# Patent

## Title
Virulence of Streptococci

## Abstract

The invention relates to the field of diagnosis of and vaccination against Streptococcal infections and to the detection of virulence markers of Streptococci. The invention discloses a method for modulating virulence of a Streptococcus comprising modifying a genomic fragment of Streptococcus wherein the genomic fragment comprises at least a functional part of a fragment identifiable by hybridization in Streptococcus suis to a nucleic acid or fragment thereof as shown in FIG. 5.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/NL01/00805, filed Nov. 6, 2001, designating the United States of America, corresponding to PCT International Publication WO 02/38597 (published in English on May 16, 2002), the contents of which are incorporated herein in its entirety.


TECHNICAL FIELD
The invention relates to the field of diagnosis of and vaccination against Streptococcal infections and to the detection of virulence markers of Streptococci.
BACKGROUND
Streptococcus species, of which there are a large variety of that cause infections in domestic animals and man, are often grouped according to Lancefield's groups. Typing according to Lancefield occurs on the basis of serological determinants or antigens that are, among others, present in the capsule of the bacterium and, thus, allows for an approximate determination. Often bacteria from a different group show cross-reactivity with each other, while other Streptococci cannot be assigned a specific group-determinant at all. Within groups, further differentiation is often possible on the basis of serotyping. These serotypes further contribute to the large antigenic variability of Streptococci, a fact that creates an array of difficulties within diagnosis of and vaccination against Streptococcal infections.
Lancefield group A Streptococcus (GAS, Streptococcus pyogenes), are common with children and cause nasopharyngeal infections and complications thereof. Among animals, cattle are susceptible to GAS, and mastitis is often found.
Lancefield group B Streptococcus (GBS) are most often seen with cattle and cause mastitis. However, human infants are susceptible as well, often with fatal consequences. Group B streptococci (GBS) constitute a major cause of bacterial sepsis and meningitis among human neonates born in the United States and Western Europe and are emerging as significant neonatal pathogens in developing countries.
Lancefield group C infections, such as those with S. equi, S. zooepidemicus, S. dysgalactiae, and others are mainly seen with horse, cattle and pigs, but can also cross the species barrier to humans.
Lancefield group D (S. bovis) infections are found with all mammals and some birds, sometimes resulting in endocarditis or septicemia.
Lancefield groups E, G, L, P, U and V (S. porcinus, S. canis, S. dysgalactiae) are found with various hosts, cause neonatal infections, nasopharyngeal infections or mastitis.
Within Lancefield groups R, S, and T (and with ungrouped types), S. suis is found and is an important cause of meningitis, septicemia, arthritis and sudden death in young pigs. Incidentally, it can also cause meningitis in man.
Ungrouped Streptococcus species, such as S. mutans, causes caries with humans, S. uberis, causes mastitis with cattle, and S. pneumonia, causes major infections in humans, and Enterococcus faecalis and E. faecium, further contribute to the large group of Streptococci. Streptococcus pneumoniae (the pneumococcus) is a human pathogen that causes invasive diseases, such as pneumonia, bacteremia, and meningitis.
Little is known about the pathogenesis of the disease caused by Streptococci. Various cellular components, such as muramidase-released protein (MRP), extracellular factor (EF) and cell membrane associated proteins, fimbriae, hemagglutinins, and hemolysin have been suggested as virulence factors. However, the precise role of these protein components in the pathogenesis of the disease remains unclear. It is, however, known and generally accepted that the polysaccharidic capsule of various Streptococci and other gram-positive bacteria plays an important role in pathogenesis. The capsule enables these microorganisms to resist phagocytosis and is, therefore, regarded as an important virulence factor or marker.
In particular, Streptococcus suis is an important cause of meningitis, septicemia, arthritis and sudden death in young pigs. It can also cause meningitis in man. Attempts to control the disease are hampered by the lack of sufficient knowledge about the pathogenesis of the disease and the lack of effective vaccines and sensitive diagnostic methods.
So far, 35 serotypes of S. suis have been described. Virulence of S. suis can differ within and among serotypes. Worldwide, S. suis serotype 2 is the most frequently isolated serotype. Within S. suis serotype 2, pathogenic, weak-pathogenic and non-pathogenic strains can be found. The pathogenic strains cause severe clinical signs of disease in pigs and large numbers of bacteria can be re-isolated from the central nervous system (CNS) and the joints after experimental infection. The weak-pathogenic strains cause only mild clinical signs of disease and infrequently bacteria can be re-isolated from the CNS and the joints after experimental infection. The non-pathogenic strains are completely avirulent in young pigs after experimental infection.
The 136-kDa muramidase-related protein (MRP) and the 110-kDa extracellular factor (EF) are generally considered as important virulence markers for S. suis serotype 2 strains isolated in Europe and the United States. However, differences in virulence between pathogenic, weak-pathogenic and non-pathogenic strains cannot exclusively be explained by differences in their MRP and EF expression patterns. In addition, it is known that the capsule of Streptococcus suis serotype 2 is an important virulence factor. However, since pathogenic, weak-pathogenic and non-pathogenic strains seem to be fully encapsulated after growth in vitro and in vivo, it is not likely that the level of encapsulation of these fully encapsulated strains is associated with their difference in virulence.
SUMMARY OF THE INVENTION
The invention discloses a method for modulating virulence of a Streptococcus comprising modifying a genomic fragment of the Streptococcus, wherein the genomic fragment comprises at least a functional part of a fragment identifiable by hybridization in Streptococcus suis to a nucleic acid or fragment thereof as shown in FIG. 5. To gather an insight in the differences between pathogenic, weak-pathogenic and non-pathogenic strains that determine their difference in virulence, the invention discloses an in vivo complementation system wherein virulence can be modified by modifying the fragment.
For example, within S. suis serotype 2, pathogenic, weak-pathogenic and non-pathogenic strains are found. A genomic library of a pathogenic strain was introduced into a weak-pathogenic strain. After infection of the library into young piglets, pathogenic transformants were selected. One specific transformant that contained a 3 kb fragment of the pathogenic strain, V10, appeared to be dominantly enriched in diseased pigs. The observed enrichment was not tissue specific. The selected fragment, when introduced into two different weak-pathogenic strains, considerably increased the virulence of these strains. In particular, the fragment described and identified as ORF2, or functional fragments thereof, was shown to be an important virulence factor. In contrast, introduction of the corresponding fragment of a weak-pathogenic strain had only minor effects on virulence.
Accordingly, the invention also described a method for assaying virulence of a Streptococcus comprising assaying a genomic fragment of the Streptococcus, wherein the genomic fragment comprises at least a functional part of a fragment identifiable by hybridization in Streptococcus suis to a nucleic acid or fragment thereof as shown in FIG. 5, in particular the ORF2 fragment.
Nucleotide sequence analysis of the selected fragment of the pathogenic. strain revealed the presence of two potential open reading frames, both of which were found to be mutated in the corresponding fragment of the weak-pathogenic strain. It was previously shown by ribotyping and random amplified polymorphic DNA analysis (RAPD) assays that pathogenic and weak-pathogenic strains of S. suis serotype 2 are genetically closely related, whereas non-pathogenic strains showed a high degree of genetic heterogeneity. A genomic library of the pathogenic S. suis strain 10 in plasmids was constructed and the plasmid library was introduced into the weak-pathogenic reference strain of S. suis serotype 2, strain S735. Pigs were inoculated intravenously with the recombinants and bacteria were recovered from the CNS and the joints of diseased pigs.
The re-isolated bacteria were subsequently analyzed for plasmid content and virulence. With this approach, a DNA fragment of a pathogenic serotype 2 strain that transformed weak-pathogenic strains into highly pathogenic strains was identified. This fragment, as described herein, comprises a genetic determinant important for virulence. The fragment is in other Streptococci identifiable by, for example, hybridization experiments such as Northern or Southern blotting, or by amplification experiments (such as PCR) using primers and/or probes derived from a nucleic acid as described herein.
With the fragment and parts thereof, such as the open reading frames identified in FIG. 3, a virulence marker is described herein. The marker is associated with an isolated and/or recombinant nucleic acid as described herein and derivable from Streptococcus and identifiable by hybridization in Streptococcus (preferably S. suis) to a nucleic acid or fragment thereof as shown in FIG. 5.
The invention also discloses a vector comprising a nucleic acid according to the invention and a host cell comprising a nucleic acid or a vector according to the invention. Such a host cell comprises an easily modifiable organism such as E. coli. However, other host cells, such as recombinant Streptococcus (such as those derived from one of the grouped or ungrouped Streptococci as identified hereinabove) comprising a vector or nucleic acid according to the invention are also herein disclosed. In particular, recombinant Streptococcus as described herein is useful for inclusion in a vaccine.
Furthermore, the invention discloses a vaccine comprising a nucleic acid, a vector or a host cell according to the invention, and the use of such a vaccine in the prevention and/or treatment of Streptococcal infections.
Also described is a protein or fragment thereof encoded by a nucleic acid according to the invention, such as a protein encoded by ORF2 or ORF3 as disclosed herein, or a functional, i.e., antigenic fragment thereof. The invention also discloses an antibody directed against a protein or fragment thereof according to the invention and an antigen reactive with such an antibody, for example, comprising a protein or fragment. Such a protein or fragment thereof need not be obtained by recombinant means since synthesis of the peptides according to the amino acid sequence is also possible. Such antigens and antibodies as described herein can be used in a diagnostic test comprising an antibody of the invention, or within a vaccine or diagnostic test comprising the antigen of the invention. Such vaccines and diagnostic tests can be used in the field of diagnosis of and vaccination against Streptococcal infections and for the detection of virulence markers of Streptococci.
The phrase “means that imparts virulence” will be used to refer to a nucleic acid that encodes a peptide corresponding to a virulence factor or to a peptide encoded by the nucleic acid that possesses a characteristic associated with the virulence factor. The phrase “means that imparts virulence” also includes, without limitation, the nucleic acids having sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, and the peptides having sequences corresponding to SEQ ID NOS: 10–13, and any functional sequences originating therefrom. For instance, the nucleic acid sequence and the amino acid sequence may have conservative changes, such as additions, deletions or substitutions that do not affect a function associated with the virulence factor. For example, since the genetic code is degenerate, i.e., an amino acid may be encoded for by more than one codon, a conservative change in an original nucleic acid may result in an altered nucleic acid that encodes the same or homologous peptide as the original nucleic acid, wherein the peptide encoded by the altered nucleic acid retains the same function as the original nucleic acid. Further, since some amino acids are similar in charge, a conservative change, such as an addition, deletion or substitution, in the original amino acid sequence may result in an altered amino acid sequence, wherein the altered amino acid sequence retains the same function as the original amino acid sequence.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the pCOM1 vector. pCOM1 contains the replication functions of pWVO1, the eythromycin-resistance gene of pE194 preceded by the promoter region of the mrp gene, as well as the SacI-PstI part of the multiple cloning site of pKUN19.
FIG. 2 shows plasmids digested with SmaI and XbaI on an 0.8% agarose gel. Library: plasmids isolated from ten randomly selected clones of the original library; clones enriched in pigs: plasmids isolated from 19 independently selected clones enriched in pigs; c: pCOM1; m: molecular size marker.
FIG. 3 is a schematic representation of the fragments V10 and V735. The arrows indicate the potential ORFs. æ P, indicates the position of the potential promoter sequence; °, indicates the positions of the potential transcription regulator sequences. Homologies (% identities) between the potential proteins encoded by the ORFs and proteins present in the data libraries are indicated.
FIGS. 4A and 4B illustrate the homology between the ORFs 2 (A) and 3 (B) enoding proteins of fragments V10 and V735. The asterisks indicate the non-identical amino acids.
FIGS. 5A–5F are nucleotide and amino acid sequences of fragments V10 and V735.



DETAILED DESCRIPTION
Bacterial strains and growth conditions. The bacterial strains and plasmids used herein are listed in Table 1. S suis strains were grown in Todd-Hewitt broth (code CM189, Oxoid), and plated on Columbia agar blood base (code CM331, Oxoid) containing 6% (v/v) horse blood. If required, antibiotics were added at the following concentrations: erythromycin, 1 μg/ml. E coli strains were grown in Luria broth and plated on Luria broth containing 1.5% (w/v) agar. If required, 200 μg/ml of erythromycin was added.
pCOM1. pCOM1 (FIG. 1) is based on the replication functions of pWVO1. Further, the vector contained the erythromycin-resistance gene of pE194 preceded by the promoter region of the mrp gene, as well as the SacI-PstI part of the multiple cloning site of pKUN19. As the result, pCOM1 contained a unique BamHI site (FIG. 1).
Construction of genomic S. suis library in pCOM1. Sau3AI partial digests of the DNA of the pathogenic S. suis serotype 2, strain 10 were size fractionated (>3 kb) by precipitation with 4.6% of PEG 6000 (BDH Chemicals, 19). The fragments were ligated to BamHI digested pCOM1 and the ligation mixtures were transformed to E. coli XL2-blue cells. Erythromycin-resistant colonies were selected. About 17,000 independent E. coli clones were obtained. Analysis of 55 of the transformants showed that 64% contained an insert of greater than 3 kb. From the pool of E. coli transformants, plasmid DNA was isolated and subsequently used for the electrotransformation of the weak-pathogenic S. suis strain S735. This resulted in approximately 30,000 independent S. suis transformants. The S. suis library was designated S735(pCOM-L). The transformants were pooled and stored at −80° C.
DNA techniques. Routine DNA manipulations were performed as described by Sambrook et al. DNA sequences were determined on a 373A DNA Sequencing System (Applied Biosystems, Warrington, GB). Samples were prepared by use of an ABI/PRISM dye terminator cycle sequencing-ready reaction kit (Applied Biosystems). Custom-made sequencing primers were purchased from Life Technologies. Sequencing data was assembled and analyzed using the McMollyTetra software package. The BLAST program was used to search for protein sequences homologous to the deduced amino acid sequences.
For PCR reaction mixtures (50 μl), the PCR Expand High Fidelity system (Boehringer, Mannheim, Germany) was used as described by the supplier. DNA amplification was carried out in a Perkin Elmer 9600 thermal cycler and the program included an incubation for two minutes at 95° C., ten cycles of 20 seconds at 95° C., one minute at 60° C. and four minutes at 68° C., 30 cycles of 20 seconds at 95° C., one minute at 60° C. and four minutes, extended with 20 seconds for each cycle, at 68° C. and ten minutes at 72° C.
Southern blotting and hybridization. Chromosomal DNA was isolated as described by Sambrook et al. DNA fragments were separated on 0.8% agarose gels and transferred to Gene-Screen Plus membranes (NEN) as described by Sambrook et al. DNA probes were labeled with [(α-32P]dCTP (3000 Ci mmol−1; Amersham) by use of a random primed labeling kit (Boehringer). The DNA on the blots was hybridized at 65° C. with the appropriate DNA probes as recommended by the supplier of the Gene-Screen Plus membranes. After hybridization, the membranes were washed twice with a solution of 40 mM sodium phosphate, pH 7.2, 1 mM EDTA, 5% SDS for 30 minutes at 65° C., and twice with a solution of 40 mM sodium phosphate, pH 7.2, 1 mM EDTA, 1% SDS for 30 minutes at 65° C.
Construction of pCOM-V10-ORF2 and pCOM-V10-ORF3. To construct pCOM-V10-ORF2, the primers 5′-CGAGCTCGGAAGAATTGGTTATTGCGCGTG-3′ (SEQ ID NO: 1) and 5′-CGGGATCCCGGGGGATGACCTGTTGCTTG-3′ (SEQ ID NO: 2) were used in a PCR reaction on chromosomal DNA of S. suis strain 10 to amplify the ORF2 encoding region. The resulting fragment was purified, digested with SacI and BamHI and cloned into SacI and BamHI-digested pCOM1.
To construct pCOM-V10-ORF3, the primers 5′-TCCCCCGGGGGACAAGCAACGGGTCATCCCC-3′ (SEQ ID NO: 3) and 5′-CGGGATCCCGGTTGAATGCCCGGCAAAGCG-3′ (SEQ ID NO: 4) were used to amplify the ORF3 encoding region. The resulting fragment was digested with SmaI and BamHI and cloned into pKUN19. The resulting plasmid was designated pKUN-ORF3. Because the ORF2 and ORF3 encoding regions are most probably co-transcribed, the promoter region of ORF2 was subsequently amplified with primers 5′-CGAGCTCGGAAGAATTGGTTATTGCGCGTG-3′ (SEQ ID NO: 1) and 5′-TCCCCCGGGGGAGTCGTGTGTATTCGACAGCGG-3′ (SEQ ID NO: 5). The fragments were digested with SacI and SmaI and cloned into SacI and SmaI digested pKUN-ORF3. The resulting plasmid was digested with SacI and BamHI, the insert fragment was purified and cloned into SacI and BamHI digested pCOM1. This resulted in pCOM-V10-ORF3.
Experimental infections. Germfree pigs, crossbreeds of Great Yorkshire and Dutch landrace, were obtained from sows by caesarian sections. The surgery was performed in sterile flexible film isolators. Pigs were allotted to groups, each including 4 or 5 pigs, and were housed in sterile stainless steel incubators. Housing conditions and feeding regimes were performed as described by Vecht et al. One week old pigs were intravenously inoculated with S. suis strains as described by Vecht et al. Pigs received erythromycin orally twice a day (Erythromycin stearate, Abbott B. V., Amstelveen, The Netherlands, 40 mg/kg body weight). Two hours after the infection, the pigs were treated with erythromycin for the first time. Pigs were monitored twice a day for clinical signs of disease, such as fever, nervous signs and lameness. Blood samples were collected three times a week from each pig. White blood cells were counted with a cell counter.
To monitor infection with S. suis, swabs of nasopharynx and feces were collected daily. The swabs were directly plated onto Columbia agar containing 6% horse blood. After the pigs were sacrificed, they were examined for pathological changes. Further, tissue specimens were collected from the central nervous system, serosa, joints, lungs, liver, kidney, spleen, heart and tonsils. The tissues were homogenized in the presence of Todd-Hewitt medium by using an Ultra-Turrax tissuemizer (Omni International, Waterbury, USA), centrifuged for five minutes at 3,000 rpm and the supernatants were frozen at −80° C. in the presence of 15% glycerol.
Results.
Complementation system. A genomic library of the pathogenic S. suis strain 10 was constructed into the weak-pathogenic strain S735 as described in Materials and Methods. The plasmid pCOM1 allowed the insertion of large DNA fragments into the unique BamHI site (FIG. 1). The plasmid carries the origin of replication of pWVO1 that functions in E. coli and in S. suis. This allowed the construction of a DNA library in E. coli first. Plasmid DNA, isolated from the pool of E. coli transformants, was subsequently electrotransformed into S. suis strain S735. 30,000 individual S. suis clones were obtained. As determined by analysis of 24 randomly selected transformants, more than 30% of the S735(pCOM-L) transformants contained an insert greater than 3 kb.
Selection of genomic fragments associated with virulence. To select for genetic determinants of the pathogenic S. suis strain 10 that could increase the virulence of the weak-pathogenic strain S735, pigs were inoculated with the S. suis library S735 (pCOM-L). A dose of either 107 or 108 cfu was used and the pigs were treated with erythromycin as described in Materials and Methods. All pigs showed specific S. suis symptoms (Table 2, A) three to seven days after the infection and except for one, all pigs died during the course of the experiment. From five of the pigs, bacteria could be re-isolated from the CNS and from two other pigs, bacteria were isolated from the joints (Table 2, A).
In previously performed experiments in which pigs were inoculated with weak-pathogenic strains, specific S. suis symptoms were observed with a very low frequency. In addition, from those pigs, bacteria could not be re-isolated from the CNS or from the joints. Therefore, the data indicated that, compared to virulence of strain S735, bacteria isolated from pigs inoculated with the S. suis library S735(pCOM-L) are more virulent due to the presence of a DNA fragment of the pathogenic strain 10. The plasmid content of 90 randomly selected clones isolated from the CNS or the joints of the seven diseased pigs was analyzed by PCR and restriction analysis. The results showed that 88 of the 90 clones analyzed (19 of which are shown in FIG. 2) contained an insert of about 3 kb and had substantially identical restriction patterns. Moreover, the inserts of ten randomly selected clones having substantially identical restriction patterns, also showed identical DNA sequences (results not shown). Plasmid DNA of ten randomly selected clones from the original S735 (pCOM-L) library showed ten different restriction patterns (FIG. 2). The data suggest that one specific clone, which was designated S735(pCOM-V10), was greatly enriched in seven different pigs. Further, this particular clone was isolated from the CNS and from the joints of the various pigs, indicating that the observed enrichment was not tissue specific.
Virulence-associated properties of the selected fragment V10. To further analyze the virulence properties of strain S735(pCOM-V10), pigs were intravenously inoculated with 106 cfu of strain S735 (pCOM1) or strain S735 (pCOM-V10). The results (Table 2, B) show that, compared to the virulence of strain S735 (PCOM1), the virulence of strain S735 (pCOM-V10) was greatly enhanced.
All pigs inoculated with strain S735 (pCOM-V10) showed specific S. suis symptoms and died within one day after infection. In contrast, except for one, none of the pigs inoculated with the control strain S735(pCOM1) showed specific clinical symptoms and these pigs survived until the end of the experiment (15 days after infection). The data proved that introduction of fragment V10 of strain 10 into S735 transformed the weak-pathogenic strain S735 into a highly pathogenic strain. This strongly suggests that the protein(s) encoded by V10 are important virulence determinants and play an important role in the pathogenesis of S. suis serotype 2 infections in pigs.
To find out whether the observed increase of the fragment V10 on virulence was specific for strain S735, pCOM1 and pCOM-V10 were introduced into another weak-pathogenic strain, strain 24. Subsequently, the virulence properties of the strains 24 (PCOM1) and 24 (PCOM-V10) were determined. As shown in Table 2 C and D, similar effects of V10 on the virulence of strains S735 and 24 were observed. Both strains 24 (pCOM-V10) and S735 (pCOM-V10) were highly pathogenic for young piglets, whereas strains 24 (PCOM1) and S735 (pCOM1) were shown to be weakly-pathogenic (Table 2, C and D). This strongly indicates that V10 has a more general ability to transform weak-pathogenic serotype 2 strains into highly pathogenic strains.
Because a plasmid system for the complementation approach was used, gene-dose effects cannot be excluded. Plasmid pCOM1 is based on the replication functions of pWVO1. In Gram-positive bacteria, the latter plasmid has a copy number of between 3 and 6. To find out whether copy effects play a role, the genomic region of strain S735 homologous to fragment V10 of strain 10 (see below) was cloned into plasmid pCOM1. This plasmid was designated pCOM-V735. The virulence of strains S735 (pCOM-V735), and 24 (pCOM-V735) was subsequently compared to that of S735 (pCOM-V10), S735 (pCOM1), 24 (pCOM-V10) and 24 (pCOM1). The results (Table 2, C and D) show that, in contrast to pCOM-V10, the plasmid pCOM-V735, did not carry virulence-enhancing activity. Pigs infected with strains S735 (pCOM-V10) and 24 (pCOM-V10) died within one or two days after infection, whereas most of the pigs infected with strains S735 (pCOM-V735), 24 (pCOM-V735), S735 (pCOM1) and 24 (pCOM1) survived until the end of the experiment (17 days after infection).
Compared to pigs infected with strains containing pCOM1, pigs infected with strains containing pCOM-V735 developed more general and specific signs of disease, but much less than pigs infected with strains containing pCOM-V10 (Table 2, C and D). From these data, it was concluded that the differences in virulence observed between the strains containing pCOM-V10 and the strains containing pCOM-VS735 are caused by differences between the fragments V10 and V735 (see below). The differences in virulence observed between the strains containing pCOM1 and the strains containing pCOM-VS735 may be due to gene-dose effects.
Sequence analysis of fragments V10 and V735. By using the fragment V10 as a probe, a 3.1 kb PstI-HindIII fragment of strain S735 (V735) was identified and cloned into pCOM1 (FIG. 3). To analyze the differences between the fragments V10 and V735, the nucleotide sequences of the fragments V10 and V735 were determined and the sequences were analyzed for homology to known genes by comparison with the GenBank/EMBL and SWISSPROT databases.
The sequence of V10 revealed two complete and two incomplete open reading frames (FIG. 3). ORF1 (nucleotides 1 to 461) coded for a polypeptide of 153 amino acids. This protein showed homology (49% identity) to the C-terminal region of acetate kinase of Clostridium thermocellum (accession number AF041841) and various other bacterial species. ORF2 (nucleotides 625 to 1327) coded for a protein of 233 amino acids. No significant similarities were found between the predicted amino acid sequence of this protein and other proteins present in the data libraries.
ORF3 (nucleotides 1382 to 2639) coded for a protein of 418 amino acids. This protein showed homology (36% identity) to FolC (folylpolyglutamate synthetase) of Bacillus subtilis. Compared to the other ORFs, ORF4 is transcribed in the opposite direction. ORF4 (nucleotides 2684 to 2972) coded for a polypeptide of 96 amino acids. This polypeptide showed homology (67% identity) to the C-terminal part of PepA (glutamyl-aminopeptidase) of Lactococcus lactis. Both ORFs 2 and 3 possessed putative initiation codons and ribosome-binding sites. Putative −35 (TGGACA) and −10 (TACAAT) sequences, which may function as promoter sequences, were found preceding ORF2. ORFs 2 and 3 were separated by 55 nucleotides. In this region, no putative promoter sequences could be observed. This could indicate that the ORFs 2 and 3 are co-transcribed. Downstream of the ORFs 1 and 3, regions of extended dyad symmetry were found which may function as transcription termination signals.
The sequence of the fragment V735 was determined and compared to the sequence of the fragment V10. No major deletions or insertions were found between the sequenced regions. The ORFs 1, 3 and 4 of strains 10 and S735 were highly homologous. The putative protein fragments encoded by the ORFs 1 differed in 2 (1.3%) amino acids; the putative proteins encoded by the ORFs 3 differed in 19 (4.5%) amino acids (FIG. 4B), whereas the putative protein fragments of the ORFs 4 were identical. However, major differences were observed between the ORFs 2 of strains 10 and S735. In the pathogenic strain 10, an ORF of 699 bases was found with a protein product of 233 amino acids. In contrast, due to a frame-shift mutation in the weak-pathogenic strain S735, an ORF of 569 bases was found and coded for a polypeptide of 183 amino acids.
Compared to the putative protein encoded by strain 10, the putative protein encoded by strain S735 lacked the N-terminal 50 amino acids (FIG. 4A). Beside these N-terminal differences, the putative proteins differed at 9 amino acid positions (4.9%). In addition, the putative −35 regions that may be part of the promoter sequences involved in the expression of ORFs 2 and 3, differed between the two strains. A TGGACA sequence was found in strain 10, whereas a TGGTCA sequence was found in strain S735. The sequence data suggest that the differences in the virulence-enhancing effects of the fragments V10 and V735 may be the result of functional differences between the putative proteins expressed by the ORFs 2 and/or 3, and/or by differences in their levels of expression.
ORF2 or ORF3.
To examine whether the observed increase of the fragment V10 on virulence resulted from ORF2 or ORF3 or both, the plasmids pCOM-V10-ORF2 and pCOM-V10 ORF3 containing the individual ORF2 and ORF3 encoding regions were constructed. Because ORF3 is probably co-transcribed with ORF2, in pCOM-V10-ORF3 the ORF3 encoding region was preceded by the promoter region of ORF2. Subsequently, the virulence properties of the strains S735 (pCOM-V10), S735 (pCOM-V10-ORF2), S735 (pCOM-V10-ORF3) and S735 (PCOM1) were determined. As shown at E in Table 2, the fragments V10 and ORF2 showed similar effects on the virulence of strain S735 while no effect of ORF3 could be observed on the virulence of strain S735. These data show that ORF2 is responsible for the observed effect on virulence and that the ORF2 protein is an important virulence factor.
Distribution of the ORF2 and ORF3 sequences among all known 35 S. suis serotypes. To examine the homology between the ORF2 and ORF3 genes and genes of other S. suis serotypes, cross-hybridization experiments were performed. DNA fragments of the ORF2 and 3 genes were amplified by PCR, labeled by 32P, and hybridized to chromosomal DNAs of the reference strains of the 35 different S. suis serotypes. As a positive control, a probe specific for 16S rRNA was used. The 16S rRNA probe hybridized with almost equal intensities with all serotypes tested (results not shown). Probes ORF2 and ORF3 hybridized with all serotypes, except for serotypes 32 and 34 (results not shown). This indicates that the proteins encoded by ORF2 and 3 are common among most Streptococcus species.
Herein, the development and the successful application of an in vivo complementation approach for the identification of important molecular determinants that determine the differences in virulence between pathogenic and weak-pathogenic strains of Streptococcus is described. Using the complementation approach, one unique clone containing a 3.0 kb fragment of pathogenic strain (V10) was selected. The selected fragment was greatly enriched in seven different pigs and the observed enrichment was not tissue specific. The selected fragment showed similar enhancing effects on the virulence of two different weak-pathogenic strains. Large differences were observed between the effects of the selected fragment V10 of the pathogenic strain 10 and the corresponding fragment V735 isolated from the weak-pathogenic strain S735 on virulence.
In contrast to V10 which had a strong virulence-enhancing effect on weak-pathogenic strains, V735 showed only minor effects. Therefore, differences between these two fragments are considered responsible for the observed differences on virulence. Sequence data showed that the fragments V10 and V735 were highly homologous. Both fragments contained two complete ORFs (ORFs 2 and 3), both of which can potentially express proteins that may further contribute to the observed effect on virulence. The ORFs 3 are highly homologous and differ in only 19 amino acids.
The proteins encoded by the ORFs 3 showed homology to FolC (folylpolyglutamate synthetase) of various pro- and eukaryotic organisms. Folylpolyglutamate synthetase catalyzes the conversion of folates to polyglutamate derivatives. Bacteria require folates for the biosynthesis of glycin, methionine, formylmethionine, thymidine, purines and patothenate. Whether the FolC proteins encoded by the fragments V10 and V735 have different enzymatic activities or different substrate specificities is unknown so far. In E. coli, a folC mutant is methionine deficient, however, so far a role of FolC in virulence has not been described. Significant differences were also observed between the ORFs 2 of the fragments V10 and V735. Compared to the putative ORF2 protein encoded by strain 10, the putative protein encoded by strain S735 lacked the N-terminal 50 amino acids. In strain S735, a strong ribosome-binding site precedes the methionine start codon of ORF2. In contrast, however, the sequence in strain 10 did not indicate the presence of a strong ribosome-binding site preceding the methionine start codon of ORF2. Therefore, although ORF2 of strain 10 is extended compared to ORF2 of strain S735, it is not clear whether the proteins expressed by these two ORFs differ in length.
In addition to the putative N-terminal differences, the putative ORF2 proteins differed at nine amino acid positions (4.9%). Except for one amino acid, these amino acid substitutions were clustered at two different positions in the putative protein. The function of the ORF2 protein is unknown so far. Not even distant or partial homologies were found between the ORF2 protein sequences and protein sequences present in the data libraries. Hydrophobicity profiles showed that the ORF2 encoded protein(s) are very hydrophobic thus suggesting a role of the ORF2 protein in the cellular membrane. The putative −35 region preceding the ORFs 2 and 3 differed between strains S735 and 10. Therefore, differences in the expression Levels rather than functional differences responsible for the observed effects on virulence are not excluded.
In previous experiments, it was found that pigs infected with weak-pathogenic strains showed only mild clinical signs of disease and that bacteria could never be re-isolated from the CNS or the joints. Surprisingly, in the experiments described herein in which weak-pathogenic strains containing the control plasmid pCOM1 were used, bacteria could (with a low frequency) be re-isolated from the CNS as well as from the joints. Several possible explanations for these observed differences exist. One explanation is that the presence of the plasmid somehow affects the (virulence) properties of the strains. Another possibility is that the treatment of the pigs with erythromycin makes the pigs more sensitive for S. suis infections and a third possibility is that compared to the pigs previously used, the pigs used for the current experiments were more sensitive for S. suis infections.
REFERENCES


    Anson, K. J., S. Movahedi, H. G. Griffin, M. J. Gasson, and F. Mulholland. 1995. A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk. Microbiol. 141: 2873–2881.
    Arends, J. P., and H. C. Zanen. 1988. Meningitis caused by Streptococcus suis in humans. Rev. Infect. Dis. 10: 131–13.
    Awad-Masalmeh, M., J. Köfer, M. Schuh, and F. Hinterdorfer. 1999. Serotypen, virulenzfaktoren und empfindlichkeit gegenuber antibiotika von Streptococcus suis stammer isoliert aus klinisch gesunden und erkrankten schweinen in Österreich. Wien. Tierärtztl. Mschr. 86: 262 269.
    Bogner, A. L., C. Osborne, and B. Shane. 1987. Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J. Biol. Chem. 262: 12337–12343.
    Chalettier, S., M. Gottschalk, R. Higgins, R. Brousseau and J. Harel. 1999. Relatedness of Streptococcus suis serotype 2 isolates from different geographic origins as evaluated by molecular fingerprinting and phenotyping. J. Clin. Microbiol. 37: 362–366.
    Clifton-Hadley, F. A. 1983. Streptococcus suis type 2 infections. Br. Vet. J. 139: 1–5.
    Galina, L., U. Vecht, H. J. Wisselink, and C. Pijoan. 1996. Prevalence of various phenotypes of Streptococcus suis isolated from swine in the USA based on the presence of muramidase-released protein and extracellular factor. Can. J. Vet. Res. 60: 72–74.8.
    Gottschalk, M., R. Higgins, M. Jacques, R. K. Mittal, and J. Henrichsen. 1989. Description of 14 new capsular types of Streptococcus suis. J. Clin. Microbiol. 27: 2633–2636.
    Gottschalk, M., R. Higgins, M. Jacques, M. Beaudain, and J. Henrichsen. 1991. Characterization of six new capsular types (23–28) of Streptococcus suis. J. Clin. Microbiol. 29: 2590–2594.
    Higgins, R., M. Gottschalk, M. Jacques, M. Beaudain, and J. Henrichsen. 1995. Description of six new capsular types (29–34) of Streptococcus suis. J. Vet. Diagn. Invest. 7: 405–406.
    Horinouchi, S., and B. Weisblum. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide and streptogramin type B antibiotics. J. Bacteriol. 150: 804–814.
    Kakuda, H., K. Honoso, K. Shiroishi, and S. Ichihara. 1994. Identification and characterization of the ack (acetate kinase A)-pta (phosphotransacetylase) operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli. J. Biochem. 116: 916–922.
    Kok, J., J. M. B. M. van der Vossen, and G. Venema. 1984. Construction of plasmid cloning vectors for lactic acid streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl. Environ. Microbiol. 48: 726–731.
    Konings, R. N. H., E. J. M. Verhoeven, and B. P. H. Peeters. 1987. pKUN vectors for the separate production of both DNA strands of recombinant plasmids. Methods Enzymol. 153: 12–34.
    Luo, D., J. Leautey, M. Grunberg-Manago, and H. Putzer. 1997. Structure and regulation of expression of the Bacillus subtilis valyl-tRNA synthetase gene. J. Bacteriol. 179: 2472–2478.
    Luque, I., C. Tarradas, R. Astorga, A. Perea, H. J. Wisselink, and U. Vecht. 1998. The presence of muramidase released protein and extracellular factor protein in various serotype of Streptococcus suis isolated from diseased and healthy pigs in Spain. Res. Vet. Science 66: 69–72.
    Margolis, P. S., A. Driks, and R. Losick. 1993. Sporulation gene spoIIB from Bacillus subtilis. J. Bacteriol. 175: 528–540.
    Miller, J. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
    Paithankar, K. R., and K. S. N. Prasad. 1991. Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res. 19: 134.
    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    Salasia, S. I. O., and C. Lämmler. 1995. Distribution of serotype, virulence markers and further characteristics of Streptococcus suis isolates from pigs. J. Vet. Med. Series B 42: 78–83.
    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    Smith, H. E., M. Damman, J. van der Velde, F. Wagenaar, H. J. Wisselink, N. Stockhofe-Zurwieden, and M. A. Smits. 1999. Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infect. Immun. 67: 1750–1756.
    Smith, H. E., M. Rijnsburger, N. Stockhofe-Zurwieden, H. J. Wisselink, U. Vecht, and M. A. Smits. 1997. Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile. J. Clin. Microbiol. 35: 1049–1053.
    Smith, H. E., U. Vecht, A. L. J. Gielkens, and M. A. Smits. 1992. Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of Streptococcus suis type 2. Infect. Immun. 60: 2361–2367.
    Smith, H. E., U. Vecht, H. J. Wisselink, N. Stockhofe-Zurwieden, Y. Biermann, and M. A. Smits. 1996. Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs. Infect. Immun. 64: 4409–4412.
    Smith, H. E., H. J. Wisselink, U. Vecht, A. L. J. Gielkens, and M. A. Smits. 1995. High-efficiency transformation and gene inactivation in Streptococcus suis type 2. Microbiol. 141: 181–188.
    Staats, J. J., B. L. Plattner, G. C. Stewart, and M. M. Chengappa. 1999. Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates. 1999. Vet. Microbiol. 70: 201–211.
    Stockhofe-Zurwieden, N., U. Vecht, H. J. Wisselink, H. van Lieshout, and H. E. Smith. 1996. Comparative studies on the pathogenicity of different Streptococcus suis serotype 1 strains, p. 299. In P. G. Monetti and G. Vignola (ed.), Proceedings of the 14th International Pig Veterinary Society Congress, Bologna, Italy.
    Vecht, U., J. P. Arenda, E. J. van der Molen, and L. A. M. G. van Leengoed. 1989. Difference in virulence between two strains of Streptococcus suis type 2 after experimentally induced infection of newborn germfree pigs. Am. J. Vet. Res. 50: 1037–1043.
    Vecht, U., L. A. M. G. van Leengoed, and E. R. M. Verheyen. 1985. Streptococcus suis infections in pigs in The Netherlands (part one). Vet. Q. 7: 315–321.
    Vecht, U., H. J. Wisselink, M. L. Jellema, and H. E. Smith. 1991. Identification of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun. 59: 3156–3162.
    Vecht, U., H. J. Wisselink, J. E. van Dijk, and H. E. Smith. 1992. Virulence of Streptococcus suis type 2 strains in newborn germfree pigs depends on phenotype. Infect. Immun. 60: 550–556.
    Vecht, U., H. J. Wisselink, N. Stockhofe-Zurwieden, and H. E. Smith. 1995. Characterization of virulence of the Streptococcus suis serotype 2 reference strain Henrichsen S735 in newborn germfree pigs. Vet. Microbiol. 51: 125–136.
    Wisselink, H. J., H. E. Smith, N. Stockhofe-Zurwieden, K. Peperkamp and U. Vecht. 2000.


Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European Countries. Vet. Microbiol. 74: 237–247.







TABLE 1







Bacterial strains and plasmids









strain/plasmid
relevant characteristics*
source/reference





Strain




E. coli


XL2 blue

Stratagene


S. suis


10
pathogenic serotype 2 strain
Vecht et al.


S735
weak-pathogenic serotype 2
Vecht et al.



reference strain


24
weak-pathogenic serotype 2 strain
Vecht et al.


Plasmid


pKUN19
replication functions pUC, AmpR
Konings et al.


pE194
EmR
Horinouchi et al.


pMR11
pKUN19 containing S. suis mrp gene
Smith et al.


pCOM1
replication functions pWVO1, EmR
this work


pCOM-L
pCOM1 containing random
this work



sequences of S. suis strain 10


pCOM-V10
pCOM1 containing S. suis strain 10
this work



fragment selected in pigs


pCOM-V735
pCOM1 containing a 3.1 kb PstI-
this work



HindIII fragment from S. suis 



strain S735 (homologous to V10)





*SpcR: spectinomycin resistant


AmpR: ampicillin resistant


EmR: erythromycin resistant













TABLE 2







Virulence of S. suis library and strains in germfree pigs






















clinical index


no. of pigs



No.


mean no.

of the group


in which S. suis


















of

moralitya
of days
morbidityb
specificc
non-specifiedd
fever
leukocyte
was isolated from



















strains
pigs
dose
(%)
till death
(%)
symptoms
symptoms
indexe
indexf
CNS
serosa
joints






















A














S735(pCOM-L)
4
107
100
4
100
69
91
25
n.a.
3
2
3


S735(pCOM-L)
4
108
75
7
100
50
69
20
17
2
1
2


B


S735(pCOM-V10)
5
106
100
1
100
100
100
54
4
5
5
5


S735(pCOM1)
4
106
25
12
25
2
11
6
80
1
1
2


C


S735(pCOM-V10)
5
106
100
1
100
100
100
60
n.a.
5
5
5


S735(pCOM-V735)
5
106
20
15
100
40
26
17
52
1
1
1


S735(pCOM1)
5
106
20
16
60
11
9
11
20
1
0
0


D


24(pCOM-V10)
5
106
100
2
100
50
66
42
29
3
3
5


24(pCOM-V735)
4
106
25
15
100
40
30
17
18
1
0
0


24(pCOM1)
5
106
20
15
20
2
14
6
21
1
0
0


E


S735(pCOM-V10)
4
106
100
1
100
100
100
57
n.d.
4
4
4


S735(pCOM-V10-ORF2)
4
106
100
1
100
100
84
50
n.d.
4
4
4


S735(pCOM-V10-ORF3)
4
106
0
11
0
6
4
3
n.d.
0
0
0


S735(pCOM1)
4
106
0
11
0
0
9
5
n.d.
0
0
0





aPercentage of pigs that died due to infection or had to be killed for animal welfare reasons


bPercentage of pigs with specific symptoms


cPercentage of observations for the experimental group in which specific symptoms (ataxia, lameness of at least one joint and/or stillness) were observed


dPercentage of observations for the experimental group in which non-specific symptoms (inappetite and/or depression) were observed


ePercentage of observations for the experimental group of a body temperature of >40° C.


fPercentage of blood samples for the experimental group in which the concentration of granulocytes was >1010/liter


n.a.: not applicable


n.d.: not determined

## Claims

1. A method for modifying a Streptococcus suis, said method comprising:
introducing a nucleic acid sequence encoding a virulence factor into the Streptococcus suis, said nucleic acid sequence selected from the group consisting of SEQ ID NO:14, and a conservatively substituted variant of SEQ ID NO:14, wherein said conservatively substituted variant encodes a virulence factor having the virulence-modulating activity of the virulence factor encoded by SEQ ID NO:14.

2. A method of modulating virulence of a Streptococcus suis comprising:
introducing into said S. suis a nucleotide sequence comprising SEQ ID NO:14 or a conservatively substituted variant of SEQ ID NO:14, wherein said conservatively substituted variant encodes a virulence factor having the virulence-modulating activity of the virulence factor encoded by SEQ ID NO:14.