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Journal of Clinical Microbiology, October 2008, p. 3201-3207, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.02309-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Differentiation of Highly Virulent Strains of Streptococcus suis Serotype 2 According to Glutamate Dehydrogenase Electrophoretic and Sequence Type

Russell Kutz¹ and Ogi Okwumabua^2,3*

Department of Bacteriology,¹ Department of Pathobiological Sciences, School of Veterinary Medicine,² Wisconsin Veterinary Diagnostic Laboratory, Microbiology Section, University of Wisconsin, Madison, Wisconsin 53706³

Received 30 November 2007/ Returned for modification 6 January 2008/ Accepted 24 July 2008

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The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala²⁹⁹-to-Ser, Glu³⁰⁵-to-Lys, and Glu³³⁰-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr⁷²-to-Asp and Thr²⁹⁶-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

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Streptococcus suis is a world-wide pathogen of swine and an important zoonotic agent (21, 22, 24, 26, 27). Strains of this bacterium are divided into 35 serotypes (types 1/2 and 1 through 34) according to polysaccharide capsular antigens. Type 2 is the type most frequently associated with disease and also the type most often isolated. The disease conditions in swine include meningitis, arthritis, septicemia, endocarditis, encephalitis, abortions, polyserositis, and bronchopneumonia (22). S. suis has been isolated from other animal species, including horses, dogs, cats, birds, and wild boars, suggesting that it may be pathogenic for more than one animal species (22). In infected humans, S. suis type 2 can cause meningitis, endocarditis, septicemia, toxic shock, and permanent hearing loss (21, 22, 24, 26, 27). A recent outbreak in China, which involved 39 human and >200 swine deaths, has caused elevated concern (27). Attempts to effectively control S. suis infection have been problematic due to genetic heterogeneity within and between serotypes, a lack of effective vaccine and diagnostic reagents, inability to effectively distinguish virulent from nonvirulent isolates, a paucity of information on the genetics of S. suis relative to its mechanism of pathogenesis, and the evolution of multiple-drug-resistant strains (6, 13, 22).

Although a few S. suis proteins that are associated with virulent strains, such as the muramidase released protein (MRP), capsule (CPS), hemolysin (suilysin [SLY]), and extracellular protein factor (EPF), have been identified, evidence has shown that factors other than these proteins apparently are also involved in the pathogenic mechanism (1, 3, 9, 19, 20, 22, 25).

Three levels of virulence have been shown to exist among the strains belonging to S. suis serotype 2: high virulence, moderate virulence, and no virulence (22, 23). Highly virulent strains produce gross pathological findings and moderate to severe clinical signs in naturally infected animals and experimental pig challenge models, resulting in the death of the animals in most cases, whereas moderately virulent strains produce minimal lesions and mild clinical signs. In comparison, nonvirulent strains produce no visible lesions or clinical signs (22, 23, 24). The lack of virulence of these strains may be related to decreased or absent expression of specific virulence factors or to changes in bacterial metabolic processes that decrease the ability of the bacteria to survive and multiply in the host. Several reports in the past decade have shown that housekeeping enzymes perform a variety of functions, including acting as putative virulence factors for pathogens (14). Other investigators have also reported that Clostridium botulinum group I strains, which are highly virulent and are responsible for most cases of human botulism, are distinguishable from group II strains, which are nonvirulent or weakly virulent, on the basis of their glutamate dehydrogenase (GDH) activity (7). In addition, an enzyme electrophoresis method has been employed in the characterization of various species of bacteria to estimate genetic relationships and structure and to distinguish strains during outbreaks for epidemiological tracing (17).

In this study, our aim is to determine whether the GDH of S. suis can be used to distinguish highly virulent from moderately virulent and nonvirulent strains. For this purpose, we obtained strains with known degrees of pathogenicity from a geographically varied collection and characterized their GDH enzymes. We observed that highly virulent strains of S. suis type 2 exhibited a different electrophoretic type (ET) on a nondenaturing gel following activity staining from those of moderately virulent and nonvirulent strains and that the ETs are due to amino acid sequence types (STs). A spectrophotometric assay of the S. suis GDH did not yield significant differences in the rate of substrate utilization among the highly virulent, moderately virulent, and nonvirulent strains.

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Bacterial strains and media. Nineteen clinical isolates of S. suis serotype 2 originated from various regions of the United States, England, and Canada. Two of the strains (700794, from swine, and 700796, from human brain) were purchased from the American Type Culture Collection (Manassas, VA). The DNAs of two strains (05ZYH33 and 98HAH12) involved in recent Chinese human outbreaks (4) were obtained from Jiaqi Tang, Hua Dong Research Institute of Medical Biotechnics, Nanjing, China. Other than the Wisconsin strains, which we isolated from naturally infected pigs, the remaining isolates were obtained from M. M. Chengappa, Kansas State University, Manhattan. The strains were selected based on their clinical outcomes in an experimental pig challenge model, (4, 23). Todd-Hewitt broth supplemented with 0.6% yeast extract (Difco Laboratories, Detroit, MI) and sheep blood agar plates (Remel, Lenexa, KS) were used to grow the S. suis strains. Recombinant plasmids in Escherichia coli cells were grown on Trypticase soy agar or in Luria-Bertani broth containing 60 µg/ml of ampicillin. All cultures were grown at 37°C. Plasmids used in this study are listed in Table 1.

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TABLE 1. Plasmids used in this study

Chemicals and enzymes. Molecular-grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or from Fisher Scientific Co. (Pittsburgh, PA). Restriction enzymes and T4 DNA ligase were purchased from Promega (Madison, WI) or from Fermentas Inc. (Hanover, MD) and were used as recommended by the manufacturer.

Virulence studies with pigs. Piglets (age, 3 weeks; average weight, 16 lb) were obtained from a herd with no S. suis history and were grouped into independent units (7 pigs per group) 1 week prior to challenge. They were then challenged intravenously with 1 ml of phosphate-buffered saline containing 2 x 10⁶ CFU overnight cultures of S. suis strain 700794, TC, 700796, 06-17072, 06-05622, or 06-18395. Strain 1933 is a highly virulent strain of S. suis serotype 2 routinely used in our laboratory for research and was used as a positive control. The negative-control group received phosphate-buffered saline under the same conditions. Pigs were monitored twice daily for 7 days at 12-h intervals postchallenge for clinical signs of disease (2, 23). Gross pathological conditions were recorded for dead pigs. Finally, surviving pigs were sacrificed, and their gross pathological conditions were also recorded. Data were compared, and mean clinical sign scores per isolate were used to determine virulence as described elsewhere (23).

GDH activity staining. Cultures of wild-type S. suis strains and E. coli carrying recombinant gdh were grown overnight in 10 ml of broth at 37°C. The cultures were then centrifuged at 5,000 x g for 10 min at 4°C to pellet the cells. Activity staining on a nondenaturing gel was performed as previously described (11).

DNA extraction. Genomic DNA was extracted using the Bio-Rad (Hercules, CA) AquaPure genomic DNA kit for gram-positive bacteria. DNA concentration and purity were determined spectrophotometrically (Genesys 2; Thermo Spectronic) at 260 and 280 nm, respectively, using a 1:200 dilution of each sample.

Primers, PCR amplification, and amplicon purification. Oligonucleotide primers were designed by using previously published gdh nucleotide sequence data (GenBank accession number AF229683) (11). The primers and their sequences are listed in Table 2. GD21 and GD25 were designed to amplify a 1,820-bp fragment containing the gdh gene and its flanking sequences. Both primers and additional internal primers (GD22, GD23, GD24, GD26, GD27, and GD28) were used either to sequence the gdh gene or for subcloning. The primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Amplification reactions were performed as previously described (10). The products were purified after gel electrophoresis by using the QIAquick gel extraction kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions or by electroelution (16). The purified DNA was stored at –20°C until use.

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TABLE 2. Primers used in this study

Presence of suilysin. The strains were screened for the presence of the suilysin gene (sly) essentially as described previously (12).

Sequencing and analysis. The purified gdh DNA fragment of each strain used in this study was sequenced with primers GD21, GD22, GD23, GD24, GD25, GD26, GD27, and GD28. The nucleotide sequences were assembled, and multiple sequence alignment of the nucleotide and amino acid sequences and cluster analysis were performed using Vector NTI AlignX (Invitrogen, Carlsbad, CA).

Construction and screening of clones for gene exchange. Plasmid pOT411, carrying the gdh gene of S. suis strain 1933, a highly virulent isolate, has been described previously (11). The gdh gene of S. suis strain AAH6, a moderately virulent strain (23), was amplified with primers GD21 and GD25. The resulting 1,820-bp amplicon was purified and digested with DraI to produce a 1,547-bp fragment. The fragment was purified and cloned into the SmaI site of pUC19 to create pRW101. Plasmid pOT411 was digested with KpnI and XbaI, while pRW101 was digested with KpnI and BamHI to release the 1,547-bp gdh gene. The 1,547-bp fragment was then digested with TauI and BanI to release a 173-bp region spanning the region of the missense amino acid residues within the GDH open reading frames (ORFs) of strains 1933 and AAH6. The 173-bp fragment from pOT411 was exchanged with that from pRW101 to yield pRW106, and the 173-bp fragment from pRW101 was exchanged with that from pOT411 to yield pRW105.

Construction and screening of clones for GDH activity. Plasmids pRW104 and pRW114, carrying the GDH enzymes from strains AAH6 and ATCC 700796, respectively, were constructed to represent two of the three GDH genotypes. For pRW104, plasmid pRW101 was digested with KpnI and BamHI to release the gdh gene. The gdh gene fragment was purified and ligated into the KpnI and BglII site of the pBAD/Myc-His version B overexpression vector. For pRW114, the gdh gene of S. suis ATCC 700796 was amplified with primers GD21 and GD25, and the resulting 1,820-bp amplicon was purified and digested with DraI. A 1,547-bp product of the digest was purified and cloned into the SmaI site of pUC19 to create pRW103. pRW103 was then digested with KpnI and XbaI, and the fragment containing the gdh gene was ligated into the KpnI and XbaI site of the pBAD/Myc-His version B vector and transformed into competent E. coli TOP10 cells.

Overexpression of gdh and spectrophotometric activity assay. E. coli cells carrying plasmids pOT411, pRW104, and pRW114 were overexpressed with arabinose at a final concentration of 0.02% (11). The cell pellets were suspended in 500 µl of 50 mM Tris buffer (pH 8.0) and disrupted by sonication at a setting of 2.5, using six 10-s bursts with 10-s cooling intervals (Sonic Dismembrator, model F60; Fisher Scientific). Following sonication, samples were centrifuged at 14,000 rpm for 5 min at 4°C, and supernatants were placed on ice. The concentrations of the cell extracts (CFE) were determined using the bovine serum albumin standard curve, and the proteins were standardized to the concentration of the CFE of the sample with the lowest concentration. Coomassie stains of the CFE following sodium dodecyl sulfate-polyacrylamide gel electrophoresis were also used to verify concentrations. The reaction mixture for the GDH activity assay was prepared as previously described (11) and filtered through a 0.2-µm-pore-size filter. Approximately 67.5 µg of the CFE was added to 1 ml of the reaction mixture, and the GDH activity was monitored spectrophotometrically by measuring the change in absorbance at 540 nm for 30 min, with values recorded at 1-min intervals. A control run, in which L-glutamate was omitted in the reaction mixture, was also performed. Data were plotted using Microsoft Office Excel.

Nucleotide sequence accession numbers. The GenBank accession numbers for the genes encoding the GDH proteins of strains AAH6 and ATCC 700796 are EF198475 and EF198476, respectively. The accession number for the GDH of strain 1933 (AF229683) was reported previously (11).

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Virulence studies with pigs. The virulence of the S. suis strains was determined using the average clinical sign score per day per animal (23; also data not shown). In pigs, strains 1933 and 700794 were highly virulent whereas 700796, 06-17072, 06-05622, and 06-18395 were moderately virulent. Four pigs inoculated with strain 1933 died 48 to 72 h postinoculation, and an additional three pigs with severe clinical signs were euthanized. Pigs inoculated with strain 70094 developed severe clinical signs in the same time frame, resulting in the death of two pigs and the euthanasia of five. Strains 700796, 06-17072, 06-05622, and 06-18395 induced mild clinical signs. The degrees of virulence of the remaining strains used in this study have been reported previously (4, 23) (Table 3).

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TABLE 3. Summary of the GDH proteins of the 21 S. suis serotype 2 strains used in this study

Division of strains into two groups. The strains examined were found to contain two ETs forming two ET groups, I and II. ETI consisted of seven of the seven (100%) highly virulent strains tested by the GDH activity assay (Fig. 1, lanes 1, 3, 5, 6, 8, 10, 12 [upper migrating bands]; Table 3). ETII consisted of 12 strains comprising moderately virulent and nonvirulent isolates (Fig. 1, lanes 2, 4, 9, 11, 13, and 15 [lower migrating bands]; Table 3). The ETs of two strains (strains 05ZYH33 and 98HAH12) were not determined because of difficulty in obtaining the live bacteria from China (4).

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FIG. 1. Example of the S. suis serotype 2 GDH showing the ETs. Highly virulent strains are in lanes 1, 3, 5, 6, 8, 10, and 12. Lanes 1 and 8, strain 1933; lane 2, 700796; lane 3, D930; lane 4, 95-16426; lane 5, strain TC; lane 6, 95-16753; lanes 7 and 14, E. coli DH5(pOT410) recombinant expressing the GDH of S. suis type 2, strain 1933; lane 9, 94-623; lane 10, 3977B; lane 11, 89-999; lane 12, 95-8506; lane 13, AAH6; lane 15, 06-05622-1.

Examination of the strains for the presence of the suilysin gene (sly) revealed that the gene is present only in the highly virulent strains and is absent from the moderately virulent and nonvirulent strains (Table 3), thus correlating with the GDH ETs.

Molecular basis for the GDH ETs. The DNAs of all 19 S. suis strains tested by GDH activity staining, including the two Chinese strains, produced PCR products of 1,820 bp. Analysis of the nucleotide sequences of the 1,820-bp amplicons revealed that they contained an ORF of 1,344 bp with a deduced amino acid sequence of 448 residues (Table 3). The polypeptides have similar calculated molecular masses and yielded similar isoelectric points (pI) (data not shown), indicating an absence of deletions or insertions within the GDH. Analysis of the nucleotide and protein sequences revealed extensive homology among the isolates. Several base pair changes that resulted in silent mutations and a few changes that resulted in missense mutations were noted (data not shown). The missense mutations occured at nucleotide positions 214, 886, 895, 913, and 988, leading to Tyr⁷²-to-Asp, Thr²⁹⁶-to-Ala, Ala²⁹⁹-to-Ser, Glu³⁰⁵-to-Lys, and Glu³³⁰-to-Lys substitutions on the polypeptide chain (Fig. 2). Cluster analysis of the deduced amino acid sequences grouped the isolates into three groups (groups I, II, and III) based on the amino acid STs (Fig. 3). Group I consisted of the nine highly virulent strains containing the Ala²⁹⁹-to-Ser, Glu³⁰⁵-to-Lys, and Glu³³⁰-to-Lys substitutions (Fig. 2). Groups II and III consisted of the moderately virulent and nonvirulent strains, and they are separated from each other by Tyr⁷²-to-Asp and Thr²⁹⁶-to-Ala substitutions.

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FIG. 2. Alignment of the regions of amino acid substitutions in the deduced amino acid sequences of GDH showing the STs. ETI contains A, S, K, and K at positions 296, 299, 305, and 330, respectively, whereas ETIII contains T, A, E, and E, respectively, at these positions. ETII differs from ETIII by having A at position 296. Identical amino acid residues are shown on a shaded ground, and missense mutations (at positions 296, 299, 305, and 330) are on a white ground.

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FIG. 3. Cluster analysis of the deduced amino acid sequences of GDH. Strains in group I are highly virulent and produced a high band pattern (ETI) and an ST different from those of group II and III strains, which produced ETII (Fig. 1 and 2).

Gene exchange. To determine whether the missense mutations are responsible for the ETs, gene exchange studies were performed using a 173-bp TauI-and-BanI DNA region encoding the Ala²⁹⁹-to-Ser, Glu³⁰⁵-to-Lys, and Glu³³⁰-to-Lys substitutions (Fig. 2). The highly virulent wild-type isolate (strain 1933) and its gdh clone (pOT411) yielded an upper migrating band, as expected (Fig. 4, lanes 1 and 2). When the 173-bp TauI-and-BanI fragment of pOT411 was deleted and replaced with the TauI-and-BanI gdh fragment from strain AAH6, the ET changed to a lower band (Fig. 4, lane 3). Also as expected, the moderately virulent wild-type isolate (strain AAH6) and its gdh clone (pRW104) yielded lower migrating bands. When the 173-bp TauI-and-BanI gdh fragment of strain AAH6 was replaced with that of strain 1933, the ET changed to an upper band (Fig. 4, lanes 4, 5, and 6). These results demonstrated that Ala²⁹⁹-to-Ser, Glu³⁰⁵-to-Lys, and Glu³³⁰-to-Lys substitutions—and not deletions, insertions, or processing uniqueness—account for the ETs visualized on the activity staining gel.

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FIG. 4. ETs of GDH after the exchange of the 173-bp gdh fragment between a highly virulent isolate (strain 1933) and a moderately virulent isolate (strain AAH6). Activity staining was performed as described in Materials and Methods. Lanes: 1, wild type S. suis 1933; 2, pOT411 carrying the strain 1933 gdh gene; 3, pRW106, a derivative of pOT411 in which the 173-bp gdh fragment was replaced with that from S. suis strain AAH6; 4, wild-type S. suis AAH6; 5, pRW104, carrying the strain AAH6 gdh gene; 6, pRW105, a derivative of pRW104 in which the 173-bp gdh fragment was replaced with that from S. suis strain 1933.

Analysis of activity of recombinant GDH. We used spectrophotometric measurement to determine if there are differences in the rate of substrate utilization among the recombinant GDH clones from the three virulent S. suis type 2 genotypes. The standardized CFE used for the assay yielded similar GDH concentrations (Fig. 5a). The results showed that plasmid pOT411, expressing the GDH of a highly virulent isolate (strain 1933), had a slightly higher activity than pRW104 and pRW114, which express GDH proteins from two moderately virulent isolates (strains AAH6 and 700796, respectively). The pBAD/Myc-His version B plasmid cloning vector without a gdh insert (negative control) did not yield any activity, indicating that the observed results were due to the S. suis GDH (Fig. 5b). There was no activity in the reaction mixture when L-glutamate was omitted (data not shown).

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FIG. 5. (a) Coomassie-stained 12% polyacrylamide gel showing recombinant GDH protein after induction with 0.02% arabinose. Each lane contains approximately 67.5 µg of CFE. Lane M, Rainbow molecular size marker; lane 1, CFE of transformants of E. coli TOP10 in the empty pBAD/Myc-His version B overexpression vector (negative control); lane 2, pOT411; lane 3, pRW104; lane 4, pRW114 (Table 1). (b) Spectrophotometric measurement at 540 nm of the rate of substrate utilization by S. suis GDH. The assay used 67.5 µg of the CFE of recombinant clones. pOT411 represents the group I GDH genotype; pRW104 represents the group III GDH genotype; and pRW114 represents the group II GDH genotype. The pBAD/Myc-His version B cloning plasmid vector was used as a negative control.

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Three levels of virulence (high virulence, moderate virulence, and no virulence) have been shown to exist among strains of S. suis serotype 2 (22, 23). We chose the serotype 2 strains used in this study because their degrees of virulence have been verified in an experimental pig challenge model (4, 23; this study). Because the highly virulent group I Clostridium botulinum strains, responsible for most cases of human botulism, are distinguishable from group II strains, which are nonvirulent or weakly virulent, on the basis of their GDH activities (7), we were interested in determining if a GDH-based analysis could yield results that might be useful in distinguishing highly virulent from moderately virulent and nonvirulent strains of S. suis type 2, thus providing an additional tool for virulence phenotype determination. We found a relationship between the ETs and virulence phenotypes (Fig. 1; Table 3). This result is consistent with our preliminary observations, in which only two highly virulent and four moderately virulent and nonvirulent strains were tested (11).

Differences in protein size could result from insertion, deletion, or processing uniqueness. We investigated the molecular basis for the different ETs of highly virulent, moderately virulent, and nonvirulent strains by sequencing the gdh genes of all strains and analyzing the deduced amino acid sequence data. Because a majority of the strains used in this study came from North America, it was important to include strains with known degrees of pathogenicity from distant sources. Thus, we obtained the whole-genome DNAs of strains 05ZYH33 and 98HAH12 from China. Both strains were highly virulent in experimental pig challenge studies and caused recent Chinese human and swine outbreaks (4). We noted that highly virulent strains (ETI), regardless of geographic origin, shared identical amino acid sequences that were different from those of moderately virulent and nonvirulent strains (ETII) and that amino acid substitutions account for the differences in the migration pattern, as evidenced by gene exchange studies (Fig. 2, 3, and 4). Thus, the substitutions may have induced some conformational changes in the protein molecule.

S. suis encodes a hemolysin known as SLY. SLY is a cytotoxic membrane-damaging protein that belongs to the family of thiol-activated hemolysins (8). These proteins have been shown to be involved in the virulence of other bacteria, including S. suis (1, 5, 9). Other S. suis type 2 proteins, such as CPS, MRP, and EPF, are also considered to be virulence markers (3, 15, 18, 19). Reportedly, one genotype (cps2 mrp⁺ epf⁺ sly⁺) is associated with highly invasive S. suis strains, while isolates with reduced virulence are associated with cps2 mrp⁺ Epf^– Sly^– (18). Based on our data, all nine highly virulent strains possess sly, whereas the moderately virulent and nonvirulent strains lack this gene. Therefore, if one assumes that the associations of the GDH protein sequence and ETs and of sly with the virulence phenotype are not coincidental, it is safe to infer that S. suis strains with such a genetic background can be highly virulent in pigs. Thus, the GDH ETs and amino acid STs may serve as additional virulence markers to distinguish highly invasive S. suis strains. However, since a small number of strains with known virulence status in pigs were used, a more comprehensive approach would strengthen our observations when more strains with defined virulence phenotypes become available.

CFE of proteolytic strains of Clostridium botulinum types A, B, and F (group I), responsible for most cases of human botulism, reportedly have unusually high specific activities of NAD⁺-dependent L-glutamate dehydrogenase compared to nonproteolytic strains of types B, E, and F (group II), suggesting that this enzyme may have important physiological roles in group I proteolytic strains (7). For this reason, we were interested in determining whether the amino acid substitutions may lead to differences in the activity of S. suis GDH between highly virulent and nonvirulent strains, thus providing indirect evidence of a unique physiological role. Attempts to use CFE of wild-type S. suis strains for the spectrophotometric activity assay gave ambiguous results (data not shown). Also, the activity of the purified recombinant GDH with a C-terminal histidine tag was lost (data not shown), indicating a possible conformational change that affected the active site. Due to these results, CFE of clones (Fig. 5a) were used for the assay. Although the plot of the spectrophotometric activity assay (Fig. 5b) appears to suggest that the highly virulent strain has higher activity, the differences between time intervals are not significant, making it difficult to reach a conclusion at this time.

In conclusion, highly virulent strains of S. suis serotype 2 can be distinguished from moderately virulent and nonvirulent strains on the basis of their GDH ETs and STs. We also conclude that amino acid substitutions are the basis for the ETs. We have recently constructed a mutant S. suis type 2 strain lacking GDH in order to determine whether this protein has a physiological role that contributes to increased virulence.

 
We thank Michael O'Connor, John Pharo, Carman Ames, Kelly Homb, Stephanie Kutz, and Suzanne Burgener for their assistance.

 
* Corresponding author. Mailing address: University of Wisconsin, Wisconsin Veterinary Diagnostic Laboratory, 445 Easterday Lane, Madison, WI 53706. Phone: (608) 262-4168. Fax: (847) 574-8085. E-mail: ogi.okwumabua{at}wvdl.wisc.edu

Published ahead of print on 6 August 2008.

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Journal of Clinical Microbiology, October 2008, p. 3201-3207, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.02309-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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