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Infection and Immunity, April 1999, p. 1750-1756, Vol. 67, No. 4
Department of
Bacteriology1 and Department of
Immunology, Pathobiology and Epidemiology,2
DLO-Institute for Animal Science and Health, 8200 AB Lelystad, The
Netherlands
Received 12 November 1998/Returned for modification 22 December
1998/Accepted 19 January 1999
To study the role of the capsule of Streptococcus suis
serotype 2 in virulence, we generated two isogenic mutants disturbed in
capsule production. For that purpose, we first cloned and characterized a major part of the capsular polysaccharide biosynthesis
(cps) locus of S. suis serotype 2. Based on the
established sequence, 14 open reading frames (ORFs), designated Orf2Z,
Orf2Y, Orf2X, and Cps2A to Cps2K, were identified. Twelve ORFs belonged
to a single transcriptional unit. The gene products of 11 of these ORFs
showed similarity to proteins involved in polysaccharide biosynthesis
of other gram-positive microorganisms. Nonencapsulated isogenic mutants
were generated in the cps2B and cps2EF genes by
insertional mutagenesis. In contrast to the wild-type S. suis serotype 2 strain, the nonencapsulated strains were highly
sensitive to ingestion by porcine alveolar lung macrophages in vitro.
More importantly, the nonencapsulated mutant strains were completely avirulent in young germfree pigs after intranasal inoculation. These
observations indicate that the capsule of S. suis serotype 2 plays an essential role in the pathogenesis of S. suis
serotype 2 infections.
Streptococcus suis is an
important cause of meningitis, septicemia, arthritis, and sudden death
in young pigs (4, 38). It can, however, also cause human
meningitis (1). S. suis strains are identified by
their morphological, biochemical, and serological characteristics.
Serological classification is based on the presence of specific
epitopes on its polysaccharidic capsule. So far, 35 different serotypes
have been described (8, 13). Strains of S. suis
can differ in virulence. Some serotypes are more frequently isolated
from diseased pigs than others, suggesting that differences in
virulence are associated with differences in capsular polysaccharides. In Europe, S. suis serotype 2 is the type most frequently
isolated from diseased pigs, followed by serotypes 9 and 1. The idea
that the capsule of S. suis serotype 2 plays a role in the
pathogenesis was supported by the observation of reduced virulence for
transposon mutants of S. suis impaired in capsule production
(3). Moreover, it is well known that the levels of virulence
of S. suis strains within a single serotype can differ
greatly (37, 39). A number of strains of S. suis
serotypes 1 and 2 have been shown to be highly virulent in pigs,
whereas other strains of serotypes 1 and 2 are completely avirulent
(33, 37, 39). Both the virulent and avirulent strains of
either serotype seem to be fully encapsulated. This suggests that there
is only a minor contribution of the capsule to the virulence of
S. suis. Indeed, various bacterial components, such as
extracellular and cell membrane-associated proteins, fimbriae, hemagglutinins, and hemolysin, have been suggested as virulence factors
(7, 9, 10, 14, 15, 39, 41). However, the precise role of
these protein components in the pathogenesis of the disease has not
been established (29).
To provide conclusive evidence with regard to the role and contribution
of the capsule of S. suis in determining virulence, we
identified and characterized a major part of the DNA region encoding
the proteins necessary for capsule synthesis. In addition, we generated
isogenic mutants in two different capsular genes. Both isogenic mutants
were found to be resistant to phagocytosis by alveolar lung macrophages
in vitro. In addition, the nonencapsulated mutants were completely
avirulent in young germfree pigs.
Bacterial strains and growth conditions.
The bacterial
strains and plasmids used in this study 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% (vol/vol) horse blood.
Escherichia coli strains were grown in Luria broth
(23) and plated on Luria broth containing 1.5% (wt/vol)
agar. If required, antibiotics were added to the plates at the
following concentrations: spectinomycin, 100 µg/ml for S. suis and 50 µg/ml for E. coli; ampicillin, 50 µg/ml
for E. coli.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification and Characterization of the
cps Locus of Streptococcus suis Serotype 2: the
Capsule Protects against Phagocytosis and Is an Important
Virulence Factor
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
Serotyping. The S. suis strains were serotyped by the slide agglutination test with serotype-specific antibodies (36).
Selection of genes encoding exported proteins.
Chromosomal
DNA of S. suis serotype 2 was digested with AluI.
The 300- to 500-bp fragments were ligated to SmaI-digested
pPHOS2. Ligation mixtures were transformed to PhoA
E. coli CC118. Transformants were plated on Luria broth agar plates supplemented with 5-bromo-4-chloro-3-indolylphosphate (BCIP) (50 µg/ml) (Boehringer, Mannheim, Germany). Blue colonies were purified
on fresh Luria broth/BCIP plates to verify the blue phenotype.
DNA techniques and sequence analysis. Routine DNA manipulations were performed as described by Sambrook et al. (28). DNA sequences were determined on a 373A DNA Sequencing System (Applied Biosystems, Warrington, Great Britain). Samples were prepared by use of an ABI/PRISM dye terminator cycle sequencing ready reaction kit (Applied Biosystems). Sequencing data were assembled and analyzed by using the MacMollyTetra software package. Custom-made sequencing primers were purchased from Life Technologies. The BLAST software package was used to search for protein sequences homologous to the deduced amino acid sequences in the GenBank/EMBL databases.
Construction of gene-specific knock out mutants.
To
construct the mutant strains 10cps
B and 10cpsEF, we
electrotransformed pathogenic strain 10 (37, 41) of S. suis serotype 2 with pCPS11 and pCPS28, respectively. In these
plasmids the cpsB and cpsEF genes are inactivated
by the insertion of a spectinomycin resistance gene. To create pCPS11,
the internal 400-bp PstI-BamHI fragment of the
cpsB gene in pCPS7 was replaced by the 1,200-bp PstI-BamHI fragment from pIC-spc, containing the
spectinomycin resistance gene. To construct pCPS28 we have used pIC20R.
Into this plasmid we inserted the KpnI-SalI
fragment from pCPS17 (resulting in pCPS25) and the
XbaI-ClaI fragment from pCPS20 (resulting in pCPS27). pCPS27 was digested with PstI and XhoI
and ligated to the 1,200-bp PstI-XhoI fragment,
containing the spectinomycin resistance gene of pIC-spc. The
electrotransformation to S. suis was carried out as
described before (30).
Southern blotting and hybridization.
Chromosomal DNA was
isolated as described by Sambrook et al. (28). DNA fragments
were separated on 0.8% agarose gels and transferred to Zeta-Probe GT
membranes (Bio-Rad) as described by Sambrook et al. (28).
DNA probes were labelled with [
-32P]dCTP (3,000 Ci
mmol1; Amersham) by use of a random primed labelling kit
(Boehringer). The DNA on the blots was hybridized at 65°C with the
appropriate DNA probes as recommended by the supplier of the Zeta-Probe
membranes. After hybridization, the membranes were washed twice with a
solution of 40 mM sodium phosphate (pH 7.2), 1 mM EDTA, and 5% sodium
dodecyl sulfate for 30 min at 65°C and twice with a solution of 40 mM sodium phosphate (pH 7.2), 1 mM EDTA, and 1% sodium dodecyl sulfate for 30 min at 65°C.
Electron microscopy. Bacteria were prepared for electron microscopy as described by Wagenaar et al. (42). Shortly, bacteria were mixed with agarose MP (Boehringer) of 37°C to a concentration of 0.7%. The mixture was immediately cooled on ice. Upon gelling, samples were cut into 1- to 1.5-mm-thick slices and incubated in a fixative containing 0.8% glutaraldehyde and 0.8% osmium tetroxide. Subsequently, the samples were fixed and stained with uranyl acetate by microwave stimulation, dehydrated, and embedded in eponaraldite resin. Ultrathin sections were counterstained with lead citrate and examined with a Philips CM 10 electron microscope at 80 kV. In addition, we used the polycationic ferritin method as described by Quessy et al. (26).
Phagocytosis assay. Porcine alveolar macrophages (AM) were obtained from the lungs of specific-pathogen-free (SPF) pigs. Lung lavage samples were collected as described by van Leengoed et al. (35). Cells were suspended in Eagle's minimal essential medium (EMEM) containing 6% (vol/vol) SPF pig serum and adjusted to 107 cells per ml. Phagocytosis assays were performed as described by Leij et al. (20). Briefly, 107 S. suis cells were incubated with 6% SPF pig serum for 30 min at 37°C in a head-over-head rotor at 6 rpm, to opsonize the cells. We combined 107 AM and 107 opsonized S. suis cells and incubated them at 37°C under continuous rotation at 6 rpm. At 0, 30, 60, and 90 min, 1-ml samples were collected and mixed with 4 ml of ice-cold EMEM to stop phagocytosis. Phagocytes were removed by centrifugation for 4 min at 110 × g and 4°C. The number of CFU in the supernatants was determined by plating. Control experiments were carried out simultaneously by combining 107 opsonized S. suis cells with EMEM without AM.
Killing assays. Killing assays were performed as described by Leij et al. (20). AM (107/ml) and opsonized S. suis cells (107/ml) were mixed 1:1 and incubated for 10 min at 37°C under continuous rotation at 6 rpm. Ice-cold EMEM was added to stop further phagocytosis and killing. To remove extracellular S. suis cells, phagocytes were washed twice (4 min, 110 × g, 4°C) and resuspended in 5 ml of EMEM containing 6% SPF pig serum. The resuspended AM were incubated at 37°C under rotation at 6 rpm. After 0, 15, 30, 60, and 90 min, samples were collected and mixed with ice-cold EMEM to stop further killing. The samples were centrifuged for 4 min at 110 × g at 4°C, and the phagocytic cells were lysed in EMEM containing 1% saponin for 20 min at room temperature. The number of CFU in the suspensions was determined by plating.
Experimental infections. Germfree pigs, crossbreeds of Great Yorkshire and Dutch Landrace, were obtained from sows by cesarean sections. The surgery was performed in sterile flexible film isolators. Pigs were allotted to groups, each consisting of 4 pigs, and were housed in sterile stainless steel incubators. Housing conditions and feeding regimens were as described before (37, 41). Pigs were inoculated intranasally with S. suis serotype 2 as described before (37, 41). To predispose the pigs to infection with S. suis, 5-day-old pigs were inoculated intranasally with about 107 CFU of Bordetella bronchiseptica 92932. Two days later the pigs were inoculated intranasally with S. suis serotype 2 (106 CFU). Pigs were monitored twice daily for clinical signs of disease, such as fever, nervous signs, and lameness. Blood samples were collected three times a week from each pig. Leucocytes were counted with a cell counter. To monitor infection with S. suis and B. bronchiseptica and to check for absence of contaminants, we collected swabs of the nasopharynx and the feces daily. The swabs were plated directly onto Columbia agar containing 6% horse blood. After the pigs were killed, they were examined for pathological changes. Tissue specimens from the central nervous system (CNS), serosae, and joints were examined bacteriologically and histologically as described before (37, 41). Colonization of the serosae was scored positively when S. suis was isolated from the pericardium, thoracic pleura, or peritoneum. Colonization of the joints was scored positively when S. suis was isolated from one or more joints (12 joints per animal were scored).
Nucleotide sequence accession number. The nucleotide sequence data have been submitted to GenBank under accession no. AF118389.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification and isolation of capsule-encoding DNA. Initially, a part of the capsular locus of S. suis serotype 2 was isolated in an attempt to identify secreted proteins by genetic means (12, 25). For this purpose chromosomal DNA of S. suis serotype 2 was cloned in E. coli in front of a 5'-truncated alkaline phosphatase gene. To do this, we made use of the vector pPHOS2 (Table 1), which contained the truncated alkaline phosphatase gene of pPHO7 (12) as well as a spectinomycin resistance gene (31). A number of E. coli clones displayed a dark blue phenotype when plated on media containing BCIP, indicating that the cloned fragment contained a promoter, a translational start site, and a signal sequence. The deduced amino acid sequence of one of the cloned fragments (on plasmid pPHOS7) showed a high similarity (37% identity) to a protein (Cps14C) involved in capsular synthesis of Streptococcus pneumoniae (18). This strongly suggested that pPHOS7 contained a part of the corresponding cps gene of S. suis serotype 2. Subsequently, the insert of pPHOS7 (Fig. 1C) was used as a probe to identify chromosomal DNA fragments containing flanking cps genes. A 6-kb HindIII fragment was identified and cloned in pKUN19. This yielded clone pCPS6 (Fig. 1C). Sequence analysis of the insert revealed that pCPS6 contained the 5' end of the cps locus. Sequences of the 3' end of pCPS6 were, in turn, used to identify a chromosomal fragment containing cps sequences located further downstream. This fragment was also cloned in pKUN19, resulting in pCPS17. Using a similar approach, we subsequently isolated the plasmids pCPS18, pCPS20, pCPS23, and pCPS26 containing downstream cps sequences (Fig. 1C).
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Analysis of the cps operon. The complete nucleotide sequences of the cloned fragments were determined. Examination of the compiled sequence revealed the presence of 14 potential open reading frames (ORFs), which were designated Orf2Z, Orf2Y, Orf2X, and Cps2A through Cps2K (Fig. 1A). Orf2Z, located at the 5' end of the sequence, was incomplete. Compared to the other ORFs, Orf2Y is expressed in the opposite orientation. Two potential promoter sequences were identified. One was located 313 bp (positions 1885 to 1865 and 1884 and 1889) upstream of Orf2X. The other was located 68 bp upstream of Orf2Y (positions 2241 to 2236 and 2216 to 2211). Between Orf2Y and Orf2Z the sequence contained a potential stem-loop structure, which could act as a transcription terminator. Each ORF is preceded by a ribosome-binding site, and the majority of the ORFs are very closely linked. The only significant intergenic gap was that found between Cps2G and Cps2H (389 nucleotides). No obvious promoter sequences or potential stem-loop structures were found in this region. This suggests that Orf2X and Cps2A through Cps2K are part of a single transcriptional unit.
A list of all ORFs with their properties is shown in Table 2. Orf2Z showed similarity to the YitS protein of Bacillus subtilis, a protein with an unknown function. Orf2Y showed homology to the YcxD protein of B. subtilis (43), which is supposed to be a regulatory protein. Orf2X showed homology with the hypothetical YAAA proteins with unknown function of Haemophilus influenzae and E. coli. The products of the cps2A, cps2B, cps2C, and cps2D genes showed significant homologies with the CpsA, CpsC, CpsD, and CpsB proteins of several streptococci (Table 2), suggesting similar functions for these proteins. Hence, Cps2A may have a role in the regulation of the capsular polysaccharide synthesis, Cps2B and Cps2C could be involved in the chain length determination of the type 2 capsule, and Cps2C could play an additional role in the export of the polysaccharide. Cps2D is homologous to Cps proteins of streptococci involved in the polysaccharide or exopolysaccharide synthesis, but it is without a known specific function (18). The proteins encoded by the cps2E, cps2F, cps2G, cps2H, cps2J, and cps2K genes showed homology to proteins with glycosyltransferase activities of several streptococci (5, 16, 17, 18, 32), suggesting that these proteins are involved in the biosynthesis of the type 2 oligosaccharide subunit. The protein encoded by the cps2I gene showed homology to a protein of S. pneumoniae with potential polysaccharide polymerase activity (5).
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Construction of mutants impaired in capsule synthesis.
To
evaluate the role of the capsule of S. suis serotype 2 in
virulence, we constructed two isogenic mutants in which capsule production was disturbed. To construct mutants 10cpsB and
10cpsEF, the plasmids pCPS11 and pCPS28 were used. pCPS11 and pCPS28
were electrotransformed into strain 10 of S. suis serotype
2, and spectinomycin-resistant colonies were selected. Southern
blotting and hybridization experiments were used to select double
crossover integration events (data not shown).
B
and 10cpsEF was disturbed, we used a slide agglutination test
(36). The parent strain, strain 10, of S. suis
serotype 2 agglutinated only in type 2-specific serum. The mutant
strains, however, agglutinated in sera specific for all S. suis strains, but they also agglutinated in the absence of
serotype-specific serum. This indicated that in the mutant strains the
capsular structure was disturbed. To confirm this, thin sections of
wild-type and mutant strains were compared by electron microscopy.
Compared to the wild-type strain (Fig.
2A), the amount of capsule produced by
the mutant strains was greatly reduced (Fig. 2B and C). No capsular
material could be detected on the surfaces of the mutant strains.
Similar results were obtained after the polycationic ferritin method
was used (results not shown).
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Capsular mutants are sensitive to phagocytosis and killing by
AM.
The capsular mutants and the parent strain were tested for the
ability to resist phagocytosis by AM in the presence of porcine SPF
serum. As shown in Fig. 3A, the wild-type
strain (strain 10) is resistant to phagocytosis under the in vitro
conditions used (Fig. 3A). In contrast, both mutant strains were
efficiently ingested by the macrophages (Fig. 3A). After 90 min, more
than 99.7% (strain 10cpsB) and 99.8% (strain 10cpsEF) of the
mutants were ingested by the macrophages. Moreover, as shown in Fig.
3B, the ingested strains were efficiently killed by the macrophages.
From 90 to 98% of all ingested cells were killed within 90 min. No
differences in killing efficiency could be observed between wild-type
and mutant strains. Similar results were obtained after
polymorphonuclear leukocytes were used (results not shown). These data
indicate that the capsule of S. suis serotype 2 efficiently
protects the bacterium from uptake by macrophages in vitro.
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, wild-type strain,
strain 10; 
, mutant strain 10cps
, mutant strain
10cpsCapsular mutants are avirulent in germfree piglets.
The
virulence properties of the wild-type and mutant strains were tested by
experimental infection of newborn germfree pigs (37, 41).
Table 3 shows that specific and
nonspecific signs of disease could be observed in all pigs inoculated
with the wild-type strain. All pigs inoculated with the wild-type
strain died during the course of the experiment or were killed because
of serious illness or nervous disorders (Table 3). In contrast, the
pigs inoculated with strains 10cpsB or 10cpsEF showed no specific signs of disease and all of these pigs survived until the end of the
experiment. Moreover, we observed significant differences in the fever
index and in the leukocyte index between pigs inoculated with wild-type
and mutant strains (Table 3). S. suis strains and B. bronchiseptica could be isolated from the nasopharyngeal and fecal
swab samples of all pigs from 1 day postinfection until the end of the
experiment. Postmortem, the wild-type strain could frequently be
isolated from the CNS, kidney, heart, liver, spleen, serosae, joints,
and tonsils. Mutant strains could be recovered from the tonsils but
were never recovered from the kidney, liver, or spleen. Interestingly,
small numbers of the mutant strains could be isolated from the CNS, the
serosae, the joints, the lungs, and the heart. Agglutination tests and
Southern blot analyses showed that these mutant strains had the
unencapsulated phenotype and genotype (results not shown). Taken
together, these data demonstrate that mutant S. suis strains
impaired in capsule production are avirulent in young germfree pigs.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present paper we describe the identification and the
molecular characterization of a 16-kb DNA fragment containing a major
part of the genetic determinant involved in the capsular polysaccharide
biosynthesis of S. suis serotype 2. To study the role of the
capsule in resistance to phagocytosis and in virulence, we constructed
two isogenic mutants in which capsule synthesis was disturbed. In
10cpsB, the cps2B gene was disturbed by the insertion of
an antibiotic resistance gene, whereas in 10cpsEF parts of the
cps2E and cps2F genes were replaced by an
antibiotic resistance gene. By electron microscopical analysis both
mutant strains were found to be completely unencapsulated. Although
this finding confirms that the cpsB and cpsEF
genes are involved in capsular synthesis, this finding does not give
any clues to the function of these proteins, since they form part of an
operon structure and polar effects on the expression of downstream
genes cannot be excluded. The behavior of the mutants in the in vitro phagocytosis and killing assays clearly showed that the capsular polysaccharide of S. suis serotype 2 is a surface component
with antiphagocytic activity. Wild-type encapsulated bacteria were ingested by phagocytes at a very low frequency, whereas the mutant unencapsulated bacteria were efficiently ingested by porcine
macrophages. Once ingested, wild-type and mutant strains seemed to be
killed with the same relative efficiency. This suggests that the loss of capsular material is associated with loss of capacity to resist uptake by macrophages. This loss of resistance to in vitro phagocytosis was associated with an almost complete attenuation of the virulence of
the mutant strains in germfree pigs. All pigs inoculated with the
mutant strains survived the experiment and did not show any specific
clinical signs of disease. Only some nonspecific clinical signs of
disease could be observed. Moreover, small numbers of mutant bacteria
could be reisolated from the pigs. This supports the idea that, as in
other pathogenic streptococci, the capsule of S. suis acts
as an important virulence factor. Our data obtained with the isogenic
mutants are in agreement with the data recently reported by Charland et
al. (3). They reported that transposon mutants, which are
impaired in capsule production, showed reduced virulence in pigs and
mice (3). However, to construct these transposon mutants,
the authors used the serotype 2 reference strain S735. We previously
showed that strain S735 is only weakly virulent for young pigs
(40). Moreover, since the insertion sites of the transposon
in the mutants were not determined, it could not be concluded that the
observed reduction in virulence was a direct consequence of impaired
capsule synthesis.
Initially a part of the cps2B gene was cloned by screening for signal sequences. The hydrophobicity profile of the clone showed that the N-terminal part of the sequence resembled the characteristics of a typical signal peptide: a short N-terminal region is followed by a hydrophobic region of 38 amino acids. Apparently, this region was able to translocate alkaline phosphatase across the cellular membrane in E. coli. The hydrophobicity plot of the corresponding Cps14C protein of S. pneumoniae showed two hydrophobic segments, one each at its N and C termini, and a hydrophilic domain in the central part (18). The cellular location of this protein is unknown. The region homologous to the second hydrophobic domain was not cloned in pPHOS7.
The cloned and sequenced region described here contained 14 ORFs. At least 12 of these ORFs belong to a single transcriptional unit, suggesting a coordinated control of the expression of these genes. Based on sequence similarities we could assign putative functions to most of the gene products. We thereby identified gene products involved in regulation (Cps2A), chain length determination (Cps2B, C), export (Cps2C), biosynthesis (Cps2E through Cps2H, Cps2J, and Cps2K), and polymerization (Cps2I). The overall organization is similar to that of the cps and eps gene clusters of a number of gram-positive bacteria (17, 27, 32, 34). A region involved in biosynthesis is preceded by a region containing genes with more common functions. Although, based on sequence similarities, a role of most of the gene products in the polysaccharide biosynthesis could be envisaged, the role of the orf2Z, orf2Y, and orf2X genes remains unclear so far. The incomplete orf2Z gene was located at the 5' end of the cloned fragment. Orf2Z showed some similarity to the YitS protein of B. subtilis. However, because the function of the YitS protein is unknown, this did not give us any information about the possible function of Orf2Z. Because the orf2Z gene is not a part of the cps operon, a role of this gene in polysaccharide biosynthesis is not expected. The Orf2Y protein showed similarity to the YcxD protein of B. subtilis (43). The YcxD protein was suggested to be a regulatory protein. Similarly, Orf2Y may be involved in the regulation of polysaccharide biosynthesis. The Orf2X protein showed similarity to the YAAA proteins of H. influenzae and E. coli. The function of these proteins is unknown. In S. suis serotype 2 the orf2X gene seems to be the first gene in the cps2 operon. This suggests a role of Orf2X in polysaccharide biosynthesis. In H. influenzae and E. coli, however, these proteins are not associated with capsular gene clusters.
The products encoded by the cps2E, cps2F,
cps2G, cps2H, cps2J, and
cps2K genes showed similarities to glycosyltransferases of
several streptococci (5, 16-18, 32). The cps2E
gene product showed strong homology to the Cps14E protein of S. pneumoniae (16, 18). Cps14E is a glucosyl-1-phosphate
transferase that links glucose to a lipid carrier (18). In
S. pneumoniae this is the first step in the biosynthesis of
the oligosaccharide repeating unit. The structure of the S. suis serotype 2 capsule is unknown, but it is composed of glucose,
galactose, N-acetylglucosamine, rhamnose, and sialic acid in
a ratio of 1:3:1:1:1 (6). Therefore, because the capsule of
S. suis serotype 2 does contain glucose (6), we
speculate that Cps2E of S. suis could also have
glucosyltransferase activity and is probably involved in the linkage of
the first sugar to the lipid carrier. The cps2F gene product
showed homology to the Cps23fT protein, which has rhamnosyltransferase
activity, of S. pneumoniae (5). Because rhamnose
is a component of the S. suis serotype 2 polysaccharide
(6) Cps2F could have rhamnosyltransferase activity. The
cps2G gene encoded a protein that showed moderate similarity
to the epsF gene product of Streptococcus
thermophilus (32). On the basis of homology
epsF is suggested to encode galactosyltransferase activity.
Hence, a similar galactosyltransferase activity is proposed for Cps2G.
The cps2H gene encodes a protein with an N-terminal region
that is similar to the N-terminal region of the RGPEC protein of
Streptococcus mutans (D1033055). For this protein a
glycosyltransferase activity was suggested. Moreover, the
hydrophobicity plots of Cps2H and RGPEC looked very similar in these
regions (data not shown). Therefore, Cps2H could have
glycosyltransferase activity as well. Cps2J and Cps2K showed homology
to Cps14J of S. pneumoniae (17). Cps2J also
showed homology to Cps14I of S. pneumoniae. Cps14I has
N-acetylglucosaminyltransferase activity, whereas Cps14J possesses a 
-1,4-galactosyltransferase activity (17). In
S. pneumoniae Cps14I is responsible for the addition of the
third sugar and Cps14J is responsible for the addition of the last
sugar in the synthesis of the type 14 repeating unit (17).
Because the capsule of S. suis serotype 2 contains galactose
as well as N-acetylglucosamine components,
galactosyltransferase N-acetylglucoaminyltransferase activities could be envisaged for the cps2J and
cps2K gene products, respectively. The two conserved
regions, DXS and DXDD, which are conserved in several
glycosyltransferases (17) and which are proposed to be
important for catalytic activity, were also found in Cps2J and Cps2K.
The Cps2I protein showed similarity to the Cps23fI protein of S. pneumoniae, which has a capsular polysaccharide polymerase
activity (5), suggesting that Cps2I could be involved in the
polymerization of the type 2 specific oligosaccharides.
The capsule of S. suis serotype 2 is composed of glucose, galactose, N-acetylglucosamine, rhamnose, and sialic acid (6). Based on sequence homology genes encoding potential glucosyl-, galactosyl-, N-acetylglucosaminyl-, and rhamnosyltransferase activities could be identified. However, we have not found genes homologous to genes involved in the synthesis, activation, and transfer of sialic acid. Moreover, since we do not know whether the cps2K gene is the last gene in the cps2 locus, these genes can be located downstream of cps2K. Therefore, in future experiments we will concentrate on the cloning and characterization of these genes. Moreover, the analysis of isogenic mutants in which the individual genes are interrupted, without disturbing expression of the downstream genes, will give more information about the role of the individual cps2 genes in the polysaccharide biosynthesis of the S. suis serotype 2 capsule.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Bacteriology, DLO-Institute for Animal Science and Health, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Phone: 31 320 238270. Fax: 31 320 238153. E-mail: h.e.smith{at}id.dlo.nl.
Editor: E. I. Tuomanen
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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