Uncovering newly emerging variants of Streptococcus suis, an important zoonotic agent

Review

Uncovering newly emerging variants of Streptococcus suis, an important zoonotic agent Youjun Feng1,2, Huimin Zhang1, Ying Ma1 and George F. Gao1,3 1

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China 2 Department of Microbiology, University of Illinois, Urbana, IL 61801, USA 3 Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China

Streptococcus suis is recognized as a major swine pathogen and an emerging zoonotic agent. Two largescale outbreaks of severe S. suis epidemics occurred in China in 1998 and 2005 that posed serious concerns to public health and challenged the conventional conception that opportunistic infections of S. suis serotype 2 (SS2) in humans were only sporadic cases. An extensive, collaborative study on Chinese SS2 variants, which exhibit strong invasiveness and high pathogenicity, has resulted in the description of a new disease form of streptococcal toxic shock syndrome (STSS) and a putative pathogenicity island (termed 89K). The abbreviation of STSS is used for the severe disease caused by both Staphylococci and Streptococci. The main virulence factors involved in STSS caused by either Staphylococcus aureus or Streptococcus pyogenes consist of so-called superantigens or molecules that trigger a nonspecific, uncontrolled activation of T cells and massive cytokine release. However, although a collection of new virulence factors have been described, no superantigen candidates have been found for SS2 strains, implying that a different mechanism could be involved in the STSS form caused by SS2 variants. Epidemiology of Streptococcus suis in China In China, streptococcosis is a class II enzootic disease that can cause enormous economic loss in the swine industry, and thereby is in great demand of strict control and fast eradication. It is also a severe zoonosis (i.e. it can be transmitted to people) worldwide and can be clinically manifested with meningitis, arthritis and septicemia [1,2]. Streptococcus suis is an etiological agent for this infectious disease, as initially determined in Holland and England in the 1950s [2]. Since then, this pathogen has been found in over 30 countries, including the USA [3,4], Japan [5], Vietnam [6,7] and Thailand [8], and 35 known serotypes are differentiated on various capsular antigens. Since the discovery, in 1968, that an infection of S. suis serotype 2 (SS2, a highly virulent type) could cause human meningitis, more than 550 human cases worldwide have been recorded [1,6,9]. In Hong Kong, the first human case of S. suis meningitis was reported in 1983, which was a consequence of occupational exposure to pigs and pork [10,11]. Since then, Corresponding authors: Feng, Y. ([email protected]); Gao, G.F. ([email protected])

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more than 60 patients admitted to hospitals were confirmed to be as a result of S. suis infections in Hong Kong [10,12,13], and three cases of S. suis meningitis have been recorded in Taiwan [14,15]. In mainland China, the earliest S. suis infections in swine were observed in Guangdong Province, in the 1990s [1]. The collected epidemiological data showed that S. suis cases occur mainly in South China (Figure 1), in the period ranging from summer to autumn, which could be in part explained by local conditions characterized by high moisture and high temperature [16,17]. Recently, Wei et al. [18] systematically analyzed 407 strains of S. suis collected in China from 2003 to 2007 and indicated that SS2 is most prevalent (43.2%), followed by serotype 3 (14.7%) among the diseased pigs. Similar results have been obtained from samples in Europe and Canada [2]. In 1998 and 2005, two big SS2 outbreaks occurred in China (Figure 1) and raised serious concerns to global public health because of the high pathogenicity of these microbes [17,19]. In the 1998 epidemic, 25 people were infected and 14 died, and approximately 80,000 pigs were infected [17]. In the 2005 epidemic, 38 people died, out of 215 infected patients, and over 600 pigs were also confirmed to be infected, implying a possible route of SS2 transmission from pig to human [19]. More importantly, a new severe form of streptococcal toxic shock syndrome (STSS) was observed in all patients during the two SS2 outbreaks, indicating that the pathogen might be a new virulent variant [17]. In 2007, three sporadic cases of human SS2 meningitis were documented in China, and the presence of heterogeneous SS2 populations was found [20]. Collectively, SS2 infections in China are characterized by a coexistence of both outbreaks and sporadic cases. This situation contrasts with that found in other countries, such as Japan [5], Thailand [8] and the USA [3,4,16], where the occurrence of SS2 infections is only sporadic. Here, we discuss recent studies on the apparent molecular mechanisms by which the Chinese SS2 variants exhibit strong invasiveness and high pathogenicity. STSS, a new disease form caused by SS2 infections Toxic shock syndrome, originally found in Staphylococcusinfected patients [21,22], refers to a highly invasive infection of deep tissues associated with the production of

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Figure 1. Distribution of human SS2 infections in China. Two large-scale outbreaks are indicated in red, whereas all other sporadic cases are presented in yellow. Province names, year and number of human cases (in parentheses) are indicated. Adapted, with permission, from Ref. [20].

bacterial superantigens, a family of related substrates (e.g. staphylococcal and streptococcal exotoxins) that trigger an uncontrolled activation of T cells, and do not require processing and presentation by macrophage (Table 1) [23,24]. This disease was also observed in sporadic cases of human infections as a result of Streptococcus pyogenes, a group A streptococcus (GAS) [25,26]. Further studies have shown that M protein, a major virulence factor of S. pyogenes, plays a crucial role in the disease [27,28]. Thereafter, the syndrome was formally renamed as STSS. In general, STSS is defined based on the following clinical criteria: clear erythematous blanching rash, blood spots and petechia, sudden onset of high fever, hypotension diarrhea, and dysfunction of multiple organs (e.g. acute respiratory distress syndrome, liver and heart failure, disseminated intravascular coagulation and acute renal failure) [17,19,29]. Epidemiological and clinical data showed that nearly all death cases in the two SS2 outbreaks in China included the above STSS criteria [17,19]. This was unexpected because it was not known at the time that SS2 infections could cause this syndrome, and it indicated that a non-GAS Streptococcus, such as SS2, was capable of causing STSS. Retrospective studies indicated that SS2-triggered STSS cases had been recorded in Thailand [30] and France [31].

Recently, Tramontana et al. [32] also reported an Australian STSS case caused by SS2 infection. It would be of much interest to compare the SS2 isolates from the above cases to elucidate the molecular mechanism underlying STSS caused by SS2. In light of genomic analyses, Chen et al. [33] proposed that a specific pathogenicity island (PAI), known as 89K, might account for the high virulence of Chinese SS2, and even for the STSS manifestation of SS2infected patients. After immunological investigation, the Xu group presented a two-stage hypothesis to explain the process of STSS in SS2-infected human cases (Figure 2), where an early burst of proinflammatory cytokines (such as Th1 cytokines) is followed by the manifestation of disease by virulence factors, including suilysin, an important member of the thio-activated toxin family [34]. Although additional research is needed to test the above two models (i.e. the involvement of the 89K PAI [33,35] and the two-stage hypothesis [34]), these might be helpful as an initial framework to understand how the Chinese SS2 variants have the ability to cause STSS. Genomic and proteomic insights into virulence of Chinese SS2 variants Prior to the 2005 outbreak [17,19,68], the circulation of highly virulent SS2 in China was poorly understood. 125

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Table 1. Molecular mechanisms of bacterial toxic shock syndromea Bacterial agent Staphylococcus aureus Streptococcus pyogenes (GAS) Streptococcus dysgalactiae subsp. equisimilis (GCS) Streptococcus equi subsp. zooepidemicus (GGS) Streptococcus suis

Molecular determinant (superantigen) Enterotoxin M protein Unknown

Refs. [21–24,71–73] [25–27,74] [75]

Unknown

[17,19,31–33,62]

a

Abbreviations: GAS, GCS and GGS denote groups A, C and G of Streptococcus, respectively.

Therefore, the emergence of severe outbreaks not only seriously challenged public health but also shocked the scientific community, evidencing the urgent need for basic and translational studies of these SS2 strains. In 2005, functional genomics and proteomics studies of Chinese SS2 were systemically conducted after the epidemic in Sichuan Province [17]. A unique DNA fragment of 89 kb was found to be specific to the STSS-causing SS2 strains, and named 89K [33]. The 89K PAI encodes a two-component signal transduction system, SalK–SalR (Figure 3), which was shown to be required for full bacterial virulence [35]. Recent comparative genomics analyses of SS2 strains isolated from China and Vietnam showed insights into rapid evolution of virulence and drug resistance of these infectious agents, and indicated that 89K possesses a structure similar to integrative conjugative elements [36].

A proteomics approach was also employed to study SS2 [37–39]. Two-dimensional electrophoresis combined with mass spectroscopy was used to identify 373 proteins from 834 processed spots [39]. Among them, several virulence factors were determined, including muramidase-released protein (MRP) precursor, extracellular factor and suilysin (Table 2). Two enzymes (enolase and endopeptidase) were proposed as putative virulence-associated factors [39]. Zhang and Lu [40,41] reported an immunoproteomics analysis of two Chinese virulent SS2 strains from the 1998 and 2005 STSS epidemics, respectively. They identified 34 proteins, 15 of which were recognized by both hyperimmune sera and convalescent sera. In different studies, two new immunogenic proteins were identified, in addition to MRP: surface antigen one (Sao) [42,43] and glyceraldehyde-3-phosphate dehydrogenase [44,45]. Although these

Figure 2. Two-stage hypothesis for STSS caused by SS2. In stage I, SS2 enters blood vessels via an unknown mechanism, and leads to an early burst of proinflammatory cytokines, including Th1 cytokines, interleukin (IL-1b) and tumor-necrosis factor (TNF-a). These inflammatory super-responses could result in STSS with death as early as 13 h after SS2 infection. During stage II, which develops over several days, SS2 uses virulence factors such as suilysin to cause disease, particularly meningitis. Modified, with permission, from Ref. [34]. Abbreviation: CNS, central nervous system.

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Figure 3. Characterization of 89K PAI. (a) Cartoon depiction for 89K PAI. The 89K PAI is expressed with a golden rectangle. The salK–salR two-component system (in grey) is indicated with an arrow. The dashed lines represent the genomic sequences outside of 89K. (b) The value of GC% for 89K PAI. The average GC value is 36.8% for 89K, while 41.1% for the whole genome of Chinese SS2 virulent strain, 05ZYH33. Adapted from Ref. [20,35] with permission.

proteins are not specific to the studied SS2 virulent strain (05ZYH33), they might be involved in the severe infections and subsequent pathological process. Taken together, the comparative and functional genomics analyses have partially elucidated the genetic basis for SS2 virulence and established a specific link between the 89K PAI and the Chinese SS2 isolates. However, the speculation that 89K probably is of essence of an integrative conjugative element still lacks biological evidence [36]. Additionally, immunoproteomics studies suggest that some known virulence factors are also involved in the pathogenesis of these strains. Recently, a genome-wide screening of potential candidates for protective vaccine antigens against SS2 has been reported [46]. Newly identified elements related to virulence In addition to known virulence-associated factors such as MRP, capsular polysaccharide or suilysin [2], other bacterial components have been shown to be related to SS2 virulence (Table 2) [35,47–53]. SalK–SalR, a two-component signal transduction system, was identified from the 89K PAI

of a STSS-causing strain, O5ZYH33 [35]. Disruption of salK–salR attenuates greatly the virulence of this pathogen, whereas functional complementation restores it in experimental infection of piglets. The attenuated virulence of this mutant can, in part, be attributed to decreased colonization capability in susceptible tissues of piglets and lower resistance to polymorphonuclear leukocyte-mediated killing [35]. Although the molecular mechanism or the regulatory network associated with SalK–SalR is not clear yet, these studies provide direct evidence for 89K being a functional PAI [35]. Cell wall components are important virulence factors for bacterial pathogens. Fittipaldi et al. [47] showed evidence that the absence of lipoteichoic acid D-alanylation enhances S. suis susceptibility to the action of cationic antimicrobial peptides and impairs its pathogenicity (Table 2). Moreover, inactivation of the pgdA gene, encoding peptidoglycan N-acetylglucosamine deacetylase, led to mutant SS2 strains with significantly attenuated virulence in mice and piglets [54]. This suggests that modification of peptidoglycan by N-deacetylation is important for S. suis virulence. Although the pgdA gene was originally identified

Table 2. Factors associated with virulence of Streptococcus suis serotype 2 Gene Function or gene product Known virulence factors Extracellular protein factor epf Glutamate dehydrogenase gdh Capsular polysaccharide cps Suilysin, thio-activated hemolysin sly Fibronectin binding protein fbp Muramidase-released protein mrp Newly identified components Surface antigen protein sao Two-component signal transduction system in the 89K PAI salk-salR Enzyme catalyzing lipoteichoic acid D-alanylation dltA Peptidoglycan N-acetylglucosamine deacetylase pgdA Transpeptidase mediating covalent linkage of srtA surface proteins to peptidoglycan Di-peptidyl peptidase IV dppIV Orphan response regulator covR Enolase for dehydration of 2-phosphoglycerate eno to phosphoenolpyruvate Inosine 5-monophosphate dehydrogenase impdh Glutamine synthetase glnA

Origin

Refs.

Pig isolate (Netherlands) Strain 1933 (USA) Strain S735 (Netherlands) Strain P1/7 (Netherlands) Pig isolate (Netherlands) Pig isolate (Netherlands)

[76] [77–79] [80,81] [82–85] [86] [76]

Strains 89/1591 (Canada) and 05ZYH33 (China) Strain 05ZYH33 (China) Strain 31533 (France) Strain 31533 (France) Strains NCTC10234 (Canada) and 05ZYH33 (China) Strain 05ZYH33 (China) Strain 05ZYH33 (China) Strains 166 (France) and 05ZYH33 (China)

[37,42,43,52] [35] [47] [54] [48,52]

Strain SS2-Ha (China) Strain SC19a (China)

[53] [61]

[50] [51] [49,58–60]

a

Strains SS2-H and SC19 are isolates clinically from diseased pigs in China, 05ZYH33 is a human isolate from the STSS patient infected in SS2 outbreak, in China, in 2005.

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Review from S. suis 31533, a Canadian virulent strain, it is also present in the Chinese virulent isolate, 05ZYH33 [33]. Sortase A, a transpeptidase originally found in S. aureus, specifically cleaves the LPXTG amino acid sequence between the T and G residues and mediates the covalent linkage of bacterial surface proteins to the peptidoglycan [55,56]. Genetic evidence from two independent research groups demonstrated that the SS2 srtA homolog is associated with full virulence of this pathogen [48,52]. In particular, two important virulence-associated surface proteins, MRP and Sao, featuring a C-terminal LPXTG motif, were absent in an isogenic srtA mutant [52]. These findings can explain why the mutant displayed significantly reduced capacity for adherence to human cells [52]. Di-peptidyl peptidase IV (DPP IV), originally recognized as an antigenic enzyme (CD26) on the surface of eukaryotic cells, is widely distributed in microbial pathogens. Recently, a dppIV homolog in a highly invasive isolate of Chinese STSS-causing SS2 was identified, and it was shown that inactivation of dppIV attenuates greatly the virulence of this strain [50]. Interestingly, Pan et al. [51] recently reported that CovR, an orphan response regulator, negatively regulates virulence of S. suis 05ZYH33, a STSS-causing strain. Disruption of covR resulted in a mutant strain with increased hemolytic activity, enhanced adherence to epithelial cells, higher virulence in piglets and increased ability to colonize susceptible tissues of piglets. Microarray analyses suggests that CovR represses the expression of hundreds of genes, some of them encoding known or putative virulence factors. The Sao protein was first reported by Li et al. as a surface protein reacting with convalescent-phase sera from pigs infected with SS2 [42]. Later, three allelic variants of the sao gene were reported, namely sao-S, sao-M and saoL, based on their different lengths (1.5, 1.7 and 2.0 kb, respectively) among S. suis isolates [43]. These differences are caused by heterogeneity within the number of C-terminal repeat sequences (R) [43], which are related to pathogenicity in the plant pathogen Xanthomonas oryzae [57]. A Sao-based ELISA method shows promise for laboratory, clinical and even field monitoring of S. suis infection in the swine industry [43]. Indeed, it has greatly contributed to fast diagnosis of three newly recurrent cases of human SS2 infections in China [20]. Finally, enolase is generally considered to be a glycolytic enzyme, catalyzing the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. However, it has recently been shown that S. suis enolase can be exported to the bacterial surface [58,59]. Furthermore, it appears to function as a novel protective antigen conferring full protection upon mice against SS2 attack [49], although some controversy on the protective efficiency exists [60]. Surprisingly, genetic studies showed that two other enzymes associated with central metabolism also contribute to SS2 pathogenicity: glutamine synthetase [61] and inosine 5-monophosphate dehydratase [53] (Table 2). Heterogeneity of Chinese SS2 variants Multilocus sequence typing of S. suis has demonstrated that the 2005 epidemic strains in China are grouped into 128

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the same Sequence Type 7 (ST7), on the basis of systematic analyses of the following housekeeping genes: cpn60, dpr, recA, aroA, thrA, gki and mutS [62]. The ST7 strains are believed to be a single-locus variant of sequence type 1 (ST1). Ye et al. [63] further showed evidence that ST7 is spreading in China. Similarly, Mai et al. [6] reported that ST1 appears to be prevalent in the neighboring country, Vietnam, claiming 151 human cases of SS2 infections from 1996 to 2005. After comparative genomics of S. suis, it has been suggested that the 89K PAI can function as a new, specific virulence marker for Chinese SS2 clones [33], and a set of primers has been designed to identify 89K [20,33]. Remarkably, all the virulent strains carrying 89K are ST7 strains (Figure 3) [20]. By contrast, 89K is heterogeneous among the isolates from sporadic cases of human SS2 meningitis that occurred in 2007 in China [20]. Pulsed-field gel electrophoresis analyses showed variations between three isolates (07CQH01, 07SZH01 and 07SZH02), in part attributable to gene deletions in 89K [20]. Also, the sequence type of these isolates is variable, being either ST1 or ST7. Therefore, it seems that heterogeneous SS2 populations are circulating in China [20]. At present, the origin and evolution of these strains are poorly understood, and it is possible that a new virulent clone will emerge in the future. For this reason, it is urgent that a comprehensive system for surveillance and prevention is established soon. Concluding remarks and future perspectives The Chinese SS2 virulent variants have been regarded as an emerging infectious entity, posing great concerns to public health [1,9,13]. Systematic studies at multiple levels, from epidemiology [17,19], molecular genetics and immunology [35,54,64–67] to functional genomics and proteomics [33,37–39,68], have been carried out to understand SS2 pathogenesis. These studies have yielded fruitful outcomes, including the discovery of a new form of STSS that is caused by SS2 (a non-GAS Streptococcus) [17], the 89K PAI as a specific virulence marker [20,33,35] and the recognition of heterogeneity among Chinese SS2 strains [20]. Nevertheless, there are still many unresolved questions (Box 1). For a better understanding of the evolution and circulation of SS2 strains, it is necessary to conduct a Box 1. Outstanding questions  How prevalent is SS2 in China? In addition to the clonal ST1 complex (ST1 and ST7) [62], are there any other sequence types?  From the available epidemiological data [19,20], can we estimate whether a big outbreak of human SS2 infection is likely to occur? Can we predict when and where would it happen? How could we prevent or minimize the effects of such an outbreak?  Are there any superantigens involved in STSS caused by SS2? If not, is the disease mechanism better explained by the presence of the 89K PAI or by the two-stage hypothesis?  Is 89K present in all Chinese virulent SS2 strains? Is it present in all virulent isolates in neighboring countries (e.g. Thailand and Vietnam)?  What other genes, in addition to salK–salR, are associated with SS2 pathogenicity?

Review complete epidemiological survey across China. This information might help to prevent or control future epidemic outbreaks. Although the 89K DNA fragment has been proposed as a PAI to partially account for high virulence of Chinese SS2 variants, experimental evidence is limited [33]. Although the salK–salR two-component system has been shown to be essential for full virulence, all other genes encoded by 89K still lack solid functional verification. Bacterial two-component systems, consisting of a sensor protein–histidine kinase and a response regulator, enable bacteria to sense, respond and adapt to changes in their environment or in their intracellular state. Thus, it is of much interest to dissect (i) what signal is specifically detected by the SalK sensor, and (ii) which genes are directly regulated by SalR. Additionally, 14 other twocomponent systems are predicted in a Chinese SS2 strain [33] — do any of them contribute to virulence control too? Moreover, it would be interesting to identify the physiological ligand (if any) for CovR, an orphan repressor, and the genes under its control. The future resolution of the structures of virulencerelated proteins and their complexes with antibodies or ligands will provide new insights into SS2 pathogenesis. It will also establish the basis for rationale design of smallmolecule therapeutic drugs that could be useful to prevent or antagonize the adherence, virulence or growth of SS2 strains. The recently-resolved crystal structures of two SS2 enzymes may serve as a model for structure-based drug design for targeting SS2 central metabolism [69,70]. Finally, the identification of protective antigens seems a promising strategy for developing engineered subunit vaccines that might be safer than the conventional, inactivated SS2 vaccine used in the swine industry. In particular, tandem fusion expression of poly-protective antigens could result in a robust multiple-subunit vaccine candidate. For this purpose, it is necessary to screen SS2 surface proteins in search for possible protective antigens [46,49,58]. Nevertheless, searching for a naturally avirulent SS2 strain and evaluating its safety and efficacy as an improved alive vaccine candidate will also be an appealing challenge. The use of mouse or swine infection models will allow further study of the interplay between SS2 and its host cells. Hopefully, upcoming multidisciplinary research will provide us, in the coming years, with effective tools for better prevention and treatment of STSS caused by SS2. Acknowledgements This work was supported by the China Ministry of Science and Technology (‘‘973’’ Grant No. 2005CB523001). G.F.G. is a distinguished young investigator of the National Natural Science Foundation of China (NSFC) (Grant No. 30525010).

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