Critical Streptococcus suis Virulence Factors: Are They All Really Critical?

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Review

Critical Streptococcus suis Virulence Factors: Are They All Really Critical? Mariela Segura,1 Nahuel Fittipaldi,2,3 Cynthia Calzas,1 and Marcelo Gottschalk4,* Streptococcus suis is an important swine pathogen that can be transmitted to humans by contact with diseased animals or contaminated raw pork products. This pathogen possesses a coat of capsular polysaccharide (CPS) that confers protection against the immune system. Yet, the CPS is not the only virulence factor enabling this bacterium to successfully colonize, invade, and disseminate in its host leading to severe systemic diseases such as meningitis and toxic shock-like syndrome. Indeed, recent research developments, cautiously inventoried in this review, have revealed over 100 ‘putative virulence factors or traits’ (surface-associated or secreted components, regulatory genes or metabolic pathways), of which at least 37 have been claimed as being ‘critical’ for virulence. In this review we discuss the current contradictions and controversies raised by this explosion of virulence factors and the future directions that may be conceived to advance and enlighten research on S. suis pathogenesis.

Trends Streptococcus suis is a major swine pathogen – an emerging zoonotic agent whose pathogenesis of disease is partially characterized. S. suis is an encapsulated microorganism and its capsular polysaccharide (CPS) allows bacterial evasion of the host immune system and bloodstream dissemination. But, the CPS is not the only virulence factor and, under certain circumstances, its absence may also be beneficial to pathogenic strains. Indeed, the immune-pathogenesis of S. suis-induced disease is a complex, multifactorial process.

Understanding the Virulence of Streptococcus suis: An Emerging or Reemerging Pathogen The global willingness to reduce the preventative use of antibiotics in food-producing livestock in combination with the lack of effective vaccines has led to the re-emergence of animal bacterial pathogens with a high potential of zoonosis (see Glossary). This is the case of Streptococcus suis, a Gram-positive bacterium which causes meningitis, sepsis, and other diseases in swine. This infection not only results in severe economic losses and raises concerns on animal welfare, but the organism is also an agent of invasive diseases in humans, particularly among workers in the swine and pork industry, or people consuming raw or undercooked pork or pork by-products [1]. Besides this re-emergence of S. suis disease in swine, the identification of a new, highly virulent clone, responsible for human outbreaks in China [2], suggests that there is an evolution of this pathogen's virulence traits, leading to increased pathogenicity and/or better cross-species adaptation. This epidemic clone brought out the concept of S. suis as an emerging human pathogen. Either emerging or re-emerging, S. suis undoubtedly poses a threat to public health. Indeed, human S. suis cases have been reported in most of Western Europe, Canada, the United States, Chile, Argentina, Australia, New Zealand, India, Japan, and several other East and South East Asian countries (recently reviewed in [3]). In these latter countries, particularly China, Vietnam, and Thailand, S. suis has been shown to be responsible for thousands of human disease cases and has been identified as one of the most, or the most, common cause of adult bacterial meningitis [4,5]. Research on this pathogen, neglected for years, experienced an upsurge of studies aimed at dissecting the molecular components of the pathogenesis of disease and the discovery of

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Yet, the discovery of a large array of virulence factors claimed as ‘critical’ for the pathogen's virulence has brought confusion into the field of S. suis. The large genetic diversity of the S. suis species further complicated the study of the pathogenesis of the disease.

1

Laboratory of Immunology, Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, QC, Canada 2 Public Health Ontario Laboratory, Toronto, ON, Canada 3 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada 4 Streptococcus suis Laboratory, Faculty of Veterinary Medicine, University of Montreal, Saint-

http://dx.doi.org/10.1016/j.tim.2017.02.005 © 2017 Elsevier Ltd. All rights reserved.

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new virulence factors or reconsidering old virulence factors. Albeit of extreme importance in our understanding of molecular pathways and host–bacteria interactions, extrapolations from in vitro studies might have led to misinterpretation of the actual role of a putative virulence factor or bacterial component in the disease process. The use of different animal models, routes of infection, and other diverse in vivo experimental approaches added another level of [234_TD$IF]complexity to the elucidation of the pathogenesis of disease caused by S. suis, resulting in overall confusion rather than enlightenment. Finally, the advent of molecular typing and whole-genome sequencing revealed the extreme heterogeneity of S. suis isolates, making our efforts to unveil the key virulence traits of this pathogen almost unachievable. The scope of this review is to provide a critical perspective of our current knowledge of S. suis virulence factors, to highlight contradictions and controversies and, finally, to address the future directions that may be conceived to advance and enlighten research on this pathogen.

Streptococcus suis: A Highly Diverse Species The majority of porcine S. suis infections are caused by strains of a relatively small number of serotypes (Box 1). Albeit the distribution of serotypes from clinical cases differs depending on the geographic location (Box 1), serotype 2 strains are by far responsible for the majority of cases in both swine and humans worldwide, and thus have been historically considered the most frequent and virulent type [3]. Besides this serotype, recent years have seen the emergence of serotype 9 strains among swine diseases in several European countries [9]. A first human case due to this serotype has recently been described [10]. One striking observation over the years has been that the percentage of S. suis serotype 2 strains recovered from diseased pigs is lower in North America than in other parts of the world [3]. In addition, in most cases, serotype 2 disease in North America appears to be opportunistic, and develops concomitantly with other bacterial or viral infections that impair the immune system of the host. In addition, in this part of the American continent, human S. suis disease cases are rarely reported. In fact, it has been shown that circulating serotype 2 strains in North America are less virulent and genetically unrelated to those causing disease in other [235_TD$IF]parts of the world [11]. The genetic diversity of S. suis is better evidenced by multilocus sequence typing (MLST) [12]. As of October 2016, more than 704 sequence types (STs) were known in S. suis (http://ssuis.mlst. net/). Most common STs of S. suis serotype 2 are displayed in Figure 1 and further described in Box 2. The epidemiological data based on serotyping (Box 1) and MLST (Figure 1, Box 2) highlight not only the high genotypical/phenotypical diversity of S. suis strains (within the same serotype and among serotypes) but also the difficulty in correlating virulence with a set of well-defined genetic traits. The advent of whole-genome sequencing has contributed to the identification of further diversity within the S. suis species, as evidenced by 957 published genomes of various serotypes and STs (https://www.ncbi.nlm.nih.gov/genome), yet a vast majority are incomplete annotations. Indeed, only 26 of them are circularized, closed-genome sequences.

Box 1. Streptococcus suis Serotypes S. suis strains are primarily classified into serotypes on the basis of a serological reaction directed against the capsular polysaccharide (CPS). Until relatively recently, 35 serotypes (1 to 34 and 1/2) were recognized. However, serotypes 20, 22, 26, 33, 32, and 34 have been shown or hypothesized to belong to novel bacterial species [6]. Globally, the predominant S. suis serotypes isolated from clinical cases in pigs are, in decreasing order, serotypes 2, 9, 3, 1/2, and 7. Yet, in North America, both serotypes 2 and 3 are the two most prevalent serotypes isolated from clinical pig cases, followed by serotypes 1/2, 8 and 7 [3]. Nontypable S. suis strains are also frequently isolated, mainly from healthy carrier pigs. Although some of these strains are probably nonencapsulated, they may also represent new uncharacterized serotypes expressing novel capsules. Indeed, very recently, 17 novel S. suis CPS loci (NCLs), mostly from strains recovered from healthy animals, have been described [7,8].

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Hyacinthe, QC, Canada

*Correspondence: [email protected] (M. Gottschalk).

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7

1 28 1

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Japan

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1 25 28

28

1

7

Hong Kong

1 104

1 1

Vietnam Thailand Cambodia 28

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1 25

Australia

Figure 1. Most Important Sequence Types (STs) of Streptococcus suis Serotype 2 as Determined by Multilocus Sequence Typing (MLST). ST1 serotype 2 strains are mostly associated with disease in both pigs (where data are available) and humans in Europe, Asia, Africa, and South America (Argentina). ST7, a single-locus variant of ST1, is endemic to mainland China. The situation is different in North America, where few clinical ST1 cases of infection in pigs and only one human ST1 case has been described. Indeed, North American serotype 2 strains belong mainly to ST25 (human and pigs) and ST28 (pigs only). The latter ST is also associated with swine clinical cases in mainland China, Australia, and Japan. Interestingly, Japan and Thailand are the only countries also reporting ST28 human cases. Besides North America, human cases of ST25 have been reported in Australia and Thailand. Finally, ST20 is prevalent only in Europe (mostly in The Netherlands). In this figure, numbers (1, 20, 25, 28, 104) in the different hosts represent different STs (i.e., ST1, ST20, ST25, ST28, ST104) and each ST has been attributed with a different color.

Clonal complex: a group of bacterial strains, derived from a common ancestor, which share many alleles at various phylogenetically informative loci. As such, the clonal complex usually includes the ancestral genotype and strains with minor variations. As this term has a broad meaning in epidemiology and may be defined by different typing methods, here, sequence types (STs) are grouped into clonal complexes by their similarity to a central allelic profile. Multilocus sequence typing (MLST): a molecular biology technique that characterizes isolates of microbial species using the sequences of internal fragments of (usually) seven house-keeping genes. The obtained genotypes are named sequence types (ST) and correspond to different alleles at the seven housekeeping loci. Pathogenesis of disease: cellular and molecular mechanisms and events underlying the development of a clinical outcome (disease). The word comes from the Greek pathos (‘disease’) and genesis (‘creation’). Virulence factors: are molecules produced by pathogens that contribute to the pathogenicity of the organism by allowing its establishment, replication, dissemination, and persistence in the host. Virulence traits: a set of virulence strategies or factors that contributes to the pathogenesis of disease. Zoonosis: is any disease or infection that is naturally transmissible from vertebrate animals to humans.

Nevertheless, genome comparisons between different strains and/or serotypes have identified major variations in gene content [14,19–23]. Extensive genomic comparisons involving a sample of >300 strains of different serotypes and genetic backgrounds, although of limited geographic diversity (United Kingdom and Vietnam only), identified genetic differences between systemic, respiratory, and carriage S. suis isolates, and a generalized tendency that diseasecausing isolates have significantly smaller genomes than nonclinical isolates [23]. The latter were found to possess many mobile genetic elements and were more prone to recombination. It was speculated that these findings may be associated with the different ecological specialization of the different organisms, as the respiratory tract (tonsils) was more ecologically diverse than systemic locations [23]. However, it is apparent that systemic isolates of these more virulent strains with reduced genomes are also carried in the same nasopharyngeal niche. More recently, a genomic investigation compared zoonotic serotype 2 isolates belonging to clonal complex (CC)20 with several other strains belonging to either other serotypes or clonal

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Box 3. Overview of Streptococcus suis Epidemiology and Pathogenesis of Infection Pigs and wild boars are considered to be the S. suis natural reservoir, although the organism has occasionally been isolated from many animal species [24]. Healthy pigs carry multiple S. suis serotypes/strains, mainly in their upper respiratory tract (nasal cavities and tonsils) [25]. Vertical transmission from colonized sow to piglets is thought to occur in the genital tract during farrowing, while horizontal transmission appears to be mainly due to nose-to-nose contact [24]. Septicemia with sudden death, meningitis, arthritis, and endocarditis are the most common diseases observed in pigs when S. suis is the primary agent. In humans, although a large variety of infection outcomes have been described, meningitis and septic shock (including streptococcal toxic shock-like syndrome) are the most frequent clinical manifestations [26–28]. One major difference in the epidemiology of human versus swine infection is that the bacterial route of entry is mainly through skin abrasions and the oral route (after consumption of contaminated raw pork products). The respiratory route of infection has never been demonstrated for S. suis in humans [25]. Independently of the infection site, S. suis must breach host mucosal (skin) barriers in order to disseminate and persist in the bloodstream, later invading multiple body organs including spleen, liver, kidney, lung, and heart. Access to the central nervous system (CNS) is gained by crossing the blood–brain barrier (BBB) and/or the blood–cerebrospinal fluid barriers [26].

complexes. Again, it was reported that zoonotic isolates have smaller genomes than do nonzoonotic isolates, but appear to contain more ‘virulence factors’ [20].

Historical Issues with S. suis Virulence and Virulence Factor Identification As mentioned above, and even though an important diversity among S. suis strains has been observed, most studies on virulence factors and the pathogenesis of the infection are based on archetypal serotype 2 strains, either virulent ST1 European strains or the clonal, epidemic ST7 Chinese strain. Figure 2 provides a simplified schematic view of the S. suis serotype 2 pathogenesis of infection (further described in Box 3). Only few studies have investigated a limited number of virulence traits in other STs or serotypes (such as serotype 2 ST25, ST104, or ST378, serotypes 1, 9, and 14) (see Tables S1–S5 in the supplemental information online). Early investigations on S. suis virulence and discovery of virulence factors have suffered from a number of issues, several of which continue to the present. Issue #1: Definition of Virulence Based on the Clinical Condition There is no generalized agreement among investigators in the S. suis field about what defines a strain as virulent, and on many occasions, strains have been defined as virulent or avirulent solely on the basis of the clinical condition of the animal from which the strain was recovered. Yet, the site of isolation might represent a confounding factor. For example, not all strains isolated from the tonsils of clinically healthy pigs are avirulent since pigs may be carriers of virulent strains. In addition, S. suis isolates from lung are often classified as ‘clinical isolates’; though S. suis is considered a secondary (opportunistic pathogen) in pneumonia cases. Furthermore, contamination of lung samples with S. suis strains from the normal upper respiratory tract microflora is common. In the case of an isolate from a human clinical case, one could infer that the strain is virulent (as it was transmitted from the pig and was able to induce disease in humans). Once again, patient predisposing factors might also represent a confounding criterion. Anyhow, confirmation of strain virulence by experimental infection would be required, which brings us to the next issue. Issue #2: Definition of Virulence Based on Experimental Animal Models Different investigators have used different infection models to assess the virulence of S. suis strains that are not readily comparable. These models go from the natural host, the pig, to surrogate models including different strains of inbred or outbred mice, other small mammals such as rabbits and guinea pigs, vertebrates such as the zebrafish, and unicellular eukaryotes such as the amoeba Dictyostelium discoideum (see Tables S1–S5). Advantages and disadvantages of each model are displayed in Figure 3 (Key Figure). Conclusions drawn from these surrogate models are not always transferable to swine and human disease, which makes virulence comparisons difficult to achieve. Among these models, mice have been shown to closely reproduce the typical

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CNS: Meningis Oral route of infecon Contaminated raw pork meat or typical (raw blood/meat) Asian dishes

Systemic pathology

Skin lesions: Main route of infecon

Other systemic pathologies

Intesnal normal inhabitant ?

CNS: Meningis Systemic disseminaon

Tonsils: S. suis carrier state

Resistance to gastric condions?

S. suis

Upper respiratory tract: Main route of infecon; (oral route?)

Arthris

Pneumonia: Secondary agent

Other species (possible reservoirs?)

Figure 2. Pathogenesis and Epidemiological Features of Streptococcus suis-Induced Disease. The main route of entry of S. suis in pigs is the upper respiratory tract, whereas in humans this infection occurs via skin lesion (handling of infected animals) or the oral route (ingestion of contaminated pork-derived products). After breaching the mucosal (skin) barriers, S. suis invades different organs and tissues through hematogenous and/or lymphogenous dissemination, leading mainly to arthritis and/or other multiple systemic pathologies (such as septic shock, polyserositis and endocarditis). Access to the central nervous system (CNS) is gained by crossing of the blood–brain barrier and/or the blood–cerebrospinal fluid barrier, leading to severe meningitis. S. suis has been isolated from other animal (See figure legend on the bottom of the next page.)

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Box 2. The Sequence Type (ST) Diversity of Streptococcus suis Serotype 2 ST1 serotype 2 strains are mostly associated with disease in both pigs and humans in Europe, some Asian countries such as Cambodia, China, Hong Kong, Japan, Thailand, and Vietnam, as well as Africa and Argentina [13]. ST7, a single-locus variant of ST1, is mostly endemic to mainland China and was responsible for the deadly human outbreaks that occurred in China in 1998 and 2005 [2]. MLST typing also showed that North American serotype 2 strains belong mainly to ST25 and ST28 [14,15]; while ST1, albeit present, is not predominant is this part of the globe. On the other hand, ST25/ST28 strains have also been isolated in Asia and Australia. Indeed, ST25 strains are common in Thailand, along with ST104 strains [16,17]. The hypothesis of lower virulence of North American strains being based on different genetics was confirmed by experimental infection of animals which showed that ST1 strains are significantly more virulent than ST28 strains, while ST25 strains showed an intermediate virulence [14,15]. However, differences in strain virulence belonging to the same ST have also been revealed. Indeed, ST28 strains are often isolated from diseased pigs in North America, China, Japan, and Australia, but ST28 human cases were reported only in Thailand and Japan [3,14,18].

clinical signs of S. suis disease, including a systemic phase (bacteremia/septicemia and septic shock) followed by central nervous system (CNS) invasion and development of nervous system clinical symptoms [29]. Certainly, the pig remains the most valuable model; yet S. suis swine experimental models are not easily reproducible. Indeed, variations in the type (traditional or minipigs), genetic background, age (which might have an impact on susceptibility based on the presence of maternal immunity), health status of the herd, and route of infection further complicate S. suis pathogenesis studies in the natural host. A usual mistake in the literature is the categorization of pigs as originating ‘from a S. suis-free herd’ [30], considering that S. suis is a normal inhabitant of swine microflora, and the S. suis carrier state of a herd is almost 100%. Similarly, some studies report the use of pigs from specific-pathogen-free (SPF) herds [31–38]. The definition of SPF pigs is still controversial but it does not include being free from S. suis, which would be almost impossible to achieve as S. suis is vertically transmitted to piglets at birth. In fact, when using animals from a commercial herd to perform experimental infections, the appropriate claim (and choice) would be ‘a herd free of clinical signs of streptococcal disease’ [39,40]. Animals can be further tested by serotype-specific PCR to better evaluate the carrier status [41,42]. Albeit[236_TD$IF], not exempt from possible false-negative results, this strategy represents the most suitable for selecting appropriate animals for S. suis experiments. Some studies also evaluate the presence of anti-S. suis serotype 2 antibodies by enzyme-linked immunosorbent assay (ELISA) prior to experimental infection [43–46]. Although informative, the lack of a validated serological test for S. suis precludes the use of ELISA results as a hallmark of the herd health status. Indeed, different levels of anti-S. suis antibodies (either specific or crossreactive) can be found in piglets [25], which represents another obstacle to comparative and reproducible experimental infections using swine models. Finally, caesarian-derived colostrumdeprived (CDCD) animals can be used [47,48]; this is a model that allows the absence of S. suis in tonsils as well as maternal immunity. However, results obtained can hardly be extrapolated to ‘normal’ piglets since CDCD animals are in general highly susceptible. The intraperitoneal (i.p.) followed by intravenous (i.v.) routes of infection are usually used in mice. In addition, features such as age of animals and volume inoculated may change the results obtained with a single strain [49]. In pigs, the i.v. route is prioritized, while i.p. or intramuscular routes are less frequently used. Yet, all of these parenteral infection methods bypass the natural route of infection of S. suis in swine, that is, upper respiratory tract colonization with subsequent hematogenous and/or lymphogenous dissemination. In this regard, intranasal (i.n.) infection was reported in both mice and pigs, with some inconsistent results obtained especially with pigs, where pathogenic strains may behave [237_TD$IF]with either high or low virulence when using this species, including birds, rabbits, cats, dogs, horses, cattle, fallow deer, and wild boars, complicating the global epidemiology of S. suis. It has been suggested that these animal species might act as reservoirs.

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Key Figure

Larvae

ie ud st

Warm-blodded, outbred (rabbits), Inbred/outbred (guinea pigs)

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co st, Hi gh

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No No Unclear?

Natural host

Yes Yes

Variability high cost low number ethics High

ncy

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y lit

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Cold-blodded, Inbred/outbred

rs be m nu

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d) die stu ly or po ?(

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Zebrafish

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ucon of Reprod res ase featu disese

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to ed mit , Li ies tud Ss CN No d, de

Low cost , Ea se of us e, Et hi cs

Amoeba Dictyostelium discoideum

s ge nta va ad Dis

Ad va nta ge s

The Complexity of Animal Models to Evaluate Streptococcus suis Virulence

Very low Low Very low

High

?

Key characteriscs

Figure 3. Summary of the advantages, disadvantages, and key characteristics of described animal models. The pig and mouse models are the most frequently used. Other small mammals, such as rabbits and guinea pigs, have been used in the past at very low frequency (only three published studies) [102–104]. In rabbits a study reported discrimination between virulent wild-type strain and avirulent mutant strain [103]. However, another study reported failure of this model to discriminate the virulence of a given strain [104]. One study reported the use of guinea pigs as a model of hearing loss during S. suis meningitis [102]. Due to their limited use, the advantages/disadvantages of these two models are not clearly established. A limited number of studies [23_TD$IF](20) reported the use of adult or larvae zebrafish to measure the lethal dose (LD)50 [105,106]. Yet, the major limitation of this vertebrate model is that central nervous system (CNS) pathology cannot be evaluated. Finally, only two studies have addressed S. suis-induced mortality rates using the amoeba Dictyostelium discoideum, a unicellular eukaryote model [107,108] (see also Tables S1–S5 for more details on these different animal models). Very recently, a study reported the potential use of wax moth (Galleria mellonella) larvae as a model to assess virulence of S. suis strains [109]. This newly developed model allows experiments to be performed at the host temperature (37 C); yet only mortality rates can be recorded. More studies are needed to validate this new model. Lastly, it remains largely unknown if scientific findings obtained with the aforementioned models can be extrapolated to humans.

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route of infection [50,51]. Indeed, previous mucosal irritation by acetic acid (mice/pigs) or Bordetella bronchiseptica preinfection (pigs) is normally required [238_TD$IF]in order to observe disease (Tables S1–S5) [25]. Interestingly, this route of infection does not seem to be adequate to reproduce disease with virulent serotype 9 strains [52,53]. In conclusion, there is an urgent need to reach a consensus in the experimental conditions as well as the inbred/oubred mouse strain to be used, the most appropriate and reproducible swine model, the route of infection in both mice and pigs, and the clinical scores to measure disease outcome. Finally, animal models for S. suis serotypes, other than serotype 2, still require extensive standardization [25,54]. Issue #3: Definition of Virulence Based on in vitro Tests Different in vitro approaches are commonly used to evaluate the impact of a ‘virulence factor candidate’ on bacterial capacity to interact with host cells or components. These results (normally comparing a knock-out mutant versus its wild-type parental strain) are then combined or extrapolated to the in vivo situation, using one of the experimental models described above. Examples of these in vitro approaches are phagocytosis assays (with or without the presence of opsonins) using divers cell types as well as bacterial survival in blood (which represents an indirect measurement of S. suis capacity to resist blood leukocyte phagocytosis). Cells or blood from mouse, swine, or human origin are used (Tables S1–S5). In the case of whole-blood models of swine (or human, especially in Asia) origin, a drawback might be the possible presence of anti-S. suis antibodies which would bias the result obtained. Yet, in most cases, this parameter is not analyzed. A well-encapsulated S. suis wild-type strain is typically able to multiply and survive in vitro in blood (as anticipated for a systemic pathogen); a negative result should be interpreted with caution, and technical parameters, including the presence of antibodies, evaluated. Another example is the comparative evaluation of S. suis wild-type and mutant strain adhesion to/invasion of endothelial or epithelial cells (Tables S1–S5). The latter cell type is very frequently used, yet S. suis interactions with epithelial cells are poorly known. Although swine tracheal epithelial cells have been used in some cases [25], most in vitro virulence studies have been performed with human laryngeal epithelial (Hep-2) cells, despite the fact that the respiratory route of infection has not yet been demonstrated for human infections. Furthermore, in most [239_TD$IF]cases, these in vitro studies are correlated with loss of virulence in systemically infected animal models, which bypass interaction with mucosal epithelial cells. If the hypothesis is that a putative virulence factor is involved in the first steps of colonization and mucosal invasion, an i.n. infection model should be used to confirm such a theory. Recently, cultures of porcine-origin primary differentiated respiratory epithelial cells, such as porcine precision-cut lung slices and respiratory air–liquid interface cultures from the porcine lung, were successfully established [55,56]. These new model systems most closely resemble the situation of the airway epithelial cells in the host and will contribute to further advance our knowledge on the first steps of S. suis pathogenesis of infection.

The Controversy: Overabundance of Novel ‘Crucial' Virulence Factors

Bacterial virulence is ‘the ability to enter into, replicate within, and persist at host sites that are inaccessible to commensal species[240_TD$IF]’ [57]. The last comprehensive review of the putative role of S. suis virulence factors was published in 2012 [26], and the list of factors potentially involved in the pathogenesis of S. suis-induced disease rapidly expanded since. Tables S1–S5 extensively summarize all proposed S. suis virulence factors based on mutagenesis studies. One feature of more recent investigations is that many of these factors have been reported as critical for S. suis virulence. However, most of them are not yet thoroughly characterized, and the definition of critical (including clinical significance) may be challenged. Most of these critical virulence factors have been defined as a consequence of the availability of whole-genome sequencing and high-

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throughput techniques for the characterization of knock-out mutants. While virulence factors, such as the capsular polysaccharide (CPS), has been known for years and considered necessary yet probably not sufficient for infection (see below), others have been much less characterized. A factor with a precise and unique role during the pathogenesis of the infection can be considered critical, as its function cannot be performed by other bacterial components. On the other hand, some S. suis virulence factors have been categorized as critical, albeit having functions easily compensated by other factors. For example, at least 28 factors are reported as binding extracellular matrix components, and at least 34 factors are actively involved in adhesion to epithelial cells [25]. Of the latter, at least 13 factors are reported as being important virulence [241_TD$IF]factors, as evaluated through isogenic mutants. Excluding CPS knock-out mutants, at least 37 mutants deficient for a specific putative virulence factor/gene were reported as completely avirulent (Tables S2, S4, and S5). These facts obligate us to revisit the concept of redundancy in bacterial pathogenic attributes, an issue elegantly addressed by O’Connor et al. [58]. Redundancy, widespread among many organisms, can be defined as the arsenal of genes/factors that encode/perform a similar function. Redundancy equals pathogenic success and poses a tangible problem for researchers. If a redundant gene/protein is knocked out, its counterpart can still perform the function, and no clear phenotypic effects will be seen. As such, for many pathogens, redundancy complicates the analysis of virulence factors [58]. As mentioned above, S. suis is (or seems to be) a successful redundant pathogen, and the observed controversy in virulence studies using knock-out mutants might reflect, at least in part, the absence of well-standardized animal models, as discussed above. For a better characterization of S. suis critical virulence factors, mutagenesis and virulence studies should be confirmed by independent laboratories using well-characterized strains of different genetic backgrounds. This approach, which undoubtedly placed the CPS as one of the best characterized virulence factors (Table S1), also brought up contradictions. In fact, different results for the same virulence factor have been reported in the literature, and few examples are highlighted below. Immunoglobulin A (IgA), the principal antibody class in the secretions that bathe mucosal surfaces, acts as an important first line of defense. It prevents the adhesion of microorganisms to epithelial cells and facilitates their elimination from the host. Indeed, several species of pathogenic bacteria secrete IgA proteases at mucosal sites as a mechanism of immuneevasion [59]. Even if the development of a mucosal IgA response following S. suis infection has never been studied, the secretion of an active human IgA protease, encoded by the iga gene, has been reported [45,60]. A Diga mutant strain (derived from an ST7 strain) showed decreased lethality in an i.n. swine model [45]. Albeit the iga gene could be amplified from reference strains of serotypes 1 to 9 [60], another study failed to find evidence of IgA protease activity in different S. suis strains [61]. The actual IgA protease activity of the iga gene product is still controversial. It has to be noted that the IgA protease is part of the zinc metalloproteases family [61], suggesting the possibility of other functions for this factor. Further studies would thus be necessary in order to clarify these contradictory results and define the precise role of this protein. Factor H is a key negative regulator of the alternative complement pathway [62] that protects the host against complement-mediated damage. Many frank pathogens are able to recruit factor H to their cell surface and degrade C3b in iC3b in order to reduce opsonophagocytosis. Recruitment of factor H may also improve bacterial adherence to host cells as well as their invasive capacity [25]. A first factor H-binding protein (Fhb) was originally reported, and an Fhbdeficient mutant (in a ST7 background) was shown to be completely avirulent in a swine i.v. infection model and presented lower survival rates in whole human blood or in the presence of human neutrophils [36]. Yet, as recently demonstrated, S. suis possesses several redundant mechanisms to bind factor H, including a second factor H-binding protein (named as Fhbp) [63]

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and the CPS [64]. Consequently, an fhp  fhbp double knock-out mutant was shown to be resistant to clearance by opsonophagocytosis and, even a triple fhp  fhbp  cps knock-out mutant (in an ST1 background) was still able to bind human factor H, indicating that other factors, not yet described, are present in S. suis serotype 2 strains [64]. These controversies suggest that differences in strain (ST) background, experimental conditions, and, more importantly, animal models, might lead to completely different conclusions on the critical or noncritical (redundant) role for a given bacterial component.

Revisiting ‘Traditional’ Virulence Factors for S. suis CPS Is Undisputedly Considered the Major Virulence Factor but . . . S. suis survival in blood, and dissemination, depend on the production of a thick CPS which protects the bacterium against immune-recognition and immune-clearance [26]. Indeed, there is unanimity on published studies that used different in vitro and in vivo experiments with isogenic unencapsulated mutant strains, and all have conclusively shown that the absence of CPS correlates with highly increased phagocytosis and/or killing of these strains by phagocytic cells with a rapid clearance from circulation and absence of clinical signs (Table S1). The majority of these virulence studies have been performed with serotype 2 strains (ST1, ST25, and ST7 backgrounds). In fact, despite our recently increased knowledge on the CPS biochemical composition of other serotypes [65–67], findings obtained with serotype 2 unencapsulated mutants were so far only confirmed for serotype 14 [68]. [24_TD$IF]Albeit its critical role, encapsulation itself is not a determinant of survival in blood, as demonstrated by studies showing blood clearance of well encapsulated avirulent strains within 48 h, whereas a virulent encapsulated strain can persist in circulation at relatively high titers for several days [69,70]. As such, resistance to phagocytosis and immune-clearance is multifactorial, and does not exclusively rely on CPS production [26]. As important as encapsulation is for survival in blood, unencapsulation seems to confer important virulence properties on S. suis strains by enhancing bacterial adherence to, and invasion of, host cells as well as biofilm formation [25,71–73]. These strains might represent a reservoir of disease-inducing organisms in syndromes involving biofilm-like conditions, such as infective endocarditis [74]. It has been suggested that the frequent isolation of unencapsulated S. suis isolates from porcine endocarditis might result from the enhanced ability of these isolates to adhere to platelets and, consequently, to form septic vegetations [73,74]. It is unclear whether unencapsulation occurred after invasion of the bloodstream or whether CPS expression is negatively regulated by unknown mechanisms, such as phase variation. Recently, a study reported that CPS can be retrieved, and virulence recovered, after S. suis in vivo passages of an endocarditis-derived unencapsulated strain, suggesting that some unencapsulated S. suis lurking in pigs with endocarditis are still potentially hazardous [75]. Various mutations in cps genes, including large deletions and insertions, may cause CPS loss in S. suis; thus, not all mutations would be repaired [74,76]. Furthermore, coexistence of both encapsulated and unencapsulated S. suis isolates was found in persistent endocarditis lesions [21]. It has been suggested that both phenotypes ‘cooperate' by expressing both advantageous characteristics at lesions, and that each of these phenotypically distinct, but genetically very similar, clonal subpopulations provide advantages that result in persistence of the global population of the clone [21]. Hence, although unencapsulated S. suis isolates are, in general, easily phagocytized by immune cells, it is possible that they persist and proliferate in the host by the assistance of encapsulated cells. One additional important finding was that turning CPS on– off appears to be independent of the genomic background of the strains, as both ST28 and ST1 dual phenotypic pairs were identified in independent lesions. Interestingly, these pairs of strains

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differed, genome-wide, only by a relatively low number of mutations, which particularly affected cps genes involved in CPS biosynthesis [21,74,76]. Based on CPS modulation during endocarditis, it is tempting to speculate that mutations leading to unencapsulation and reversion thereof are a common mechanism associated with transition from open S. suis infection to asymptomatic carriage at the tonsillar level, and back to open infection. This type of off–on switching of CPS expression based on reversible mutations arising in cps genes is an interesting addition to the longstanding hypothesis of regulation of S. suis CPS expression by transcriptional mechanisms [25,77]. Environmental factors, such as the availability of glucose or other carbohydrates, pH, and temperature, have also been suggested to influence S. suis CPS expression [78,79]. For example, the catabolite control protein A (CcpA) was shown to exert control of CPS production at the transcriptional level under conditions of high glucose availability [80]. The hypotheses of off–on switching of CPS expression and/or transcriptional regulation remain to be further evaluated. Finally, studies described above also revealed that mutations in the cps2IJ and cps2NO regions fatally affect the viability of S. suis [76]. The only reported Dcps2J mutant in the literature is likely a result of gene misidentification [81]. Clearly, the S. suis cps locus still represents a challenging research area. Suilysin: Be (Mouse) or Not to Be (Pig) a Critical Virulence Factor Suilysin, the only S. suis toxin well characterized so far, was first reported from virulent European serotype 2 strains (ST1 background) and was thus associated with pathogenic attributes in these isolates [82–84]. Suilysin was then described to be present in the majority of invasive serotype 9 isolates from Europe, and has also been reported in serotypes 1, 1/2, 3, 4, 5, 7, 8, 10, 12, 14, 15, 18, 19, 23, and 30 [82,85]. However, the fact that many virulent serotype 2 strains (mainly from North America) as well as virulent strains belonging to other serotypes are not able to produce the suilysin, seems to indicate that this toxin does not play a critical role in virulence [15,85–87]. Indeed, several isogenic mutants lacking suilysin expression have been obtained from different serotype 2 strains (ST1 and ST7 backgrounds), whose level of virulence depended on the animal model used (Table S2). Using mouse i.p. infection models, suilysin was shown to play a critical role in virulence [88–90]. It was also reported that ST104 strains, which have reduced suilysin production due to insertions in the sly promoter region, show lower and less persistent bacterial densities in blood and brains than suilysin+[23_TD$IF] ST1 strains, as well as markedly reduced virulence in i.p. infected mice [90]. On the other hand, a mouse i.n. model using sublethal doses showed no differences in upper respiratory tract colonization between the wild-type strain and the suilysin knock-out mutant [91,92]. Yet, colonization rather than lethality/morbidity was evaluated in these studies, precluding a definitive conclusion on the actual role of suilysin in virulence when using the i.n. route in mice. In contrast, and independently of the infection route, swine i.v. or i.n. infection models revealed that suilysin was not required for virulence [39,88]. When suilysin, or genes regulating its expression, are reported as critical virulence factors, it should be considered that this may be true for the mouse model but not for the natural host (the pig). Interpretation of such studies must be carefully and critically analyzed. In spite of this discrepancy, in vitro studies have shown that suilysin plays important roles in the interactions with different host cells, in the induction of cell death, and in the inflammatory response (Table S2), as recently reviewed in [93]. Altogether, these findings indicate that suilysin cannot be considered critical for virulence; though, when present, it probably contributes to the pathogenicity traits of S. suis suilysin+ strains.

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The Mystic Muramidase-Released Protein (MRP) Earlier studies identified that most serotype 2 S. suis strains isolated from diseased pigs in Europe, later characterized as belonging to ST1, expressed two proteins known as MRP and as extracellular factor (EF) protein [94,95]. Besides serotype 2, the MRP proteins (either the 136kDa protein or the large- and small-size variants) were described in S. suis strains of nearly all serotypes investigated, although their presence is far from being constant [85,96,97]. Albeit MRP expression was originally associated with virulence [94], MRP-deficient mutants (ST1 background) were shown to be as virulent as the parent strains in a swine i.n. infection model (Table S3), changing the status of this protein from a ‘virulence factor' to a ‘virulence marker' [95]. Although this association seems to be positive for ST1 virulent strains, a recent study revealed that this is not the case for other serotype 2 ST backgrounds [15]. Indeed, intermediate-virulence ST25 strains are MRP , whereas low-virulence ST28 strains are MRP+ (or its variants) [15], indicating that mrp genotyping may only be useful in discriminating virulent ST1 strains (when expressed together with epf and sly) but not those of other genetic backgrounds. In addition, these results also confirm dispensability of this factor for S. suis full virulence. Until recently, there were no hints on the potential role (if any) of MRP in the pathogenesis of the infection [95]. A particular region within the MRP amino acid sequence showed some similarity with the fibronectin-binding protein of Staphylococcus aureus; yet binding of MRP to human fibronectin could not be confirmed [98]. In contrast to these previous findings with ST1 strains, new mutagenesis studies appear to indicate that MRP would mediate binding to human fibrinogen in the serotype 2 ST7 clone from China, facilitating both attachment to and traversal of human brain microvascular endothelial cells (BMEC) by increasing transendothelial cell permeability in vitro [99]. Although binding to mouse fibrinogen has not been demonstrated, MRP was shown to contribute to changes in the BBB permeability and to more severe brain histopathological lesions at 3 days post-infection in an i.v. infection mouse model [99]. Yet, clinical signs of meningitis or mortality were not evaluated in this model, especially at later time points, when CNS clinical signs are usually observed [29]. In fact, no differences were observed in blood bacterial loads between wild-type S. suis-infected mice and those infected with the MRP-deficient mutant. The latter observation contradicts follow-up studies reporting that human fibrinogen–MRP interaction improved S. suis survival in human blood, the bacterial antiphagocytic capacity against human neutrophils and led to the aggregation and exhaustion of these cells, suggesting that MRP might be involved in the survival of the bacteria in the host blood [100,101]. Thus, it remains unclear whether MRP actually binds mouse or swine fibrinogen and the role of this protein in both species. Although the differential role of MRP upon the genetic background (STs) of the strains needs further evaluation, it would still not be considered as a critical virulence factor.

Concluding Remarks and Future Perspectives Undoubtedly, S. suis is a very heterogonous species which complicates the characterization of virulence factors. Throughout the contradictions and controversies discussed in this review, the following issues deserve consideration in the study of virulence factor candidates (see Outstanding Questions): 1. Need of confirmation of a critical role in virulence of those candidates by independent laboratories with well characterized and completely sequenced strains of different STs (and serotypes). 2. Prescreening, hypothesis-driven in vitro studies that must be confirmed in the appropriate animal infection model. 3. A well standardized animal model (mouse for initial screening) and swine (for final confirmation) for S. suis serotype 2. 4. Well-defined clinical scores for measuring mortality (euthanasia) and morbidity. A statistically validated number of animals per experimental group.

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Outstanding Questions What is the correct definition of a ‘critical virulence factor' for S. suis and which experimental parameters should be considered in order to claim a bacterial attribute as critical for the pathogenesis process? Is it really possible to more clearly define the role played by all these proposed virulence factors by using a well-standardized infection model? Which are the clinical and pathological features to be analyzed when standardizing the optimal mouse model to reproduce disease? What is the best swine model to be used? How to choose susceptible pigs for experimental infection models? Is it possible to better outline the differential role play by putative virulence factors during mucosal colonization versus systemic dissemination? Which is the best colonization model to be used in vitro and in vivo? What is the definitive role of the CPS at each step of the pathogenesis and under different pathological conditions? How is CPS expression ultimately regulated by the pathogen?

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5. Infection route in agreement with the hypothetical role of the virulence factor candidate. 6. Meticulously show that growth rates are similar between mutant strains and their respective wild-type parent strains, especially when dissecting the effect of mutations in genes involved in metabolic/regulatory pathways. An effect on bacterial fitness might be erroneously linked to a virulence trait (Table S5). Nevertheless, it should be stressed that a gene/factor affecting growth in vitro may also be important for bacterial fitness/survival in the host by contributing to its adaptation to specific host niches which are not colonized by nonvirulent strains. Such factors are usually called virulence-associated factors. In this context, the term ‘crucial or critical virulence factor’ is often misused. 7. Finally, it would be important to better address regulation of virulence-associated factors and their impact on the pathogenesis of the disease, an aspect often not considered appropriately. Full-genome sequencing of S. suis serotype 2 strains, as well as other S. suis serotypes, will be instrumental in the identification of the molecular basis of virulence differences observed within serotype 2 strains and between the different serotypes. The establishment of animal models for each serotype remains a major challenge, but has the potential to add valuable information on how this swine pathogen and zoonotic agent causes disease. Acknowledgments Results discussed in this review and carried out in our laboratories have been funded by the Natural Sciences and Engineering Research Council of Canada [grant #154280 to MG and 342150-07 to MS] and by China–Canada Joint Health Research Initiative financed by Canadian Institutes of Health Research and the National Natural Science Foundation of China (812111251). This publication made use of the multilocus sequence typing (MLST) website (http://www.mlst.net) at Imperial College, London, developed by David Aanensen and funded by the Welcome Trust. The authors thank Mr JeanPhilippe Auger and Mr Guillaume Goyette-Desjardins for assistance with MLST database and figure editing.

[243_TD$IF]Supplemental Information Supplemental information associated with this article can be found [24_TD$IF]online at http://dx.doi.org/10.1016/j.tim.2017.02.005.

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