MAJOR ARTICLE

The Contribution of Suilysin to the Pathogenesis of Streptococcus suis Meningitis Dan Takeuchi,1 Yukihiro Akeda,1 Tatsuya Nakayama,1 Anusak Kerdsin,4 Yasuteru Sano,2 Takashi Kanda,2 Shigeyuki Hamada,5 Surang Dejsirilert,4 and Kazunori Oishi1,3 1 Laboratory for Clinical Research on Infectious Diseases, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 2Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, and 3Infectious Disease Surveillance Center, National Institute of Infectious Disease, Tokyo, Japan; 4Department of Medical Sciences, Ministry of Public Health, National Institute of Health, and 5Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections, Nonthaburi, Thailand

Keywords.

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Background. Streptococcus suis is an emerging zoonotic pathogen, and causes sepsis and meningitis in humans. Although sequence type (ST) 1 and ST104 strains are capable of causing sepsis, ST1 strains commonly cause meningitis. In this study, we investigated the role of suilysin, a member of cholesterol-dependent cytolysins, in differential pathogenicity between ST1 and ST104 strains. Methods. The levels of transcription and translation of the sly gene and messenger RNA of both ST strains were compared by means of quantitative polymerase chain reaction and Western blotting. Survival rates and bacterial densities in brain were compared between mice infected with wild-type and sly-knockout ST1 strain. ST104 infections with or without complementation of suilysin were also assessed. Results. The amounts of suilysin produced by ST1 strains were much higher than those produced by ST104 strains. Lower production of suilysin by ST104 strains were attributed to the attenuated sly gene expression, which seemed to be associated with 2 nucleotide insertions in sly promoter region. Furthermore, suilysin contributed to the higher bacterial density and enhanced inflammation in brain and increased mortality. Conclusions. Our data may explain why ST1 strains, but not ST104 strains, commonly cause meningitis and also suggest the contribution of suilysin to the pathogenesis of meningitis in humans. Streptococcus suis; meningitis; sequence type (ST) 1; ST104; suilysin.

Streptococcus suis is a gram-positive facultative anaerobic coccus known to cause swine infections and is also recognized as an agent of zoonosis. Human infection occurs when persons eat raw pork products and/or are closely in contact with pigs or raw pork products. Once S. suis invades the blood stream and causes sepsis or bacteremia, the pathogen usually develops purulent meningitis and causes severe neurological sequelae, including hearing loss [1, 2]. Although 35 serotypes have been identified on the basis of capsular polysaccharides [3], serotype 2 is the most frequently isolated from cases of human infection globally.

Received 26 July 2013; accepted 13 November 2013; electronically published 27 November 2013. Correspondence: Dan Takeuchi, MD, PhD, 3-1 Yamadaoka, Suita, Osaka, Japan, 565-0871 ([email protected]). The Journal of Infectious Diseases 2014;209:1509–19 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected] DOI: 10.1093/infdis/jit661

In our previous study in Thailand, >90% of S. suis clinical isolates were serotype 2 strains and were divided into 2 major sequence types (STs) by multilocus sequence typing; ST1, which is globally prevalent, and ST104, which is specific to Thailand. Interestingly, both ST1 and ST104 strains cause sepsis, but ST1 strains significantly more frequently cause meningitis [1]. We investigated the bacteriological characteristics of the ST1 and ST104 strains to determine the virulence factor responsible for the different clinical outcomes. Among the virulence factors of S. suis [4], suilysin is a well-known extracellular protein with a molecular weight of about 54 kDa. Suilysin belongs to the cholesteroldependent cytolysin family, exhibiting cytotoxicity to epithelial cells, endothelial cells, neutrophils, and macrophages [5–8], and antiphagocytic and antibactericidal properties in response to neutrophils and monocytes/ macrophages [4, 5, 9]. The cholesterol-dependent cytolysins include several hemolysins, such as pneumolysin, perfringolysin, listeriolysin O, and streptolysin O, Suilysin Promotes S. suis Meningitis



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produced by Streptococcus pneumoniae, Clostridium perfringens, Listeria monocytogenes, and Streptococcus pyogenes, respectively. These proteins have been shown to contribute to the pathogenesis of the diseases caused by each pathogen [10–12]. In this study, we found differential transcriptional levels of suilysin in ST1 and ST104 strains, and these differences resulted in different in vitro pathogenicity. Moreover, suilysin also contributed to the pathogenesis of meningitis in a mouse model of infection and may explain the different clinical outcomes resulting from human infection by ST1 and ST104 strains.

cultured in Luria-Bertani broth (Sigma) and used for routine DNA manipulation, negative control of in vitro assays, and the production of recombinant suilysin. Antibiotics were added to the medium at the following final concentrations: 4 µg/mL of chloramphenicol, 125 µg/mL of spectinomycin (in the reporter assay), and 100 µg/mL of spectinomycin (in the construction of sly-knockout strains) for S. suis, 50 µg/mL of kanamycin for E. coli. To measure bacterial concentrations, sheep blood agar was used for S. suis and Luria-Bertani agar for E. coli. Hemolysis Assays

METHODS Bacterial Strains and Growth Conditions

Table 1.

Bacterial Strains and Plasmids Used in Current Study

Strain or Plasmid

Genotype or Relevant Features

Streptococcus suis P1/7 Serotype 2, ST1, reference strain

Source or Reference Jacobs et al, 1996 [13]

26154 24828

Serotype 2, ST1 Serotype 2, ST1

Clinical isolate, Thailand Clinical isolate, Thailand

25018

Serotype 2, ST1

Clinical isolate, Thailand

24651 25016

Serotype 2, ST1 Serotype 2, ST104

Clinical isolate, Thailand Clinical isolate, Thailand

22713

Serotype 2, ST104

Clinical isolate, Thailand

21927 24525

Serotype 2, ST104 Serotype 2, ST104

Clinical isolate, Thailand Clinical isolate, Thailand

Escherichia coli Laboratory stock F−, Φ80lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rk−, mk+), phoA, supE44, λ −, thi-1, gyrA96, relA1, deoR Laboratory stock BL21(DE3) E. coli B, F−, dcm, ompT, hsdS (rb− mb−), gal, λ(DE3)

DH5α

Plasmids pET-28a(+) Overexpression vector for polyhistidine-tagged proteins, Kmr pBSU100 pUCori, pAMb1 ori, egfp, Specr pSET4s

Novagen

Aymanns et al [15]

Thermosensitive suicide Takamatsu et al [16] vector, Tsori, pUCori, lacZ’, Specr

Abbreviation: ST, sequence type.

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Production of Recombinant Suilysin protein and Anti-suilysin Rabbit Serum

The primer sets of RS1 and RS2 (Supplementary Table 1) were designed for amplification and cloning of the sly gene of P1/7 strain in expression vector pET-28a(+) (Novagen). The following procedures were performed as described elsewhere [9], with some modifications. Purification procedure of polyhistidinetagged suilysin followed the manufacture’s instruction of Ni-NTA Agarose (Qiagen). The production of recombinant suilysin was confirmed by Western blotting using mouse anti-polyhistidine tag antibody (Sigma). A female rabbit (New Zealand White, 8 weeks old) was immunized with subcutaneous injection of 100 µg of recombinant suilysin once a week for 4 weeks to produce anti-suilysin rabbit serum. The suilysin-binding ability of the collected serum was confirmed by Western blotting to detect recombinant suilysin and secreted suilysin in the culture supernatant of P1/7 strain. Cytotoxicity and Adhesion of S. suis to Human Brain Microvascular Endothelial Cells

Immortalized human brain microvascular endothelial cells with SV40 large T antigen (TY09 cells) were used. The culture method for the cells followed procedures reported elsewhere [17, 18]. TY09 cells were inoculated with ST1 or ST104 strains at a multiplicity of infection of 500 and were assayed for cytotoxicity using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega), according to the manufacturer’s instructions. To inhibit cytotoxicity, 20-fold-diluted anti-suilysin rabbit serum was simultaneously added to the cells with the bacterial inoculation. Rabbit anti-VPA1027 (against the putative protein of Vibrio parahaemolyticus) serum was used as a negative control. To measure the adhesion to the endothelial cells, TY09 cells were inoculated with each strain at a multiplicity of infection of 5 so as not to cause cytolysis and incubated

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All S. suis strains used in this study were serotype 2 and clinical isolates other than P1/7 [13] were from cases of human infection in Thailand (Table 1). Each strain was cultured in ToddHewitt broth with yeast extract (Becton Dickinson) at 37°C with 5% carbon dioxide (CO2). For the preparation of competent cells of S. suis, we followed methods reported elsewhere [14]. Escherichia coli DH5α and BL21 (DE3) strains were

ST1 and ST104 strains were cultured overnight and adjusted to an optical density at 600 nm (OD600) of 0.9–1.0. The filtered culture supernatants were mixed with 100 µL of a 10-folddiluted sheep red blood cells and incubated for 1 hour at 37°C with 5% CO2. After brief centrifugation, the OD570 of each supernatant was measured using a microplate reader SH-9000 (Corona Electric).

for 2 hours at 37°C with 5% CO2. After incubation, cells were washed 3 times with phosphate-buffered saline and collected along with supernatants of reaction wells for the measurement of bacterial concentrations. Comparison of the sly Gene Sequences and Its Promoter Region in ST1 and ST104 Strains

To compare the sly gene and its promoter regions, DNAs were extracted from ST1 and ST104 clinical isolates. Polymerase chain reaction (PCR) amplification was performed with the primer sets (RS1, RS2, SP1, and SP2) described in Supplementary Table 1. The sequences of the PCR products were confirmed using an ABI Prism 3100 Genetic Analyzer. Levels of Transcription of the sly Gene and Translation of sly Messenger RNA in ST1 and ST104 Strains

Reporter Assay for the ST1 and ST104 Strain sly Promoter Region

Approximately 500 base pairs of the region flanking the 5′ side of the sly gene was amplified by PCR using genomic DNA of ST1 or ST104 strain. The primer sets used (RA1 and RA2) are described in Supplementary Table 1. The vector pBSU100 carrying egfp gene was provided by Barbara Spellerberg, PhD (Institute of Medical Microbiology and Hygiene, University of Ulm, Germany) [15]. The PCR products were inserted into the plasmid to construct reporter plasmid, which produce enhanced green fluorescent protein (EGFP) fused with the first 7 amino acids of suilysin. The ST104 (21927) strain was transformed with the reporter plasmid and was cultured for 17 hours, and fluorescence of enhanced green fluorescent protein was measured using a microplate reader set with the excitation wavelength at 489 nm and emission wavelength at 508 nm. The ST104 strain was transformed with pBSU100 as a negative control. The transformed strains showed normal growth rates. Construction of the sly-Knockout ST1 Strain

This strain was constructed as described elsewhere [16]. The plasmid, pSET4s, was provided by Daisuke Takamatsu, DVM,

Mouse Model of S. suis Infection

SPF 7-week-old female A/J mice were purchased from Japan SLC and were intraperitoneally inoculated with each strain. To compare the survival rate after ST1 (25018) and ST104 (21927) infection, we inoculated the mice with 1 × 108 colony-forming units (CFU) of each ST strain. To compare bacterial densities in blood and brains of mice infected with ST1 and ST104 strains, we inoculated mice with 5 × 107 CFU of the ST1 strain or 5 × 108 CFU of the ST104 strain. To examine the contribution of suilysin to the clinical manifestations, survival, bacterial densities in blood and brains, and histopathological changes in brains of mice infected with the ST1 strain, we inoculated mice with 1 × 108 CFU of wild-type ST1 strain or the corresponding slyknockout ST1 strain. To examine the contribution of suilysin to those of mice infected withST104 strain, we inoculated mice with 5 × 108 CFU of ST104 strain followed by 5 × 105 CFU of the wild-type ST1 strain to complement suilysin or 5 × 105 CFU of the corresponding sly-knockout ST1 strain as a control. Mice survival was observed for 9 days. Bacterial densities in blood and brains were measured at 24 and 72 hours after infection with the detection limits of 10 CFU/mL or CFU/g. To measure the bacterial densities of only the ST104 strain in the mixed samples with the ST1 strain, sheep blood agar supplemented with 0.3 µg/mL of erythromycin was prepared, because only the ST104 strain, not the ST1 strain, showed resistance to erythromycin. All animal experiments were performed in accordance with institutional guidelines for the Osaka University animal facility. Histopathological Studies

Brain samples were taken at 24 and 72 hours after infection from the models of ST1 infection and ST104 coinfection. Other procedures were performed as described elsewhere [20]. Statistical Analysis

All data were analyzed using GraphPad Prism software (version 5 for Windows) and SPSS software (version 15.0 J for Windows). Each experiment was repeated ≥3 times. Dunnett tests were used to compare of hemolytic activity, cell adhesion, and transcriptional levels of the sly gene; Tukey tests for cytotoxicity; and Student t tests for inhibition and reporter assays. In all mouse experiments, Kaplan-Meier estimator method was used Suilysin Promotes S. suis Meningitis



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ST1 and ST104 strains were grown to a midexponential phase (OD600, 0.3), because the transcription and translation of the sly gene and messenger RNA (mRNA) is known to occur in the growth phase [19]. RNAs were extracted from cultured cells using an RNeasy Mini Kit (Qiagen), and complementary DNAs were synthesized using SuperScript III Reverse Transcriptase (Invitrogen). The CT (Cycle threshold) method of quantitative PCR was performed using a 7900HT Fast Real Time PCR System (Applied Biosystems) to measure the relative quantity of sly mRNA for each strain, using the result for strain 22713 (ST104) as a control value. The primer sets used (16s1, 16s2, SU1, and SU2) are described in Supplementary Table 1. The levels of translation of sly mRNA were compared by means of Western blotting using anti-suilysin serum.

PhD (Molecular Bacteriology Section, National Institute of Animal Health, Tsukuba, Ibaraki prefecture, Japan). The primer sets used for the construction of the insert (SK1, SK2, SK3, SK4, CH1, and CH2) are described in Supplementary Table 1. The constructed recombinant plasmid was used for the electrotransformation of the ST1 strain (25018). A lack of suilysin expression in the culture medium was confirmed by Western blotting with anti-suilysin rabbit serum and hemolytic assays. The sly-knockout strain showed a normal growth rate.

to draw survival curves and a log-rank test was used to compare survival rates. Mann–Whitney U tests were used to compare bacterial densities in blood and brain samples. RESULTS Hemolytic Activities

26154 strain (ST1) demonstrated the highest hemolytic activity of all 7 strains, and the other 3 ST1 strains exhibited high activities of >50% hemolysis. By contrast, the hemolytic activities of all ST104 strains were 50% cytotoxicity, that of the 3 ST104 strains was negligible. The cytotoxicity of ST1 strains was significantly higher than that of ST104 strains (P < .05; Figure 1B). The cytotoxicity of all ST1 strains was significantly inhibited by anti-suilysin rabbit serum (P < .05; Figure 1D). On the other hand, no difference was found in the rates of cell adhesion between the ST1 and ST104 strains, although that of 21927 strain (ST104) was significantly higher than that of the other ST1 and ST104 strains (P < .05; Figure 1C). Similar adhesion was observed in the presence of 20-fold-diluted anti-suilysin rabbit serum for the external inhibition of cytotoxicity of suilysin (data not shown).

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Figure 1. In vitro assays of sequence type (ST) 1 and ST104 strains. A, Comparison of the hemolytic activities of culture supernatants from ST1 and ST104 clinical isolates. A mixture of red blood cells and Todd Hewitt Yeast (THY) broth was used as a blank control and complete hemolysis was achieved by adding 1% Triton X-100 to the mixture. Hemolytic activity was defined as 100 × (OD570 of the reaction mixture − OD570 of the blank)/(OD570 of complete hemolysis − OD570 of the blank), where OD570 indicates optical density at 570 nm. Data are compared using the Dunnett test. B, Comparison of the cytotoxicity of ST1 and ST104 clinical isolates for human brain microvascular endothelial cells. Data are compared using the Tukey test. C, Comparison of the cell adhesion characteristics of ST1 and ST104 clinical isolates to human brain microvascular endothelial cells. The percentage adhesion is defined as 100 × (No. of bacteria adherent to cells)/(total No. of bacteria in reaction wells). Data are expressed as percentage adhesion relative to one of the results of the 25018 strain and are compared using the Dunnett test. D, Inhibition of the cytotoxicity of ST1 strains for human brain microvascular endothelial cells using anti-suilysin and control rabbit serum Student’s t test is used for statistical analysis. All data in Figure 1 are expressed as means ± standard deviations. *P < .05. 1512



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Differential Expression of Suilysin

fluorescence intensity than that of the ST104 strain (P < .05; Figure 2D). Mouse Infection by the ST1 or ST104 Strains

The survival rate of mice inoculated with the ST104 strain was significantly higher than that of mice inoculated with the ST1 strain (Figure 3A). Bacterial densities in blood of mice inoculated with the ST104 were significantly higher than those inoculated with the ST1 strain at 24 hours after infection (Figure 3B). By contrast, the bacterial densities in the blood and brains were significantly higher in mice inoculated with the ST1 strain than in those inoculated with the ST104 strain at 72 hours after infection (P < .05; Figure 3C). Contribution of Suilysin to the Pathogenesis of the ST1 Infection Model

The survival rate of mice inoculated with the sly-knockout ST1 strain was significantly higher than that of mice inoculated with

Figure 2. Comparison of the level of transcription of the sly gene and translation of sly messenger RNA (mRNA) and sly-promoter activity. A, Transcriptional levels of the sly gene in sequence type (ST) 1 and ST104 strains. Data are expressed as means ± standard deviations and are compared using the Dunnett test. B, Protein expression of suilysin in ST1 and ST104 strains. Concentrated culture supernatants were analyzed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis followed by Coomassie G-250 staining. Suilysin in culture supernatant was detected by means of Western blotting using anti-suilysin rabbit serum. C, Nucleotide sequences from regions upstream of the sly gene from the ST1 and ST104 strains. RNA polymerase-binding site (underlined) and 2 nucleotide insertions in the ST104 strain sequence (rectangle) are shown. D, Reporter assay for sly-promoter regions of the ST1 and ST104 strains. Relative fluorescence intensity was calculated by subtracting values for the negative control from that of each sample. Abbreviation: EGFP, enhanced green fluorescent protein. Data are expressed as means ± standard deviations and are compared using Student t tests. *P < .05. Suilysin Promotes S. suis Meningitis



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Because the amino acid sequence encoded by the sly gene was confirmed as identical for ST1 and ST104 strains, we performed quantitative measurement of sly mRNA expression by these strains. The transcriptional levels of the sly gene by ST1 strains were significantly higher than those of ST104 strains (P < .05; Figure 2A). Moreover, Western blotting confirmed the higher translational levels of sly mRNA in ST1 strains compared with ST104 strains (Figure 2B). The investigation of the nucleotide sequences upstream of sly genes in ST1 and ST104 strains revealed that the sly-promoter region of both STs contains the consensus sequence 5′-TTGACA-3′ of the RNA polymerase binding site of bacteria [21–27] at 104 base pairs upstream of the start codon of the sly gene (Figure 2C). In ST104 strains, 2 nucleotide insertions were found in the region flanking the 5′ side of the consensus sequence. In a reporter assay using ST1 and ST104 sly promoter regions, the promoter region of the ST1 strain demonstrated significantly higher

the wild-type ST1 strain (P < .05; Figure 4A). Mice inoculated with the wild-type ST1 strain exhibited several neurological signs, including weakness in grabbing and paralysis of extremities, whereas mice inoculated with the sly-knockout ST1 strain exhibited no signs other than lethargy and shaggy fur. No significant difference was found in bacterial densities in blood and brains at 24 hours after infection between mice inoculated with the wild-type or sly-knockout strains (Figure 4B). At 72 hours after infection, however, the bacterial densities in blood and brains were significantly higher in mice inoculated with the wild-type ST1 strain than in mice inoculated with the slyknockout ST1 strain (P < .05; Figure 4C). Few inflammatory cells were found in the brain parenchyma or ventricles of mice inoculated with the wild-type ST1 strain at 24 hours after infection (Supplementary Figure 1B, 1E and 1H), although 1 mouse showed accumulation of inflammatory cells in the cingulate gyrus and hemorrhage in the third ventricle surrounding the choroid plexus (insets of Supplementary 1514



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Figure 1E and 1H). At 72 hours after infection, however, intense accumulation of neutrophils and monocytes/macrophages in the cerebral neocortex, meninges, and third ventricles, a hemorrhage at the corpus callosum, and dilatation of blood vessels were found in the brain tissues of mice infected with the wild-type ST1 strain (Figure 5B, 5E, 5H, and 5K ). By contrast, fewer inflammatory cells and no apparent hemorrhagic foci were found in the brain tissues of mice infected with the sly-knockout ST1 strain (Figure 5C, 5F, 5I and 5L). Contribution of Suilysin to the Pathogenesis of the ST104 Coinfection Model

The survival rates did not differ significantly between mice coinfected with the ST104 and wild-type ST1 strains and those coinfected with the ST104 and sly-knockout ST1 strains (Figure 6A). At 24 hours after infection, no significant differences were found between the bacterial densities in the blood and brains of mice from the 2 experimental groups (Figure 6B).

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Figure 3. Comparison of sequence type (ST) 1 and ST104 infection in mice. A, Survival curves for infected mice. Mice were inoculated with 1 × 108 colony-forming units (CFU) of the ST1 (circles) or ST104 (squares) strain. Curves were drawn using the Kaplan-Meier estimator method, and a log-rank test was used to compare survival rates. B, C, Bacterial concentrations in the blood and brains of mice at 24 (B) and 72 (C) hours after infection. Mice were inoculated with 5 × 107 CFU of the ST1 strain (circles) or 5 × 108 CFU of the ST104 strain (squares) to achieve similar bacterial concentrations in the blood after 24 hours. Each point represents a single sample, and solid horizontal lines denote median concentrations and interquartile ranges. Dashed lines represent detection limits, and numbers of mice infected are in parentheses. Outcomes for the 2 treatments were compared using Mann–Whitney U tests. *P < .05.

At 72 hours after infection, however, the densities were significantly higher in the brains of mice coinfected with the ST104 and wild-type ST1 strains than in those of mice coinfected with the ST104 and sly-knockout ST1 strains (P < .05; Figure 6C). Histopathological examination showed little or no accumulation of inflammatory cells or hemorrhage in brains at 72 hours after infection in mice from either experimental group (Supplementary Figure 2). DISCUSSION A difference in the hemolytic activities of culture supernatants from the ST1 and ST104 strains (Figure 1A) suggested the involvement of suilysin in their different bacteriological characteristics, because hemolytic activities of S. suis are attributed to suilysin [28]. Inhibition of the cytotoxicity of ST1 strains to the levels of ST104 strains by anti-suilysin serum confirmed the contribution of suilysin to the different in vitro characteristics

of these 2 types of ST strains (Figure 1D). Detailed comparison of the mechanisms of suilysin production in these strains revealed a negligible level of transcription of sly gene and undetectable sly promoter activities in ST104 strains (Figure 2A and 2D). These findings seem to be attributed to the 2 nucleotide insertions in the sly promoter region of the ST104 strains, because the upstream region of the consensus sequence, 5′TTGACA-3′, is considered an operator region (Figure 2C) [21– 27]. The 2 insertions may result in the reduced sly promoter activity, suilysin production, hemolytic activities, and cytotoxicity of the ST104 strains (Figures 1A and 1B, 2B, and 2D). To assess the pathogenicity of the ST1 and ST104 strains in vivo, a mouse model of infection was established (Figure 3). A significant difference in survival was found between mice inoculated with the ST1 strain and those inoculated with the ST104 strain (Figure 3A). Surprisingly, not only the ST1 strains but also the ST104 strains seemed to have central nervous system (CNS) tropism, which was suggested by the bacterial densities Suilysin Promotes S. suis Meningitis



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Figure 4. Comparison of wild-type and sly-knockout sequence type (ST) 1 infection in mice. Mice were inoculated with 1 × 108 colony-forming units (CFU) of the wild-type ST1 strain (ST1wt; circles) or 1 × 108 CFU of the sly-knockout ST1 strain (ST1ko; squares). A, Survival curves for infected mice, drawn using the Kaplan-Meier estimator method, with a log-rank test used to compare survival rates. B, C, Bacterial concentrations in the blood and brains of mice at 24 (B ) and 72 (C) hours after infection. Each point represents a single sample, and solid horizontal lines denote median concentrations and interquartile ranges. Dashed lines represent detection limits, and numbers of mice infected are in parentheses. Outcomes for the 2 treatments were compared using Mann–Whitney U tests. *P < .05.

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Figure 5. Histopathological study of brain tissue from mice infected with the wild-type or sly-knockout sequence type (ST) 1 strain at 72 hours after infection. Phosphate-buffered saline was used as a control. A, D, G, J, Brain tissue from control mice. B, E, H, K, Brain tissue from mice infected with 1 × 108 colony-forming units (CFU) of the wild-type sequence type (ST) 1 strain (ST1wt). C, F, I, L, Brain tissue from mice infected with 1 × 108 CFU of the sly-knockout ST1 strain (ST1ko). The tissue sections were stained with hematoxylin-eosin. Insets show expanded images extended from the areas marked by rectangles (magnifications, ×100 [insets, ×1000]; bars, 100 µm [insets, 20 µm]). Arrowheads show accumulation of inflammatory cells at brain surfaces (B, C), cerebral neocortex (E), cerebral falx (H), and the third ventricle (K). Asterisks show hemorrhage at the corpus callosum; cc, corpus callosum; cg, cingulate gyrus; cn, cerebral neocortex; cp, choroid plexus; hp, hippocampus; ss, subarachnoid space; tv, third ventricle. Representative images are shown for each group.

in brains at 24 hours after infection (Figure 3B) and the results of in vitro adhesion assay (Figure 1C). These findings suggest that suilysin has a minor role in the bacterial invasion into the 1516



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CNS at the early stage of infection. However, at 72 hours after infection, higher bacterial densities were found in the brains of mice infected with the ST1 strain than in those of mice infected

with the ST104 strain (Figure 3C). This finding is resonant with the findings in human diseases that both ST1 and ST104 strains can cause sepsis but ST1 strains commonly cause meningitis [1]. To assess the contribution of suilysin to the pathogenesis of ST1 infection, we employed a mouse model of infection using the wild-type and sly-knockout ST1 strains (Figure 4). A higher mortality rate and higher bacterial densities in brains at 72 hours after infection were found in mice infected with the wildtype ST1 strain, compared with sly-knockout strain (Figure 4A and 4C). Moreover, mice infected with the wild-type ST1 strain exhibited strong neuronal signs, including hyperexcitation, bending of head toward one side, walking in circles, and locomotive problems, as reported elsewhere [20]. In agreement with these clinical manifestations, intensive infiltration of inflammatory cells into the brain parenchyma and meninges and destruction of physiological structures was found in the

histopathological studies (Figure 5). Suilysin is known to resist the phagocytic and other bactericidal effects of neutrophils and monocytes/macrophages [5, 9, 29]. These properties of suilysin in CNS may contribute to the higher bacterial densities and the prolonged, exacerbated, and uncontrolled inflammation in brain tissue of mice infected with the wild-type strain. In addition, suilysin is suggested to partially contribute to the stimulation of the astrocytic Toll-like receptor 2 [30]. A role for suilysin as an activator of resident immune cells in CNS may explain the marked influx of inflammatory cells into the brain tissue. We also examined the effect of passive transfer of antisuilysin serum on the survival and bacterial densities in blood and brain of mice at 24 and 48 hours after infection with the ST1 (25018) strain (data not shown). Treatment with antisuilysin serum significantly decreased the bacterial densities in blood of infected mice, compared with those of control mice at Suilysin Promotes S. suis Meningitis



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Figure 6. Mouse model of coinfection. Mice were coinfected with 5 × 108 colony-forming units (CFU) of the sequence type (ST) 104 strain and 5 × 105 CFU of the wild-type ST1 strain (ST104 + ST1wt; circles) or 5 × 108 CFU of the ST104 strain and 5 × 105 CFU of the sly-knockout ST1 strain (ST104 + ST1ko; squares). A, Survival curves for infected mice, drawn using the Kaplan-Meier estimator method, with a log-rank test used to compare survival rates. B, Bacterial concentrations in the blood and brains of mice at 24 and 72 hours after infection. Each point represents a single sample, and solid horizontal lines denote median concentrations and interquartile range. Dashed lines represent detection limits, and numbers of mice infected are in parentheses. Outcomes for the 2 treatments were compared using Mann–Whitney U tests. Abbreviations: Δsly, sly-knockout; wt, wild type. *P < .05.

Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes Acknowledgments. The plasmid pSET4s was kindly provided by Daisuke Takamatsu, DVM, PhD, National Institute of Animal Health, Japan. The plasmid pBSU100 was kindly provided by Barbara Spellerberg, PhD, Institute of Medical Microbiology and Hygiene, University of Ulm, Germany. We are also grateful to K. Ichida, M. Takahashi, and M. Tsujimoto, Laboratory Diagnostic Service Division, Research Foundation of Microbial Diseases, Osaka University, for their technical assistance and comments on the histological material. Financial support. This work was supported by research grants from the Department of Medical Sciences, Ministry of Public Health of Thailand; from Japan Society for the Promotion of Science (grant 21406027); and the

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program of Research Centers for Emerging and Reemerging Infectious Diseases launched by a project commissioned by the Ministry of Education, Science and Culture and the Ministry of Health, Labor and Welfare of Japan. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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72 hours after infection, although no significant effects by treatment with anti-suilysin serum were found on the mouse survival and bacterial densities in brain of mice. Based on the neutralizing effect of anti-suilysin serum, our data support antiphagocytic and antibactericidal properties of suilysin [5, 9, 29]. To assess further the contribution of suilysin to the pathogenesis of ST104 infection, we employed a coinfection mouse model (Figure 6); because complementation of ST104 strain with a plasmid carrying sly gene was unsuccessful. Significantly higher bacterial densities were found in the brains of mice coinfected with ST104 and wild-type ST1 strains than in those of mice infected with ST104 and sly-knockout ST1 strains at 72 hours after infection, supporting the hypothesis that suilysin has antiphagocytic and antibactericidal properties in the environment of the CNS and contributes to the prolonged infection of the CNS by ST104 in a coinfection mouse model (Figure 6C). Distinct from the results of the wild-type ST1 infection, mice coinfected with ST104 and wild-type ST1 strains did not show higher mortality rates or severe inflammation in their brains (Figure 6A, and Supplementary Figure 2). Lower bacterial density in the brain and lower suilysin production by the ST104 strain in vivo, which was confirmed by immunohistochemistry with anti-suilysin rabbit serum (data not shown), might not in themselves be able to cause inflammation in the CNS or increased mortality. In conclusion, we demonstrated that reduced sly promoter activity was responsible for the reduced suilysin production by S. suis ST104 strains. In mouse infection models, suilysin was confirmed to contribute to the development of bacterial meningitis and the resulting increased mortality. Our data illustrate the contribution of suilysin to the common feature of meningitis caused by ST1 strains and the common lack of meningitis caused by ST104 strains in human cases of infection.

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Suilysin Promotes S. suis Meningitis



JID 2014:209 (15 May)



1519

The contribution of suilysin to the pathogenesis of Streptococcus suis meningitis.

Streptococcus suis is an emerging zoonotic pathogen, and causes sepsis and meningitis in humans. Although sequence type (ST) 1 and ST104 strains are c...
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