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Streptococcus suis infection

Streptococcus suis (S. suis) is a family of pathogenic gram-positive bacterial strains that represents a primary health problem in the swine industry worldwide. S. suis is also an emerging zoonotic pathogen that causes severe human infections clinically featuring with varied diseases/syndromes (such as meningitis, septicemia, and arthritis). Over the past few decades, continued efforts have made significant progress toward better understanding this zoonotic infectious entity, contributing in part to the elucidation of the molecular mechanism underlying its high pathogenicity. This review is aimed at presenting an updated overview of this pathogen from the perspective of molecular epidemiology, clinical diagnosis and typing, virulence mechanism, and protective antigens contributing to its zoonosis.


Introduction
Streptococcosis is regarded as a leading infectious disease in the swine industry, that clinically features with meningitis, septicemia, or arthritis and annually results in significant economic loss worldwide. 1 Streptococcus suis (S. suis) that was initially reported in 1954 2 has been demonstrated as an etiological agent for this kind of frequently-occurring bacterial infection. 1,3 Indeed, S. suis, a complex population consisting of heterogeneous strains, 4 can be classified into 35 serotypes (1-34, 1/2) based on the differentiation of capsule antigens. 1,3 Based on the varied virulence of these bacteria, they may be categorized into highlypathogenic, weakly-pathogenic (hypo-virulent), and nonpathogenic (avirulent) strains. 1 Generally, serotype 2 of S. suis (SS2) is considered to be the most virulent, and is frequently isolated from clinically-diseased piglets. 1 In fact, serotype 9 of S. suis is also one of the most important serotypes in several countries.
Of particular note, SS2 seems to be a previously neglected but recently emerging human pathogen, 5 whose infection has become increasingly potent, especially in the southeast Asian countries like Thailand, 6 Vietnam, 7 and China. 8,9 As the primary agent of meningitis, septicemia, arthritis and as an opportunistic pathogen in the case of pneumonia, 1,5 S. suis have been reported to have spread over 30 countries and/ or regions (Fig. 1) and has claimed no less than 1600 human cases, some of which were fatal. 2 Also, similar clinical symptoms including bacterial meningitis, septicemia, and arthritis are frequently observed in human SS2 infections. 2,3 Occasionally, serotypes other than SS2, including SS1, 10 SS4, 10 SS5, 11,12 SS14, 13,14 SS16, 15,16 and SS24 11 can also be found to function as the causative agents responsible for sporadic cases of human S. suis infection. 3 Of note, two big outbreaks of human SS2 endemics which occurred in China, in 1998 and 2005, respectively, 9,17,18 have raised serious concerns in public health and have challenged the conventional opinion that human SS2 infections are only present in sporadic cases. 2,8,19 Unfortunately, no specific/effective human therapeutics or vaccine against SS2 infections is available thus far. Considering the severity (high mortality and modality) of SS2 infection in humans, 5,8 it is important to develop a method for convenient and quick diagnosis, which can be applied toward local SS2 detection. 4,18 Over the past four decades, significant progress has been made toward better understanding the highly infectious clones of S. suis. At the time of formulating this review, 1104 articles were available in PubMed regarding S. suis (http://www.ncbi.nlm.nih. gov/pubmed/?term=Streptococcus+suis).Totally, over 20 bacterial virulence-associated factors have been identified that include capsular polysaccharides (CPS), 20 Muramidase-released protein (MRP), 21 and Suilysin (SLY). 22 To date, genomic sequences of a collection of S. suis strains are available (Fig. 2), the majority of which are derived from SS2 species, 23,24 except two newly-released genomes which correspond to SS3 25 and SS14, 26 respectively. Genomic mining combined with bacterial genetics have elucidated that Chinese epidemic strains of highly pathogenic S. suis 2 carry a specific 89K PAI (pathogenicity island). 23,27 Further studies suggested that 89K PAI with a transposon-like essence can Streptococcus suis (S. suis) is a family of pathogenic grampositive bacterial strains that represents a primary health problem in the swine industry worldwide. S. suis is also an emerging zoonotic pathogen that causes severe human infections clinically featuring with varied diseases/syndromes (such as meningitis, septicemia, and arthritis). Over the past few decades, continued efforts have made significant progress toward better understanding this zoonotic infectious entity, contributing in part to the elucidation of the molecular mechanism underlying its high pathogenicity. This review is aimed at presenting an updated overview of this pathogen from the perspective of molecular epidemiology, clinical diagnosis and typing, virulence mechanism, and protective antigens contributing to its zoonosis.
undergo GI-type T4SS-mediated horizontal transfer in epidemic SS2 species. 28 The systematic elucidation of the of S. suis pathogenesis in the Omics Era was illustrated by functional definition of a collection of other new genes or putative orthologs (such as Zur, a zinc uptake regulator, 29 CovR, an orphan response regulator, 30 and Rgg-like transcription factor 31 ) following the release of the genome sequence of SS2 (e.g., 05ZYH33). 23 Although we have gained a partial glimpse of the molecular mechanism underlying the high pathogenicity of SS2 itself, we are still lacking further insights into the interface between the SS2 pathogen and the host it infects. 3,8 In this review, we aim to describe an updated but partial picture of SS2 as an emerging infectious agent, which centers on five aspects: global epidemiology/distribution, clinical diagnostics/ typing, pathogenesis, protective antigen/candidate vaccine, and zoonotic potential.

An Overview of S. suis
General microbiology of S. suis S. suis is a group of heterogeneous gram-positive bacteria that were earlier classified into Lancefield groups R, S, and T (Fig. 3). 1 These bacteria are facultative anaerobes with a spherical/ovoid shape which exist in pairs and/or short chains ( Fig. 3B and C). Generally, these microorganisms show either α-hemolysis when growing on selective plates of horse blood agar 1 (Fig. 3A). Given the variation in their CPS antigens, 35 serotypes have been proposed for S. suis population. 1 Very few studies of pathogenicity have been done for serotypes other than serotypes 2, 1, and 7. 1 Among them, SS2 is recognized as the most virulent species that is frequently associated with diseased pigs and often causes an opportunistic infection of adults having occupational contact with pig carcass or pork-related products. 2,5 S. suis, an important animal pathogen, naturally inhabits in the upper respiratory (particularly the tonsils and nasal cavities), genital, and alimentary tracts of piglets. 1,32 In addition to the natural host swine (Fig. 3D), this pathogen has been suggested to be isolated from a wide range of other animals, such as horses, dogs, and cats. 1,33 Of note, some variants of S. suis probably have evolved into highly infectious zoonotic agents that can cause meningitis, septicemia, arthritis, and even streptococcal toxic shocklike syndrome (which can cause rapid death) in humans. 9,17,18,34 Soon after the big outbreak of human SS2 infections in China, in 2005, serious concerns from both the public health and scientific community have been raised. 18 Toward better understanding and prevention/control of SS2 infections, multiple lines of new bacterial virulence determinants (such as salK-salR two component system, 27 FeoB transporter, 35 and Rgg regulator 31 ) have been identified, and fast assays using PCR-based molecular detection 18 as well as ELISA (enzyme-linked immunosorbent assay)-guided diagnostics were established 36 (Table 1). Most recently, epidemiological investigations conducted in Vietnam proposed that (1) pig population in slaughterhouses is a major reservoir of SS2 with a capacity to cause human infections 37 and (2) the most important risk factors of human S. suis infections are consecutively eating "high risk" pork-derived dishes, occupational exposure to pigs and pork-related products, and preparation of pork in the presence of skin lesions. 38 The availability of the epidemiological knowledge on human SS2 infections is critical to improve the current situation of public awareness of SS2 infections and to effectively prevent the potential occupational infections caused by SS2.
Genomics/proteomics-based glimpse of S. suis Genomics/proteomics approaches to probe S. suis have yielded unprecedented comprehensive information/knowledge about this pathogen. 8,39 At the time of writing this review, no less than 14 genomes of S. suis strains have been available in PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gen ome&cmd=DetailsSearch&term=streptococcus+suis&save_ search=false), most of which were completed by the research groups in China (Fig. 2). Genomic sequence analyses of S. suis showed that (1) all the sequenced genomes feature with the nearly same average GC content, ~41%, indicating slight evolutionary conservation; (2) genome size varies markedly from 1 640 446 nt (05HAS68, NZ_AARD00000000) to 2 146 229 nt (BM407, NC_012926), reflecting the genomic flexibility present in these species (Fig. 2B); (3) the number of putative protein-encoding genes is dramatically different (1559 for 05HAS68, and 1932 for BM407), suggesting that nearly 1/5-1/6 of total genes are not essential (redundant) for bacterial viability, and might confer some new/unknown functions to adapt to varied environmental niches. Functional/ comparative genomics of S. suis has defined a series of new infection/virulence-related determinants such as ArgR regulator, 40 SspA, a subtilisin-like protease, 41 CiaRH two-component regulatory system, 42 and so on.
On the other hand, proteomics analyses of Chinese epidemic SS2 strain 05ZYH33 identified 373 proteins in total from 834 processed spots. 43 Using an immune-proteomic approach, Lu's group revealed 11 membranerelated proteins from Chinese vaccine strain SS2-HA9801 44 and 9 extracellular antigenic proteins from the virulent Chinese SS2 strain ZY05719, 45 respectively. Using a similar strategy, Wu and coauthors from the same group addressed two strains of SS9, another prevalent serotype of S. suis (GZ0565 and SH040197), and observed 13 candidate proteins, five of which are virulence-associated factors. 46 Additionally, eight immunogenic proteins localized on bacterial surface were determined in strain GZ0565, including extracellular solute-binding protein. 47 These findings might provide a solid/reasonable basis for development of subunit vaccine candidates. 39

Current situation of human S. suis infections in Europe and North America
During the past 40 years, around 100 cases of human S. suis infections were estimated in European countries. Among them, the top three countries in the history of human SS2 infections recorded are Netherlands (41 cases), United Kingdom (15 cases), and Denmark (12 cases). 2 The rest of the European countries (such as France and Germany) had less than ten sporadic cases of human infection. 2 Moreover, the prevalent type of clinical disease caused by S. suis infections in these countries is bacterial meningitis. Although human S. suis infections are considered sporadic cases in most countries, 2,5,8 it is unbelievable that its zoonotic infectious events are rarely reported in North America (Canada and USA), two huge countries with numerous, large, and frequent swine operations. 3,5 To the best of our knowledge, only three human SS2 meningitis cases were confirmed in USA, 49,50,89 and three cases were diagnosed in Canada (one case was due to SS14, and the other 2 cases were caused by SS2 51,52 ). Gottschalk and coworkers 3 believe that this small number of reported cases from these two huge countries with a big industry for pig cultivation might be attributed to the following two major reasons: (1) Clinical under-diagnosis and/or misdiagnosis of S. suis infection, rather than true absence of this infectious disease, (2) S. suis isolates in North America are less virulent, relative to those from Europe and Asia. A pilot study conducted by Smith et al. 90 recently indicated that human infection with S. suis is more common in the United States than what it is generally thought. The reason might lie in underdiagnosis or misdiagnosis, rather than a real lacking of disease. 3,91,92 On this issue, we doubt the current situation of S. suis infections as well, though we agree that good hygiene conditions and prevention strategies during the whole process of pork operation (e.g., wearing gloves) might secure the major route of bacterial entry into blood by small cuts in the skins, and greatly decrease the incidents of S. suis infections in North America. Therefore, we believe that it is necessary to employ combined specific approaches (like multiplex PCR plus ELISA) for the re-evaluation of the epidemiological aspects of human S. suis infections in North America. Interestingly, Schmid et al. 93 recently demonstrated that American SS2 isolates do not carry 89K PAI, a DNA fragment present in Chinese epidemic strain, which might be helpful for development of an American SS2 strain-specific PCR detection assay.

Situation of human S. suis infections in southeast Asia
Accumulated epidemiological data suggests that over 85% of total cases have occurred in Asian countries. 2 The cases of human SS2 infections in the mainland of China and Vietnam are comparable. 2,8,16 Relative to the above 2 countries, the human cases clinically infected by S. suis are second. 6,14,87 Of being noteworthy, SS2 has been recognized as a pathogen with the mostly-relevance to human bacterial meningitis in southern Vietnam. 16 In Hongkong, the specialized administrative region of China, the first human case of S. suis infection was recorded in 1983, 78,80 and the accumulated number of S. suis infections were estimated to be about 60 cases. 75,78 In particular, two big outbreaks of human SS2 infections in China (1998 and 2005) seriously challenged public health. 9,18 In the 1998 epidemic, 14 out of 25 SS2infected persons died along with an estimated 80 000 pigs. 18 In the 2005 epidemic, totally 215 patients had SS2 infections, 38 of which are dead. A similar scenario was also observed when more than 600 pigs were demonstrated to be infected by SS2. 9,18 The causative agents of the both epidemics were subsequently determined to be highly invasive clones of strong virulent SS2 strain that seemed to have acquired a new pathogenicity island 89K. 8,23,94 Additionally, sporadic human meningitis cases caused by SS2 infections were observed in three other cities of China (Shenzhen City, Chongqing City, and Nanjing City) in 2007, 4,95 implying that the situation of SS2 infection in China is complicated. This could be due to variants of S. suis 2 identified in subsequent investigations. It might be of much interest to unveil the possible evolutionary relationship of the Chinese epidemic strain with those of the neighboring country like Vietnam through comparative genomics.

Detection and Typing of S. suis
Fast/effective detection and analyses of S. suis is critical for the prevention and/or diagnosis of endemic S. suis 2 infection in the swine industry as well as for S. suis infected patients. Three types of experimental approaches are available thus far that consist of (1) selective media-based microbiological cultivation, (2) molecular tests, and (3) immunological assays ( Table 1). Given its advantage in sensitivity and fastness, the second method has been experimentally developed into two subgroups (PCR-based detection plus typing-oriented analyses like pulsed-field gel electrophoresis [PFGE] and restriction fragment length polymorphism [RFLP]) and might be potential in clinical re-confirmation and/ or re-valiadtion. Generally, PCR assays can be classified into six kinds among which multiplex-PCR (using sets of specific primers including epf, mrp, and gdh) is appreciably-valid approach to assay SS2 and applied in some countries ( Table 1). The major four kinds of typing methods include PFGE, RFLP, multi-locus sequence typing (MLST) and random amplified polymorphic DNA (RAPD), some of which have derivatives such as ISR-RFLP ( Table 1). In general, the three experimental methods (PFGE, RFLP, and RAPD) can return clues about genomic differences between different strains/serotypes. Given direct sequencing of multiple loci (cpn60, dpr, recA, aroA, thrA, gki, and mutS), MLST can directly capture the nucleotide sequence deviation used for typing purpose. Among the immunological approaches, ELISA could be the most popular way to address S. suis infection in some experimental tests ( Table 1). In fact, different versions of ELISA have been developed that are based on various capture antigens identified to be specifically against S. suis 2. We have also established two kinds ELISA assays (one is based on SAO protein, 4,36 the other is based on Enolase surface antigen 96 ), both of which work well in our trials during field screening and clinical detections. Recently, two more new derivative methods were reported, which are immunochromatographic strip 97,98 and electrochemiluminescence immunosensor, 99 respectively ( Table 1).

Bacterial virulence determinants
The clinical consequence of S. suis infection is determined by the complicated interplay between this zoonotic pathogen and its host. A series of bacterial components as well as a collection of host cell factors contribute to this pathogenesis-related process. So far, almost 60 bacterial components have been identified to be involved in the infection and/or pathogenicity of S. suis ( Table 2). Of particular note, Wilson and coworkers 100 developed a powerful signature-tagged mutagenesis (STM) system for S. suis and identified nearly 20 potential virulence associate elements through screening the library consisting of approximately 2600 mutants ( Table 2). However, exact roles of these genes needed further verification. According to their general roles in the context of bacterial life cycles, these bacterial virulence-associated factors were temporarily classified into the following three sub-groups (of note some genes probably can be attributed to two different classifications due to their dual characteristics): (1) surface/secreted elements; (2) enzymes/proteases; (3) transcription factors/regulatory systems; and (4) others (transporters/secretion systems) ( Table 2).
Surface/secreted components For Subgroup 1, a total of 17 genes/gene clusters have been determined thus far to contribute to bacterial pathogenicity (  105,106 plus implication into bacterial meningitis, 107 11 more members have been supplemented into this group that include SspA, the surface-associated subtilisin-like serine protease, 41,108,109 HtpS, a novel immunogenic cell surface-exposed protein, 110 and Sat surface protein 111,112 (Table 2). Like FBP, 103 the gene SSU05_1311 with unknown function was determined to be one more surface anchored fibronectin-binding protein. More importantly, this surface protein functions in vivo in crossing the mucosal epithelia to disseminate, suggesting it is a novel virulence factor. 113 Two independent research groups (one from Canada 41 and another from China 114 ) identified the subtilisin-like protease (SspA)-encoding gene (sspA, SSU0757) from the S. suis organism by screening the mutant library and genomic expression library, respectively. SspA proteinase possesses a typical cell wall anchoring signal, LPXTG, at its C-terminus, implying it is a cell surface-displayed protein. 108 Infection assays have demonstrated that SspA protein plays critical roles in SS2 pathogenicity. 108,109,114 A subsequent study further revealed that S. suis SspA protease might modulate cytokine secretion by macrophages, and thus trigger central nervous system inflammation associated with bacterial meningitis, frequently observed in SS2-infected patients as well as piglets. 41 HP0197 is a new a surface protective antigen that was originally identified by Jin's group in 2009. This immunogenic antigen can elicit obvious humoral antibody response and confer efficient protection against SS2 challenge in the infection models of both mice and pigs. 115 Subsequently, HP0197 protein was found to interact with host cell surface glycosaminoglycans (GAGs), and the binding sites were further proved by solving the X-ray structure of N-terminal GAG-binding domain combined with site-directed mutagenesis plus indirect immunofluorescence assay. 116 Very recently, the same research group reported that HP0197 is involved in bacterial virulence by evaluating the performance of the isogenic mutant Δhp0197 in both mice and pigs. 117 A reasonable interpretation would be that virulence attenuation is due to easier clearance of the Δhp0197 mutant by host immunological system during the stage of infection, and the fast clearance is attributed to the reduced CPS thickness and decreased resistance to phagocytosis. 117 Further comparative transcriptomics-based analyses elucidated that expression of CPS synthetic operon is downregulated in the Δhp0197 mutant relative to the wild-type strain. 117 Also the introduction of plasmid-borne hp0197 gene into the Δhp0197 mutant can restore the decreased expression level of cps operon to those seen with the wild type strain. 117 This observation is consistent with the fact that CcpA, a global carbon catabolite regulator, contributes to bacterial infectivity/pathogenicity. 118,119 Preliminary evidence pointed out that HP0197 might determine the Ser-46 phosphorylation level of phospho carrier protein (HPr-46), a partner protein of CcpA in binding the catabolite-responsive elements (cre) of the target operons. Together, it suggested that integration of the posttranslational modification of HPr-46 and CcpA-mediated transcriptional regulation is linked to regulatory network of bacterial virulence.
The serum opacity factor of S. suis (OFS) is a putative member belonging to the family of MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules). 120 N-terminal region of OFS exhibits similarity to the serum opacity factor of Streptococcus pyogenes and fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae, and its C-terminus harbors repetitive sequence elements. Crude extract of S. suis and the recombinant OFS protein both possessed serum opacification activity. Experimental infections demonstrated that the deletion of the ofs gene severely impairs S. suis virulence. 120 Sao is a surface antigen first identified by Li et al., 121 which can react with convalescent-phase sera from pigs clinically infected by S. suis type 2. Subsequently recombinant Sao formulated with Quil A was found to induce potent opsonizing antibody responses, and confer cross-protection of mice against challenges of virulent heterogeneous S. suis. 122 We also discovered that three allelic variants of the sao gene (namely sao-S, sao-M, and sao-L) are present in S. suis population, and we have developed an efficient ELISA method in which Sao-M protein serves as the capture antigen. 36 Recent further genetic study suggested that SAO protein is only a minor virulence factor 123 Sat (HP272) is another newly-identified surface protein from the S. suis serotype 2, 111,112 and two different research groups have shown evidence that recombinant Sat protein can confer effective immuno-protection of mice against SS2 infections, implying that it is a vaccine molecule candidate. 111,112 In addition, HtpS, a putative member of histidine triad protein family, was determined to be cell surface-associated protein that was expressed during the infection of Chinese SS2 strain 05ZYH33. 110 Moreover, recombinant HtpS protein was demonstrated to function as a protective antigen against SS2 infections. 110 Similarly, Zhang et al. 124 recently defined another new infection-associated  antigen protein, Trag, using in vivo-induced antigen technology (IVIAT). An inactivation of the trag gene was observed to attenuate full virulence of Chinese SS2 strain in the experimental model of Zebrafish. 124 As a surface protein, PAPI-2 was found to constitute an ancillary pilus subunit and exhibit ability to confer protection of mice against serious challenge of S. suis. 125 Although we have never reported evidence that Zur, a zinc uptake regulator is essential for S. suis virulence, 29 Aranda et al. very recently reported that the zinc-binding lipoprotein 103, the structural component of zinc uptake system is associated with the infectivity of Streptococcus suis, implying that zinc uptake system might be involved into bacterial pathogenesis. 126 This discrepancy may be attributed to the fact that the deletion of zur only partially affect expression of zinc uptake system genes, whereas inactivation of "103" completely impairs zinc uptake system. That is why the latter could more seriously disrupt the normal zinc metabolism, and in turn lead to virulence attenuation in this sick bacterium.

Enzymes and proteinases
Accumulated data has suggested that no less than 20 bacterial enzymes might be implicated in the manifestation of S. suis virulence ( Table 2). Among them, eight enzymes are proposed to be virulence factors by Wilson et al. 100 using the system of signature-tagged mutagenesis ( Table 2). Four of them are generally regarded as enzymes of central metabolism, which separately correspond to (1) GlnA, glutamine synthetase, 127 (2) Gdh, glutamate dehydrogenase, 128 (3) enolase catalyzing dehydration of 2-phosphoglycerate to phosphor-enolpyruvate, 96,129,130 and (4) Impdh, inosine 5-monophosphate dehydrogenase. 131 Five of these enzymes are directly or indirectly related to synthesis and/ or modification of bacterial surface structure, including DltA, an enzyme catalyzing lipoteichoic d-alanylation, 132 PgdA, peptidoglycan N-acetylglucosamine deacetylase, 133 ApuA, a bifunctional amylopullulanase, 134 DPP IV, di-peptidyl peptidase IV, 135 and sortase A, a transpeptidase. 136,137 One of the remaining two enzymes is IgA1 protease, which has highly immune-reactive activity to convalescent sera, 138 and the other is S-ribosylhomocycteinase (LuxS) catalyzing synthesis of auto-inducer 2 (AI-2) utilized in interspecies quorum sensing. 139,140 As follows, we will discuss the roles of these enzymes in S. suis pathogenesis.
With the exceptions of neuB and neuC, the remaining six among the eight enzymes proposed by Wilson 100 NeuB is a sialic acid synthase that catalyzes the last committed step of the de novo biosynthetic pathway of sialic acid, a major element of bacterial surface structure. Recently, we systematically addressed its molecular and immunological role in bacterial virulence and claimed that an altered architecture of S. suis surface attenuates its virulence. 100,141 Similarly, neuC that encodes UDP N-Acetylglucosamine 2-Epimerase with an involvement in sialic acid biosynthesis is also found to be essential for capsule production and required for virulence in a mouse infection model. 142 Gdh, the glutamine dehydrogenase-encoding gene, was originally known as a virulence factor. It has been widely applied  128 Si et al. 127 demonstrated that glutamine synthetase, the glnA-encoding product, is associated with S. suis virulence using a mouse model. We and two other research groups 96,129,143 have reported that S. suis enolase acts as an octamer, 144 and can exported to the bacterial surface with capability of binding to host fibronectin, indicating its possible role in crosstalk between pathogen and host. However, the protective efficiency of recombinant enolase with different bacterial origins does not seem consistent. 96,129,130 Similarly, the gene encoding Inosine 5-monophosphate dehydrogenase, a nucleotide metabolism-related enzyme, was initially cloned by Lu's research group, and was subsequently suggested to be involved in full virulence of SS2-H, a Chinese strain in the infection model of piglets. 131 Modification of the bacterial surface is critical for successful invasion and entry of pathogens into host cells. Gottschalk's group systemically evaluated contributions of two kinds of modification systems to S. suis pathogenicity: lipoteichoic acid (LTA)-d-alanylation, and peptidoglycan (PG) N-deacetylation, respectively. 132,133 The ΔdltA mutant with defection in LTA d-alanylation was found to be attenuated in its virulence, which can be correlated with its diminished adherence/invasion of porcine brain microvascular endothelial cells, and decreased capacity to escape immune clearance or killing by porcine neutrophils. 132 Fittipaldi et al. 133 observed that the expression level of the pgdA gene can be induced upon interaction of SS2 with neutrophils in vitro and infected mice in vivo, implying that PG N-deacetylation is tightly involved in SS2 infections. This hypothesis was further validated by virulence attenuation of the ΔpgdA mutant of SS2. Not only does ApuA behave like a bacterial surface protein with a LPKTGE cell-wall-anchoring motif at C-terminus, but it also functions as a bi-functional amylopullulanase. 134 Further genetic study showed that the multifunctional α-glucan-degrading enzyme ApuA can promote adhesion to porcine epithelium and mucus, which might link bacterial carbohydrate utilization to its capability of colonization and invasiveness into hosts. We and the other research group demonstrated that Sortase A (SrtA, originally referred to as a transpeptidase in Staphylococcus aureus) is essential for full virulence of SS2. 136,137 However, other sortase paralogs SrtBCD are not associated with bacterial virulence. 145 In addition, we reported the functional definition of di-peptidyl peptidase IV (DPP IV) in S. suis 2, and also confirmed that it does contribute greatly to bacterial virulence. 135 Quorum sensing is a method of bacterial cell density-dependent communication, using secreted chemical molecules (like the auto-inducer) as a form of "language". The luxS gene product is the synthetase of autoinducer 2 (AI-2) that is required for interspecies communication. Recently, we and Lu's group both defined a functional LuxS member present in Chinese isolates of S. suis 2, and demonstrated its relevance to bacterial virulence. 139,140 Zhang et al. identified that IgA1 protease, an immune-dominant antigen, is necessary for full virulence of S. suis 2. 138 Additionally, Gruening et al. 146 proved that the arginine deiminase system (ADS), encoded by arcABC operon, is necessary for S. suis survival in acidic stress, indicating its possibility of correlating with bacterial successful infection. Very recently, it was discovered that the adenosine synthase functions as an effector in evasion of PMNs-mediated innate immunity 147 . It might suggest possibility that cyclic AMP (cAMP)-dependent signaling is linked to streptococcal infectivity. Given the fact that CadD enzyme, cAMP deaminase from Leptospira, 148 can quench cAMP-dependent signaling, it would be of interest to test the hypothesis that in vivo expression of CadD protein in S. suis modulate bacterial pathogenesis or not?
AdcR is a regulator controlling zinc transport in S. suis. Aranda and coauthors observed that disruption of this transcription factor can attenuate bacterial virulence in mouse model. 149 In contrast, Zur, the other zinc uptake regulator from 05ZYH33 strain of S. suis 2 is not essential for strong pathogenicity in porcine models. 29 Driven by the idea that host environment is critical for expression of bacterial virulence factors during the process of infection, Willenborg et al. 118 evaluated the effect of the sugar metabolism regulator catabolite control protein A (CcpA) on S. suis pathogenesis. As anticipated, expression levels of several virulence factors (such as ArcB, Sao, and Enolase) were altered in the ΔccpA mutant. Of particular note, a recent study implied that Sao protective antigen plays a limited role in bacterial virulence. 123 Moreover, the deletion of ccpA led to significant reduction of both capsule thickness and resistance to killing by porcine neutrophils 118 and impaired bacterial virulence. 119 ArgR, a member of ArgR/AhrC arginine repressor family, was recently proved to regulate expression of arcABC operon encoding an arginine deiminase system that is recognized as a putative virulence factor. 40,146 Therefore, it is of interest to test the role of argR in S. suis virulence. Similar to the scenario observed with Rgg regulators present in other gram-positive pathogen, Zheng et al. defined an rgg-like ortholog in S. suis 05ZYH33, and observed its multiple roles in bacterial metabolism. More importantly, it was verified to be a virulence determinant of S. suis 2 in experimental models of piglets. 31 Interestingly, an H 2 O 2 -responsive Fur-like regulator, PerR was confirmed to determine bacterial virulence through regulating expression of both dpr, a Dps-like peroxide resistance protein-encoding gene, and metQIN encoding a methionine transporter. 150 Among the 15 putative two-component signal transduction systems in Chinese virulent strain of S. suis 2, 23 five have been proposed to be correlated with manifestation of strong virulence. 27,42,152,153 In 2008, we reported the salK-salR system present in the 89K pathogenicity island. The deletion of this TCS system resulted in significant downregulation of 26 genes' expression level, and increased its susceptibility to polymorphonuclear leukocyte (PMN)-mediated killing. Consequently, the virulence of the ΔsalK-R mutant was seriously attenuated. 27 Shen et al. 158 conducted a follow-up study to address the regulatory network of SalK-SalR TCS system. As expected, proteomics-based investigation revealed 14 downregulated proteins and 1 upregulated protein, which partially agreed with our former microarray analysis. 158 To our much surprise, Zhong's group very recently elucidated the physiological role of this SalK-SalR TCS in regulating a bioactive lantibiotic suicin production, thereby replaced it with SuiK-SuiR. 151 Li and coworkers 42 reported the second TCS system, ciaK-ciaR, which is required for pathogenicity of SS2 in the infection models of both CD1 mice and piglets. Han et al. showed that Ihk/Irr TCS contributes to full virulence of Streptococcus suis serotype 2 strain 05ZYH33 via regulating bacterial cell metabolism. 152 Very recently, two more TCS (VirR/VirS 153 and NisK/ R 154 ) was demonstrated to be essential for SS2 pathogenicity.
Also, an orphan response regulator revSC21 was determined by the same research group. 155 This regulator revSC21 positively modulates expression levels of virulence factors (e.g., mrp, sly, and cps) and is required for bacterial pathogenesis. 155 In contrast, Pan et al. reported another orphan response regulator CovR with an opposite effect on S. suis pathogenicity. 30 The covR-defective (ΔcovR) mutant displayed thicker capsules and increased hemolytic activity. Furthermore, adherence of this mutant to epithelial cells was greatly increased, as well as its resistance to phagocytosis and killing by neutrophils and monocytes. Eventually, the removal of covR gene was found to be correlated with increased lethality of piglets, relative to those inoculated with its parent virulent strain 05ZYH33. 30 Others VirA is a newly-determined virulence factor that is exclusively present in virulent SS2 strain. 122 Trigger factor is also a virulence determinant which acts by controlling expression of a collection of known virulence factors, such as cps, mrp, and sly. 159 FeoBA encoding an iron transporter system might represent the first example of the fact that the machinery of a transporter can be involved in S. suis pathogenicity. 35 After Li et al. 28 defined transposal characteristics of 89K PAI in epidemic strain of Chinese virulent SS2, Hu's group recently obtained further insights into 89K PAI. In addition to virulence-determining element, salK-salR TCS system, 89K PAI also carried a second virulence factor, virD4-virB4, a type IV-like secretion system. Unexpectedly, this T4SS-like system is responsible for stimulating host immune reaction observed in mice infected with Chinese streptococcal toxic shock-like syndrome (STSLS)-causing strain of S. suis 2 160 , raising a possible relationship of 89K to molecular/immunological machinery by which this non-group A streptococcus (GAS), SS2 to cause STSLS in infected patients (Fig. 4). Finally, Wilson et al. also suggested that 05SSU0660, an uncharacterized protein homologous to S. pneumonia spr1018 might be a virulence factor. 100

Host Immunological/Inflammatory Factors
Pathogens have evolved capacity to recruit/hijack host cell factors for their successful infections/invasions. So far, the majority Clinical visualization of a representative SS2-infected patient with streptococcal toxic shock-like syndrome. it clearly shows purpura and gangrenous changes on this patient's legs. Partially adapted from reference 9 with permission. (B) Genetic evidence that 89K pathogenicity island carries elements associated with bacterial virulence and the clinical consequence of streptococcal toxic shock-like syndrome. PFGe assay reveals Chinese epidemic strain 05ZYH33 is distinct from the international strain S10. 4 The two genetic elements of the transposable 89K pathogenicity island 23,28 have been functionally defined: one is SalK-SalR (renamed as SuiK-SuiR 151 ) TCS with requirement for full virulence, 27 and other is virD4-virB4 T4SS-like system associated with clinical manifestation of STSLS in mouse model. 160 (C) The proposed model of the two-stage hypothesis for STSLS. SS2 gets into blood vessels in stage i and results in an early burst of proinflammatory factors and Th1 cytokine storms. Such kind of inflammatory super-responses lead to STSLS with death as early as 13 h after SS2 infection. in stage ii (i.e., several days post-infection), SS2 might use virulence factors like suilysin to cause disease, particularly meningitis. 94 CNS, central nervous system. integrated and modified from references 4, 23, 27, 28, 94, and 160 with permission.
of host cell factors identified to be involved in S. suis pathogenesis are immunological/apoptotic/inflammatory factors.
Nearly ten years ago, Gottschalk et al. 161 had observed that cell wall components of S. suis can induce releases of interleukin-1 (IL-1), IL-8, monocyte chemotactic protein-1 (MCP-1) in human brain microvascular endothelial cells (BMEC), which might increase the permeability of the blood-brain barrier. Using the experimental model of CD1 mice, the same group observed that (1) ~20% animals with sudden death exhibit high levels of systemic TNF-α, IL-6, IL-12, IFN-γ, CCL2, CXCL1, and CCL5 24 h after infection; (2) infected mice that survived the early sepsis later developed clinical signs of meningitis present in the transcriptional activation of TLR2, TLR3, CD14, NFκB, IL-1β, CCL2, and TNF-α, mainly in myeloid cells located in affected cerebral structures. 162 Apparently, the inflammatory response plays important roles in S. suis infection of CD1 mice. Similar observation was also reported by Scherk et al. 163 that S. suis infections are correlated with the release of pro-inflammatory cytokines and chemokines (e.g., IL6 and IL8). Gottschalk et al. found that the toll-like receptor 2 (TLR-2)-deficient exhibit significantly reduced production of astrocytes, 164 and proposed that bacterial polysaccharides probably modulates TLR2-dependent recognition of S. suis entry/invasiveness into CD1 mice. 165 Using a transcriptomic approach, de Greeff et al. 166 identified macrophage-specific genes (IL-1-β, MIP-2-α, and TNF-α) with significantly different expression upon S. suis infection, suggesting that MAP-kinase signaling pathway and NFκB signaling are implicated into response of porcine alveolar macrophages to S. suis infections. Additionally, Wu et al. 167 verified that a novel murine ribonuclease, angiogenin inhibitor 1 (AI1) can bind to S. suis hyaluronidase (Hyl), and hypothesized that this interaction between host AI1 partner and bacterial Hyl protein might contribute to S. suis meningitis.

Protective Antigens
Development of a safe and efficient vaccine is a useful strategy to combat against S. suis infection. In contrast to conventional killed/live whole-bacteria vaccines, [168][169][170] engineering of subunit vaccine exhibits significant advantage in its safety and its largescale producibility. 171 Identification of protective antigens is a prerequisite for identifying candidate vaccine molecules. Totally, there are no less than 15 protective antigens identified thus far ( Table 3). Among them, four molecules of protective antigen are well-known virulence associated factors and are MRP, EF, 38 kDa, and SLY ( Table 3). Of note, most of the remaining 10 protein antigens were elucidated by research groups in China. In 2009, in addition to identification of the known protective antigen SLY, Liu's report verified three more new protective antigens that are RTX family exoprotein A (RfeA), epidermal surface antigen (ESA), and immunoglobulin G (IgG)-binding protein (IBP). 172 Two different research groups from China confirmed that enolase, an enzyme of central metabolism, acts as a protective antigen displayed on bacterial surface. 96,129 However, Esgleas and coworker 130 reported an opposite result regarding the protective efficiency of enolase in mice. This discrepancy could be due to different versions of recombinant protein, different strains plus deviations in animal vaccination protocols. In particular note, all the three newly-identified immunogenic antigens (6PGD, HP0197, and HP245) are elucidated by the same Chen's research group in China (Table 3).
Interestingly, proteomics also facilitated to discover three new immunogenic proteins SsPepO, 173 Sat (HP0272), 112,174 and the pilus subunit PAPI-2b 125 ( Table 3). In disagreement with their former observation, 121 Li and coworkers 122 demonstrated that SAO surface antigen can confer efficient protection in both mice and piglets against virulent SS2 infection ( Table 3).

Clinical consequence of human SS2 infections
It is accepted that S. suis has developed into a significant human pathogen, especia in southeast Asia, posing a great challenge to public health. 2 Clinically, a collection of disease types can be observed in those patients with S. suis infections, including meningitis, septicemia, and pneumonia. In terms of epidemiological/clinical statistics, bacterial meningitis is the most prevalent symptom caused by S. suis infection.
Meningitis Meningitis is medically defined as an inflammation of the lining that covers the brain and spinal cord (the meninges). 10,16,60 This kind of inflammation usually initiates with a brief influenza-like prodroma and results in hearing impairment or loss. 77,175 Generally, meningitis can be grouped into two types, bacterial and non-bacterial (e.g., viral or fungal meningitis). 77 For the former, no less than 10 kinds of bacterial pathogens (such as Mycobacteria tuberculosis, Streptococcus pneumonia, and Staphylococcus aureus) have been determined as the causatives of development of human meningitis. 33,77 Although SS2 is a swine pathogen, we have come to know that SS2 can also be a severe agent for human bacterial meningitis. 60,88 The first case of human SS2 infection worldwide was recorded in Denmark in 1968. 1 Hui et al. declared that SS2 would be another leading cause of the so-called communityacquired meningitis, which is only inferior to M. tuberculosis and S. pneumonia. 77 A systematic survey of bacterial meningitis in Vietnamese adults recently suggested that (1) SS2 with multiple virulence factors (e.g., EPF, SLY, MRP) is the most common pathogen and (2) its mortality is relatively low (2.6%), but hearing loss occurs at high percentage (66.4%). 16 Moreover, Hoa et al. 176 addressed the antimicrobial susceptibility profile of S. suis strains from meningitis patients, an important question, using comprehensive approaches to analyze bacterial isolates in Vietnam from 1997 to 2008. As result, they found that (1) multidrug resistance in S. suis 2 causing meningitis in southern Vietnam has increased over the 11-y period studied; (2) the tet(L) carried in these bacteria is functionally expressed, and multiple other genes are probably co-expressed. 176 These findings alerted us again that it is important to minimize abuse of antibiotics in treatment of bacterial infectious diseases, and that the development of new therapeutics against SS2 infections are in great demand. Fortunately, Ho et al. 177 reported the largest prospective epidemiological survey of human SS2 meningitis in Vietnam, and pointed out that three important risk factors associated with human SS2 meningitis included (1) eating "high risk" dishes popular in southeast Asia, (2) occupational exposure to pigs and/or pork-related products, and (3) preparation of pork in the presence of skin lesions. Further investigations from the same research group demonstrated that slaughterhouse pigs are a major reservoir of SS2 that led to human infections of SS2 meningitis in southern Vietnam. 178 It highlighted (1) the importance of an improved hygiene at pork processing facilities, and (2) the necessity for education programs concerning food safety and proper handling of pork. 178 Similar findings were also noted in the Netherlands 59 and New Zealand. 86,179 In the Netherlands, researchers have clearly indicated that the risk of developing SS2 meningitis among abattoir workers, butchers, and pig breeders are above 1000 times higher than that among persons without close contact with pigs or their unprocessed pork products. 59 To our surprise, a study performed in New Zealand revealed that a high ratio of farmers and meat inspectors in markets were sero-positive to SS2, suggesting the presence of human sub-clinical SS2 infections to a small extent. 86,179 Septicemia Septicemia is a serious and life-threatening infection, in which a large amount of bacteria are present in the blood. It is commonly referred to as "blood poisoning" or "bacteremia with sepsis". 1 This disease often causes multi-organ failure by reducing the amount of blood reaching vital organs such as the liver and kidneys. 1,48,75 In addition to the known bacterial pathogens (such as Staphylococcus) with capability of leading to septicemia with about 25% lethal rate, 1,180 SS2 is also a causative agent of this disease. 1 In general, SS2-caused septicemia arises as a result of the localized infection in the body, especially cut skin. 1 In patients with septicemia, some of the following symptoms can be observed: fever and chills, rising heart/respiratory rate, cold and clammy feeling, fallen blood pressure, paled ,and petechial skin, and ultimately even unconsciousness. SS2 most likely releases some toxins into the blood that break down the walls of blood vessels which allows blood to leak out under the skin (it is this leaking that causes the rash or petechiae). 33 In some cases, SS2 infects the bloodstream and the meninges at the same time, causing both septicemia and meningitis. 33,181 Due to the rapidly progressive condition of septicemia, it can evolve into an irreversible toxic shock and even acute death if sufferers do not receive urgent treatment. 33,181,182 Certainly, an antibiotic remedy could be altered quickly to treat the target bacteria agents, when medical tests have identified which bacteria cause the septicemia and which antibiotics are best effective. 33,182 Of note, some medical treatments themselves are the inducers of septicemia, e.g., dental treatment, long-term use of intravenous needles, a colostomy, and so on. 33,175 Collectively, septicemia is so serious and complex that it deserves a lot of attention worldwide.
Others In addition to the above two major forms of SS2-caused diseases, there are some other clinical types that occur less frequently in the cases of human SS2 infections, including arthritis, endocarditis, streptococcal toxic shock-like syndrome (STSLS), etc. Since STSLS is a newly-described disease form of SS2 infections (Fig. 4), here we have discussed it in detail. Toxic-shock syndrome refers to a highly invasive infection of deep tissues, and can be correlated with production of bacterial super-antigens (e.g., staphylococcal and streptococcal exotoxins). 183,184 Before Tang et al. 18 formally proposed that SS2 is another bacterial non-GAS agent responsible for STSLS, we had been aware that Staphylococcus 185,186 and Streptococcus pyogenes, a group A streptococcus (GAS) 183,184 are both leading pathogens with a capability of causing STSLS. The clinical criteria for diagnosis of STSLS disease can be described as follows: (1) clear erythematous blanching rash (Fig. 4), (2) sudden onset of high fever, (3) hypotension diarrhea, (4) blood spots and petechiae, and (5) dysfunction of multiple organs (e.g., disseminated intravascular coagulation and acute renal failure). 8,9,18 According to above clinical criteria, two independent research groups in China reported two big outbreaks of human SS2-caused STSLS, which occurred in Jiangsu Province, 1998, and Sichuan Province, 2005, respectively. 9,[17][18][19]34 Two models have been proposed to explain the molecular mechanism by which SS2 triggers STSLS in its infected patients, one of which is a two-stage hypothesis, 94 the other is a specific 89K PAI. 23,160 Although we came to know that similar findings were also recorded in Thailand, 187 France, 188 and Australia, 84 we did not gain any information on difference of these SS2 strains abroad in comparison with Chinese STSLS-causing isolates. Given that the above two models are based on evidence from studying the Chinese SS2 strains, we can't conclude presently that they represent common or strain-specific mechanisms.
Two kinds of non-SS2 serotypes have been determined in sporadic cases of human S. suis infections: One of these is SS14, 13 and the other is SS16. 16 Poggenborg and coworker 189 reported a pig-farm worker with SS14 meningitis and septicaemia complicated with thoracal and lumbar spine spondylodiscitis. Similarly, Ahmed et al. 13 also documented a meningitis case caused by SS14 infections, in a female patient with occupational exposure to piglets each day. Of particular note, totally 12 cases of human SS14 infections were identified in Thailand during 2006-2008, and their clinical presentations included meningitis, septic arthritis and sepsis. 14 Further analyses demonstrated that 11 of the 12 SS14 isolates from these patients belonged to the multilocus sequence types (ST) 105, suggesting clonal dissemination of ST105 strains in Thailand. In another neighboring Asian country, Vietnam, a fatal case of human SS16 infection was revealed by Schultsz's research group, 16 indicating multiple serotypes of S. suis are developing into human pathogens circulating in this country. Together, non-SS2 serotypes of S. suis have also exhibited its zoonotic potential, highlighting a great demand for monitoring their epidemiology and development of relevant approaches applied toward prevention and therapeutics.

Concluding Remarks and Perspectives
As a zoonotic agent, S. suis is gathering increasing accumulated attention from public health officials as well as the relevant academic community. 2,3,8 With respect to S. suis epidemiology, we believe that current situation of human SS2 infections should be re-evaluated using comprehensive approaches. To minimize the occupational infections by S. suis, it is suggested that advances be made to improve public awareness of S. suis infection by science education and popularization. Although progress has been obtained toward understanding S. suis pathogenicity (especially identifying a group of bacterial virulence factors), it still sounds fragmentary, and lacks an insightful dissection of integrated regulatory networks of bacterial virulence. In the future, an important direction would be to link posttranscriptional regulation (e.g., ncRNA and riboswitch) and modification (such as acetylation and de-acetylation) to the bacterial virulence of Streptococcus suis. 190 Moreover, structural information on these virulence factors is very limited ( Table 2). We therefore believe it is necessary to strengthen the study of pathogenesis-related structural biology, which could establish a solid basis for design of small molecule drugs targeting bacterial virulence factors.
It is of interest to search for other virulence-related or immunological regulatory elements, different from 89K PAI, because of recent exciting findings on this issue. 160 Safe and effective vaccines that can be used for patients is still not available, which is due to lack of comprehensive knowledge of S. suis infections. Description of a full picture of S. suis surface antigen proteins might be helpful to screen and/or design vaccine molecules candidate. The fact that non-SS2 serotypes of S. suis can affect humans and even lead to fatal infections, has put the situation of S. suis infection in a much more complicated and serious status in public health. This might be an alternate way to decode the genome sequences of all 35 kinds of different serotypes, providing new insights into evolution and diversity of heterogeneous species, as well as distinct clues for preventing and controlling severe infections by these pathogens. 191,192 Systematic proteomic approaches are also encouraged to further address this question, which can complement genomics-based explorations. 138 In summary, our understanding and response to the situation of S. suis infections occurring especially in southeast Asia is not satisfactory. It would be helpful to integrate representative virulent/avirulent strains from different countries/regions for collaborative investigations. Therefore, there is a long way to go toward the complete conquest of S. suis, an emerging human pathogen.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.