umbilical vein endothelial (HUVE) cells and (ii) isogenic mutations in GBS capsule synthesis would influence HUVE invasion. Confluent HUVE monolayers were infected for 0.5, 2, or 6 h. Media with penicillin plus gentamicin were added and incubated for 2 h to kill extracellular bacteria. Cells were washed and lysed, and the number of live intracellular bacteria was determined by plate counting. COH-1 invaded HUVE cells in a time-dependent manner at levels 1,000-fold higher than those of the noninvasive Escherichia coli strain but significantly lower than those of Staphylococcus aureus. There was no evidence for net intracellular replication of GBS within HUVE cells. COH-1 infection of HUVE cells caused the release of lactate dehydrogenase activity. GBS invasion was inhibited by cytochalasin D in a dose-dependent manner; GBS-induced lactate dehydrogenase release was attenuated by cytochalasin D. The isogenic strains COH 1-11, devoid of capsular sialic acid, and COH 1-13, devoid of all type III capsule, invaded HUVE cells three- to fivefold more than the parent COH-1 strain. The type III capsular polysaccharide and particularly the capsular sialic acid attenuate GBS invasion of HUVE cells. Electron micrographs of lung tissue from a GBS-infected newborn Macaca nemestrina also showed GBS within capillary endothelial cells. We conclude that endothelial cell invasion and injury are potential mechanisms in the pathogenesis of GBS disease.
Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1
Authors: Gerlach G F , Anderson C , Klashinsky S , Rossi-Campos A , Potter A A , Willson P J
Published: January 2020

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping. One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis. It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A. pleuropneumoniae. This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA. Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A. pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains. Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 1 isolate.
Production of mucoid exopolysaccharide during development of Pseudomonas aeruginosa biofilms
Authors: Hoyle B D , Williams L J , Costerton J W
Published: January 2020

Production of mucoid exopolysaccharide by planktonic, chemostat-derived, and adherent Pseudomonas aeruginosa 579 bacteria was separately monitored for 7 days by using a lacZ-algD promoter-reporter gene and assays of total carbohydrate and metabolic activity. Mucoid exopolysaccharide production was transiently elevated following adherence but declined to planktonic levels by day 7.
Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins
Authors: Jagusztyn-Krynicka E K , Clark-Curtiss J E , Curtiss R
Published: January 2020

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.
Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381
Authors: Washington O R , Deslauriers M , Stevens D P , Lyford L K , Haque S , Yan Y , Flood P M
Published: January 2020

Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381.
Characterization of a conserved helper-T-cell epitope from group A Streptococcal M proteins
Authors: Robinson J H , Case M C , Kehoe M A
Published: January 2020

We have previously defined major histocompatibility complex (MHC) class II-restricted T-cell epitopes from the carboxy-terminal region of group A streptococcal type 5 M protein. In this report, T-cell responses to one of these epitopes have been characterized in detail. T-cell clones from recombinant M5-immunized mice and popliteal lymph node cells from peptide-immunized mice were used to show that sM5[300-319] is recognized in the context of I-A alleles of four of nine independent MHC class II haplotypes: I-Ad, I-Af, I-Ak, and I-As. This epitope was also recognized by both helper (Th2) and inflammatory (Th1) subsets of murine T cells. The I-Ad-restricted epitope recognized by BALB/c mice was mapped to the 12-amino-acid peptide sM5[308-319] and was shown to provide helper function for an immunoglobulin G anti-peptide antibody response in BALB/c mice. Anti-peptide antibody was shown to be specific for M5[304-315] but failed to recognize intact rM5, suggesting that the conformation of the epitope differed between peptide and protein. However, the results demonstrate that overlapping epitopes can be the focus for both immunoglobulin G antibodies and the T cells which augment their production. Comparison of the available sequences for M proteins indicated that the T-cell epitope within M5[300-319] was highly conserved between M types and hence may elicit helper function for protective antibody responses to a wide range of M types. T-cell epitopes from conserved regions of M proteins which are recognized in the context of multiple MHC haplotypes have potential for the design of multivalent streptococcal vaccines.
Effect of modulation of polymorphonuclear leukocyte migration with anti-CD18 antibody on pathogenesis of experimental otitis media in guinea pigs
Authors: Patel J , Chonmaitree T , Schmalstieg F
Published: January 2020

Guinea pigs were treated with anti-CD18 antibody (M8), and subsequently middle ears (ME) were infected with nontypeable Haemophilus influenzae. Forty-eight hours after infection, the ME washes of these animals had significantly lower polymorphonuclear leukocyte numbers but higher bacterial counts compared with washes of animals treated with control antibody (M11) or saline. The edema and epithelial damage of ME tissues correlated directly with polymorphonuclear leukocyte numbers and not bacterial counts.
Pathogenicity of an Escherichia coli O115:K"V165" mutant negative for F165(1) fimbriae in septicemia of gnotobiotic pigs
Authors: Ngeleka M , Jacques M , Martineau-Doizé B , Daigle F , Harel J , Fairbrother J M
Published: January 2020

To evaluate the role of the F165(1) fimbrial system in the pathogenesis of septicemia, 2-day-old germfree pigs were inoculated intragastrically with Escherichia coli O115:K"V165":F165 wild-type strain 5131, its F165(1)-negative TnphoA mutant M48, or E. coli O115:K(-):F165(-) wild-type strain 862B. Pigs were sacrificed at different times (3, 6, 12, 24, 48, and 96 h) postinfection (p.i.). Pigs inoculated with strain 5131 developed clinical signs (anorexia, lameness, reluctance to move, or lack of motor coordination) and were moribund within 48 h p.i., and, at necropsy, infecting bacteria were isolated in various extraintestinal organs. Strain 5131 was isolated as early as 6 h p.i. from the blood of inoculated pigs. Pigs inoculated with mutant M48 developed only mild clinical signs at 96 h p.i. Mutant M48 colonized extraintestinal organs of pigs but to a lesser extent than the parent strain did. In contrast to the parent strain, this mutant was not isolated in the blood of inoculated pigs. Pigs inoculated with strain 862B remained normal during the experiment. All of the strains colonized the mucus layer of the intestine, but no histological changes of intestinal mucosa were observed by either light or electron microscopy. The parent strain, but not the mutant M48, expressed F165(1) in vivo. In a competitive study in which the parent strain and its afimbrial mutant were inoculated simultaneously, clinical signs of septicemia developed 24 h after inoculation, and only the parent strain 5131 was isolated from the blood of inoculated pigs. Our results suggest that the F165(1) fimbrial system of E. coli O115:K"V165" strains may play an important role in the ability of the bacteria to survive in the blood and spread systemically through the porcine host.
In vitro killing of Ehrlichia risticii by activated and immune mouse peritoneal macrophages
Authors: Williams N M , Timoney P J
Published: January 2020

Normal resident murine peritoneal macrophages inoculated in vitro with Ehrlichia risticii readily phagocytized the organism but were unable to suppress ehrlichial replication as determined by indirect fluorescent-antibody staining of the inoculated cells. In contrast, macrophages from Corynebacterium parvum-inoculated and E. risticii-recovered mice rapidly eliminated the ehrlichiae. Macrophages from E. risticii-recovered mice were as effective as the C. parvum-activated cells in phagocytizing and eliminating the organism. Opsonization of E. risticii with homologous antiserum prior to inoculation of macrophage cultures resulted in enhancement of phagocytosis and greater suppression of E. risticii replication in all macrophage groups. These findings indicate that the pathogenesis of E. risticii infection centers on the ability of the organism to enter and replicate within the macrophage with avoidance of macrophage antimicrobial effects. An immune response results in macrophage activation with enhancement of the macrophage's ability to eliminate E. risticii. Opsonization of E. risticii with anti-E. risticii serum renders E. risticii more susceptible to macrophage destruction.
Murine peritoneal macrophages activated by the mycobacterial 65-kilodalton heat shock protein express enhanced microbicidal activity in vitro
Authors: Peetermans W E , Langermans J A , van der Hulst M E , van Embden J D , van Furth R
Published: January 2020

After an intraperitoneal (i.p.) injection of purified protein derivative, peritoneal macrophages from mice infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) show an enhanced respiratory burst, inhibit the intracellular proliferation of Toxoplasma gondii, and kill Listeria monocytogenes more efficiently than peritoneal macrophages from normal mice. One of the immunodominant antigens of Mycobacterium spp. is the 65-kDa heat shock protein (Hsp 65), and in the present study, we determined whether injection of this protein into mice leads to activation of their peritoneal macrophages. After an i.p. injection of Hsp 65, peritoneal macrophages from BCG-infected CBA/J mice also released more H2O2, inhibited the proliferation of T. gondii, and killed L. monocytogenes faster than peritoneal macrophages from normal mice, although Hsp 65 was less effective than purified protein derivative. When normal mice were injected with Hsp 65 suspended in saline after a booster injection with Hsp 65, their macrophages did not display enhanced antimicrobial activity, indicating that an adjuvant was required for a cellular immune response against Hsp 65. In the present study, the adjuvant dimethyl dioctadecylammonium bromide (DDA) was preferred because it contains no endotoxin or mycobacterial antigens and because it has been reported that DDA does not induce the production of gamma interferon. Peritoneal macrophages from C57BL/6 and CBA/J mice that had received a subcutaneous injection of Hsp 65 suspended in DDA followed by an i.p. booster injection of Hsp 65 suspended in saline were activated, as indicated by the enhanced production of H2O2, inhibition of the intracellular proliferation of T. gondii, and increased rate of intracellular killing of L. monocytogenes in vitro relative to that by resident peritoneal macrophages and peritoneal macrophages obtained from mice that had received ovalbumin instead of Hsp 65. The rate of phagocytosis of L. monocytogenes was not affected by Hsp 65 treatment. Despite the in vitro expression of enhanced microbicidal activity of peritoneal macrophages, no difference in the growth of L. monocytogenes in the liver and spleen between Hsp 65-treated and control mice was found.
Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum
Authors: Markham P F , Glew M D , Whithear K G , Walker I D
Published: January 2020

A hemagglutinin with an M(r) of 67,000 (pMGA) from Mycoplasma gallisepticum S6 was purified by using monoclonal antibody affinity chromatography. Purified pMGA was treated with a number of enzymes, the resultant peptides were purified, and their amino acid sequence was determined by using an Applied Biosystems (model 471A) protein sequencer. The DNA sequence encoding two peptides was used to dictate the sequences of synthetic oligonucleotides which were used to screen a library of EcoRI-cut M. gallisepticum DNA in pUC18. A clone reactive to both probes was isolated and found to contain a recombinant insert of 10 kb. The clone was mapped by using restriction endonucleases and fragments subcloned into pUC18 for DNA sequencing. Analysis of part of the DNA sequence revealed an open reading frame containing 1,941 nucleotides which encoded 647 amino acids. The amino terminus was preceded by a putative leader sequence of 25 amino acids. A promoter region preceding the putative start codon GUG was also located. This gene would encode a mature protein of 67,660 Da. There were a number of differences between the predicted amino acid sequence and that determined by direct peptide sequencing. Also, two tryptic peptides of pMGA were not found in the DNA sequence. This suggested that the cloned gene did not encode pMGA but did encode a homolog (pMGA1.2). Furthermore, downstream of pMGA1.2 was a region of DNA encoding a leader sequence followed by an amino acid sequence with high homology to that encoded by the pMGA1.2 gene. The presence within M. gallisepticum of a family of pMGA genes is inferred from the DNA sequence and Southern transfer data. A possible role for this gene family in immune evasion is discussed.
Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes
Authors: Ohara N , Matsuo K , Yamaguchi R , Yamazaki A , Tasaka H , Yamada T
Published: January 2020

The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes.
Utilization of the mouse large intestine to select an Escherichia coli F-18 DNA sequence that enhances colonizing ability and stimulates synthesis of type 1 fimbriae
Authors: Burghoff R L , Pallesen L , Krogfelt K A , Newman J V , Richardson M , Bliss J L , Laux D C , Cohen P S
Published: January 2020

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18 Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Random sequences of E. coli F-18 DNA were cloned into pRLB2, a par-B-stabilized derivative of pHC79. The entire gene library was transformed into E. coli F-18 Col- and fed to streptomycin-treated mice. The mouse large intestine selected a predominant clone which contained a recombinant plasmid (pRLB7) that enhanced E. coli F-18 Col- colonizing ability 100-fold but did not stimulate colicin synthesis. Moreover, pRLB7 simultaneously improved the survival of E. coli F-18 Col- in stationary phase in vitro, utilizing nutrients derived from mouse cecal mucus, and stimulated synthesis of both type 1 fimbriae and three E. coli F-18 Col- outer membrane proteins (74, 71, and 69 kDa). The 6.5-kb E. coli F-18 DNA sequence in pRLB7 does not contain either the fim operon or pilG (hns), both known to be involved in type 1 fimbrial synthesis. The sequence encodes six proteins, all smaller than the three E. coli F-18 Col- outer membrane proteins whose synthesis it stimulates. Collectively, the results suggest that the cloned E. coli F-18 DNA sequence contains one or more regulators of E. coli F-18 Col- operons expressed in the mouse large intestine in vivo and in isolated mouse cecal mucus in vitro.
Antibodies to Haemophilus influenzae type b outer membrane proteins in children with epiglottitis or meningitis and in healthy controls
Authors: Johnson P D , MacInnes S J , Gilbert G L
Published: January 2020

The two most common manifestations of Haemophilus influenzae type b (Hib) infection in Western communities are meningitis and epiglottitis. The role of antibodies against outer membrane proteins (OMP) in the pathogenesis of these diseases was investigated by Western blotting (immunoblotting) with an OMP antigen prepared from a local Hib strain. Acute- and convalescent-phase serum samples from 25 children with epiglottitis and 20 with meningitis and single serum samples from 19 control children in the same age group were tested. Western blots were evaluated quantitatively by use of graphs generated from a densitometer. OMP antibody was detected in all sera from patients and controls. There was no significant difference between the mean antibody level in acute-phase sera from children with meningitis (336 +/- 143 arbitrary units) and those from children with epiglottitis (286 +/- 134 arbitrary units). However, the mean OMP antibody level in sera from healthy controls, with no known history of Hib disease, was significantly higher than that in sera from patients with Hib disease within 2 days of admission to the hospital (patients [n = 35], 282 +/- 144; controls [n = 19], 425 +/- 236; P = 0.007). The difference was due mainly to higher levels, in control sera, of antibody against four proteins, one of which is either P1 or a comigrating protein of 49 kDa. The finding of higher levels of OMP antibody in healthy controls suggests a protective role for antibodies directed against one or more OMP. This information could be exploited in future vaccine development.
Cloning and sequence analysis of the structural pilin gene of Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius
Authors: Whitney A M , Farley M M
Published: January 2020

We have cloned and sequenced the Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (Hae) pilin gene. The sequence contained a 648-bp open reading frame encoding a mature pilin protein of 191 amino acids with a calculated mass of 20.5 kDa. There was 82% homology between the open reading frames of the BPF strain F3031 and H. influenzae type b (Hib) (strain M43) pilin genes and 71% homology at the amino acid level between the mature pilin proteins. However, areas of diversity were noted throughout the gene. A 17-bp probe corresponding to an area of diversity in the N-terminal region of the BPF-associated gene hybridized with other BPF strains but not with non-BPF Hae or Hib. In summary, the pilin protein of BPF-associated Hae is highly homologous to Hib pilin yet remains structurally distinct.
Presence of a capsule in Vibrio vulnificus biotype 2 and its relationship to virulence for eels
Authors: Biosca E G , Llorens H , Garay E , Amaro C
Published: January 2020

Strains of Vibrio vulnificus biotype 2, isolated from internal organs of diseased European eels as pure cultures of opaque cells, together with some reference strains from Japanese eels, were used in this study. Spontaneous translucent-phase variants were obtained from the corresponding parent strains and compared for a variety of phenotypic traits related to virulence for eels. The rate of colony dissociation from opaque to translucent cells was higher (around 10(-2)) than that observed for translucent to opaque cells (10(-3) to 10(-4)). Electron microscopy with ruthenium red revealed the presence of a capsule of variable thickness on opaque cells, whereas translucent-type colonies had no observable capsular materials. No differences in plasmid profiles were detected between the two cell types so that plasmids do not seem to be implicated in the mechanism of phase shift of biotype 2 strains. No apparent difference in outer membrane protein and lipopolysaccharide patterns could be observed between the cell types. Both isogenic morphotypes were able to grow in eel serum and minimal medium supplemented with ethylenediamine di(O-hydroxyphenyl-acetic acid) or transferrin. Therefore, the presence of capsule was not required for the acquisition of iron from iron chelators or for resistance to serum bactericidal action. Both morphotypes were highly virulent for elvers, although the 50% lethal dose for translucent cells was higher than that for the corresponding opaque cells. The latter observation, together with the overall data, suggests that the production of capsular materials by biotype 2 of V. vulnificus is not essential for the development of vibriosis in eels, at least when cells are injected intraperitoneally.
Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea
Authors: Whittam T S , Wolfe M L , Wachsmuth I K , Orskov F , Orskov I , Wilson R A
Published: January 2020

The genetic relationships among 1,300 isolates of Escherichia coli representing 16 serotypes associated with enteric disease, including O157:H7 strains recovered from patients with hemorrhagic colitis and hemolytic uremic syndrome and O26:H11, O55:H6, O55:H7, O111:H2, and O128:H2 strains, many of which were isolated originally from infants with diarrhea, were estimated from allelic variation among 20 enzyme-encoding genes detected by multilocus enzyme electrophoresis. Multiple electrophoretic types were observed among isolates of each serotype, with isolates of the same O serogroup differing on average at 28% of the enzyme loci. Comparisons of the multilocus enzyme profiles revealed that 72% of the isolates belong to 15 major electrophoretic types, each of which corresponds to a bacterial clone with a wide geographic distribution. Genetically, the O157:H7 clone is most closely related to a clone of O55:H7 strains that has long been associated with worldwide outbreaks of infantile diarrhea. We propose that the new pathogen emerged when an O55:H7-like progenitor, already possessing a mechanism for adherence to intestinal cells, acquired secondary virulence factors (Shiga-like cytotoxins and plasmid-encoded adhesins) via horizontal transfer and recombination.
Lipopolysaccharide from Brucella abortus behaves as a T-cell-independent type 1 carrier in murine antigen-specific antibody responses
Authors: Betts M , Beining P , Brunswick M , Inman J , Angus R D , Hoffman T , Golding B
Published: January 2020

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice. In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-1 carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development.
The mycobacterial secreted antigen 85 complex possesses epitopes that are differentially expressed in human leprosy lesions and Mycobacterium leprae-infected armadillo tissues
Authors: Rambukkana A , Burggraaf J D , Faber W R , Harboe M , Teeling P , Krieg S , Das P K
Published: January 2020

The granulomatous skin lesions in leprosy are thought to be initiated by the immune response to certain antigens of the causative agent, Mycobacterium leprae. The antigen 85 complex is one of the major targets in the immune response to M. leprae infection. In the present study, a panel of previously characterized monoclonal antibodies (MAbs) (3A8, Rb2, A4g4, A2h11, Pe12, and A3c12) reacting with different epitopes of the 85 complex proteins of Mycobacterium tuberculosis and M. leprae was employed in a comparative immunohistological analysis to demonstrate the in situ expression of 85 complex antigenic epitopes in leprosy lesions across the clinical spectrum and in M. leprae-infected armadillo liver tissues. These MAbs showed a heterogeneous staining pattern in a given leprosy lesion. In highly bacilliferous borderline and lepromatous leprosy lesions, MAbs Rb2, A4g4, A2h11, and Pe12 stained clear rod-shaped M. leprae bacilli within macrophages, and the degree of staining correlated with the bacillary index of the lesion. On the other hand, MAbs 3A8 and A3c12 staining was mostly seen as a diffuse staining pattern within interstitial spaces and on the membranes of the infiltrated cells but not the bacilli. In paucibacillary borderline and tuberculoid leprosy lesions, only 3A8, Rb2, and A3c12 showed distinct staining in association with infiltrates in the granuloma. None of these MAbs showed any detectable reaction with control nonleprosy skin lesions, while MAb A3c12 positively stained the granulomas of both leprosy and control specimens. In situ reactivity of these MAbs with M. leprae-infected armadillo liver tissues also showed a heterogeneous staining pattern. Interestingly, a clear difference in expression of these epitopes was observed between armadillo tissues and human leprosy lesions. By immunogold ultracytochemistry, we further showed the differential localization of these MAb-reactive epitopes on the cell surface, in the cytosol, and at the vicinity of M. leprae within Kupffer cells of armadillo liver tissues. Our results indicate that these antigenic epitopes of the antigen 85 complex are differentially expressed in leprosy lesions and infected armadillo tissues and that they could be target determinants in the immunopathological responses during M. leprae infection.
Protective salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal immunization with S. mutans antigen I/II coupled to the B subunit of cholera toxin
Authors: Katz J , Harmon C C , Buckner G P , Richardson G J , Russell M W , Michalek S M
Published: January 2020

The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/II), was chemically coupled to CTB (Ag I/II-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 micrograms) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag I/II and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/II response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag I/II and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag I/II responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/II-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.
Induction of early gene expression in murine macrophages by synthetic lipid A analogs with differing endotoxic potentials
Authors: Perera P Y , Manthey C L , Stütz P L , Hildebrandt J , Vogel S N
Published: January 2020

Numerous lipid A analogs have been synthesized in an attempt to dissociate endotoxic activities from beneficial immunomodulatory activities. In the present study, we have evaluated select lipid A analogs in macrophages for their ability to induce a panel of lipopolysaccharide (LPS)-inducible genes to gain insights into the molecular mechanisms which underlie endotoxicity. We evaluated three monosaccharide lipid A analogs: SDZ MRL 953, an agonist with an improved therapeutic margin over endotoxin; SDZ 281.288, a more toxic analog; and SDZ 880.431, an analog with proven LPS-inhibitory activity. In addition, three disaccharide lipid A analogs (i.e., lipid IVA, SDZ 880.611, and SDZ 880.924) that differ in acylation and phosphorylation patterns were also examined and compared with synthetic lipid A. With the exception of SDZ 880.431, each of these structurally diverse analogs was able to induce the complete panel of LPS-inducible genes, specifically genes which encode tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, 75-kDa type 2 TNF receptor (D7), IP-10, D3, and D8. These results underscore that macrophage stimulation by lipid A analogs is permissive to considerable structural diversity. Structures with favorable therapeutic indices (SDZ MRL 953, SDZ 880.611, and SDZ 880.924) were not different from structures with poor therapeutic indices (lipid A, lipid IVA, and SDZ 281.288) with regard to gene induction. Nonetheless, the nontoxic SDZ MRL 953 was approximately 1,000-fold less potent than synthetic lipid A at inducing TNF-alpha secretion, and perhaps this contributes to the lack of toxicity exhibited by this compound. The ability of compound SDZ 880.431 to inhibit TNF-alpha secretion induced by both SDZ MRL 953 and smooth LPS suggests that the monosaccharide and smooth LPS share a receptor or a portion thereof. A pattern of protein tyrosine phosphorylation similar to that induced by LPS was stimulated by the monosaccharide SDZ MRL 953 and SDZ 281.288 and disaccharides lipid IVA, SDZ 880.924, and SDZ 880.611, providing evidence for a common signalling pathway.
Biochemical and immunological properties of two forms of pertactin, the 69,000-molecular-weight outer membrane protein of Bordetella pertussis
Authors: Gotto J W , Eckhardt T , Reilly P A , Scott J V , Cowell J L , Metcalf T N , Mountzouros K , Gibbons J J , Siegel M
Published: January 2020

Two apparent isoforms of the virulence-associated 69,000-molecular-weight protein pertactin were purified from Bordetella pertussis. Mass spectrometry showed a difference of 2,060 Da, which may result from differential C-terminal cleavage of a larger precursor. Both forms were protective in a mouse model, eliciting bactericidal antibodies and reducing respiratory tract colonization.
Characterization of an aromatic amino acid-dependent Listeria monocytogenes mutant: attenuation, persistence, and ability to induce protective immunity in mice
Authors: Alexander J E , Andrew P W , Jones D , Roberts I S
Published: January 2020

A transposon insertion mutant of Listeria monocytogenes was shown to be deficient in prephenate dehydratase, an enzyme acting late in the pathway for biosynthesis of phenylalanine. This mutant had reduced virulence in mice. The mutant and parent strains persisted to the same extent in the tissues of infected mice and elicited similar degrees of splenomegaly. Mice vaccinated with the mutant were protected significantly from subsequent challenge with virulent L. monocytogenes.
Chemotaxis of leptospires to hemoglobin in relation to virulence
Authors: Yuri K , Takamoto Y , Okada M , Hiramune T , Kikuchi N , Yanagawa R
Published: January 2020

A guinea pig-lethal line of Leptospira interrogans serovar copenhageni strain Shibaura, but not an avirulent line of the same strain, moved in larger numbers toward hemoglobin than toward distilled water (control) in a U-shaped polypropylene tube. L. interrogans serovar lai strains 017 and KH-1, which were also guinea pig lethal, showed a similar move to hemoglobin. No such move toward hemoglobin was shown by 14 avirulent strains of L. interrogans (with one exception) or any of the 8 strains of L. biflexa tested.
Characterization and immunolocalization of a Cryptosporidium protein containing repeated amino acid motifs
Authors: Ranucci L , Müller H M , La Rosa G , Reckmann I , Morales M A , Spano F , Pozio E , Crisanti A
Published: January 2020

The oocyst wall is one of the components that permits cryptosporidia both to survive in the environment and to retain infectivity. With the aim of identifying Cryptosporidium proteins specifically expressed at the oocyst stage, we screened lambda gt11 genomic libraries of Cryptosporidium parvum with both an oocyst antiserum and a specific genetic probe. We isolated, from distinct libraries, two overlapping clones containing an open reading frame encoding a 1,252-amino-acid polypeptide. The analysis of the deduced amino acid sequence revealed unusually high contents of cysteine, proline, and histidine. The sequence was also characterized by two distinct amino acid motifs, each repeated several times. The DNA sequences coding for the amino acid repeats showed a high frequency of synonymous mutations, a result suggesting that the repeated motifs may be functionally and/or structurally important to the parasite. Antisera and monoclonal antibodies developed against a recombinant polypeptide encompassing the first 786 amino acids revealed that the corresponding protein in C. parvum had an apparent molecular weight of 190,000. Moreover, confocal microscopy analysis with immunofluorescence indicated that the protein was localized on the oocyst wall as a uniform stain and within the oocyst itself as bright granules in close association with the residual body.
Structure and copy number analyses of pap-, sfa-, and afa-related gene clusters in F165-positive bovine and porcine Escherichia coli isolates
Authors: Maiti S N , Harel J , Fairbrother J M
Published: January 2020

Pathogenic F165-positive Escherichia coli isolates of porcine and bovine origin possess gene clusters related to extraintestinal E. coli fimbrial operons pap, sfa, and afaI. Probes from different segments of the pap, sfa, and afaI operons were used in Southern hybridization to analyze 18 F165-positive, mannose-resistant hemagglutinating E. coli isolates possessing pap- and sfa-, pap- and afa-, or pap-related sequences. Only single copies of the pap-, sfa-, or afa-related sequences were found among the isolates, and the position of each sequence with respect to those of adjacent sequences was variable. Expression of the P and F adhesins individually or concurrently was associated with the presence of a single copy of pap-related sequences. Gene clusters related to pap were structurally similar to the pap operon in certain isolates or the prs operon in others. sfa-related sequences showed few internal structural polymorphisms and were structurally similar to the foc rather than the sfa operon. afa-related sequences showed many internal structural polymorphisms compared with the afa-related sequences from prototype strain KS52. These results demonstrate that the pap- and sfa-related sequences in F165-positive isolates are closely related to the prototype pap operon and foc operon of the P family and Sfa family, respectively. afa-related sequences, on the other hand, display heterogeneity and differ from the prototype afaI operon.
Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme
Authors: Sriwanthana B , Mobley H L
Published: January 2020

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.
Release of stress proteins from Mesocestoides corti is a brefeldin A-inhibitable process: evidence for active export of stress proteins
Authors: Ernani F P , Teale J M
Published: January 2020

Substantial evidence indicates that molecules released by infectious organisms affect virulence and influence immunity to infection. The characterization of extracellular molecules and their mechanism of release is therefore critical to understanding host-parasite relationships. The cestode parasite Mesocestoides corti is known to release at the larval stage several molecules including the heat shock proteins hsp70 and hsp60. In this report, it is shown that several molecules released by M. corti, including 70- and 60-kDa proteins, are induced by a temperature shift from room temperature to 37 degrees C. Such a shift is comparable to the thermal stress of parasites transmitted from insect vector to vertebrate host. By drug inhibition studies and Western blot (immunoblot) analyses, it is shown that M. corti hsp70 and hsp60 as well as other released molecules are actively exported. The active release of stress proteins by parasites has not been described and may play a critical role in the disease process.