Interleukin-1 signaling induced by Streptococcus suis serotype 2 is strain-dependent and contributes to bacterial clearance and inflammation during systemic disease in a mouse model of infection.

Streptococcus suis serotype 2 is an important porcine pathogen and zoonotic agent causing sudden death, septic shock and meningitis, with exacerbated inflammation being a hallmark of the infection. A rapid, effective and balanced innate immune response against S. suis is critical to control bacterial growth without causing excessive inflammation. Even though interleukin (IL)-1 is one of the most potent and earliest pro-inflammatory mediators produced, its role in the S. suis pathogenesis has not been studied. We demonstrated that a classical virulent European sequence type (ST) 1 strain and the highly virulent ST7 strain induce important levels of IL-1 in systemic organs. Moreover, bone marrow-derived dendritic cells and macrophages contribute to its production, with the ST7 strain inducing higher levels. To better understand the underlying mechanisms involved, different cellular pathways were studied. Independently of the strain, IL-1ß production required MyD88 and involved recognition via TLR2 and possibly TLR7 and TLR9. This suggests that the recognized bacterial components are similar and conserved between strains. However, very high levels of the pore-forming toxin suilysin, produced only by the ST7 strain, are required for efficient maturation of pro-IL-1ß via activation of different inflammasomes resulting from pore formation and ion efflux. Using IL-1R-/- mice, we demonstrated that IL-1 signaling plays a beneficial role during S. suis systemic infection by modulating the inflammation required to control and clear bacterial burden, thus promoting host survival. Beyond a certain threshold, however, S. suis-induced inflammation cannot be counterbalanced by this signaling, making it difficult to discriminate its role.

Enolase and dipeptidyl peptidase IV protein sub-unit vaccines are not protective against a lethal Streptococcus suis serotype 2 challenge in a mouse model of infection.

BACKGROUND: Streptococcus suis is a major swine pathogen causing arthritis, meningitis and sudden death in post-weaning piglets and is also a zoonotic agent. S. suis comprises 35 different serotypes of which the serotype 2 is the most prevalent in both pigs and humans. In the absence of commercial vaccines, bacterins (mostly autogenous), are used in the field, with controversial results. In the past years, the focus has turned towards the development of sub-unit vaccine candidates. However, published results are sometimes contradictory regarding the protective effect of a same candidate. Moreover, the adjuvant used may significantly influence the protective capacity of a given antigen. This study focused on two protective candidates, the dipeptidyl peptidase IV (DPPIV) and the enolase (SsEno). Both proteins are involved in S. suis pathogenesis, and while contradictory protection results have been obtained with SsEno in the past, no data on the protective capacity of DPPIV was available. RESULTS: Results showed that among all the field strains tested, 86 and 88% were positive for the expression of the SsEno and DPPIV proteins, respectively, suggesting that they are widely expressed by strains of different serotypes. However, no protection was obtained after two vaccine doses in a CD-1 mouse model of infection, regardless of the use of four different adjuvants. Even though no protection was obtained, significant amounts of antibodies were produced against both antigens, and this regardless of the adjuvant used. CONCLUSIONS: Taken together, these results demonstrate that S. suis DPPIV and SsEno are probably not good vaccine candidates, at least not in the conditions evaluated in this study. Further studies in the natural host (pig) should still be carried out. Moreover, this work highlights the importance of confirming results obtained by different research groups.

Characterization of the zinc metalloprotease of Streptococcus suis serotype 2.

Streptococcus suis is a swine pathogen and zoonotic agent responsible for meningitis and septic shock. Although several putative virulence factors have been described, the initial steps of the S. suis pathogenesis remain poorly understood. While controversial results have been reported for a S. suis serotype 2 zinc metalloprotease (Zmp) regarding its IgA protease activity, recent phylogenetic analyses suggested that this protein is homologous to the ZmpC of Streptococcus pneumoniae, which is not an IgA protease. Based on the previously described functions of metalloproteases (including IgA protease and ZmpC), different experiments were carried out to study the activities of that of S. suis serotype 2. First, results showed that S. suis, as well as the recombinant Zmp, were unable to cleave human IgA1, confirming lack of IgA protease activity. Similarly, S. suis was unable to cleave P-selectin glycoprotein ligand-1 and to activate matrix metalloprotease 9, at least under the conditions tested. However, S. suis was able to partially cleave mucin 16 and syndecan-1 ectodomains. Experiments carried out with an isogenic Δzmp mutant showed that the Zmp protein was partially involved in such activities. The absence of a functional Zmp protein did not affect the ability of S. suis to adhere to porcine bronchial epithelial cells in vitro, or to colonize the upper respiratory tract of pigs in vivo. Taken together, our results show that S. suis serotype 2 Zmp is not a critical virulence factor and highlight the importance of independently confirming results on S. suis virulence by different teams.

Serotype-specific role of antigen I/II in the initial steps of the pathogenesis of the infection caused by Streptococcus suis.

Streptococcus suis is one of the most important post-weaning porcine bacterial pathogens worldwide. The serotypes 2 and 9 are often considered the most virulent and prevalent serotypes involved in swine infections, especially in Europe. However, knowledge of the bacterial factors involved in the first steps of the pathogenesis of the infection remains scarce. In several pathogenic streptococci, expression of multimodal adhesion proteins known as antigen I/II (AgI/II) have been linked with persistence in the upper respiratory tract and the oral cavity, as well as with bacterial dissemination. Herein, we report expression of these immunostimulatory factors by S. suis serotype 2 and 9 strains and that AgI/II-encoding genes are carried by integrative and conjugative elements. Using mutagenesis and different in vitro assays, we demonstrate that the contribution of AgI/II to the virulence of the serotype 2 strain used herein appears to be modest. In contrast, data demonstrate that the serotype 9 AgI/II participates in self-aggregation, induces salivary glycoprotein 340-related aggregation, contributes to biofilm formation and increased strain resistance to low pH, as well as in bacterial adhesion to extracellular matrix proteins and epithelial cells. Moreover, the use of a porcine infection model revealed that AgI/II contributes to colonization of the upper respiratory tract of pigs. Taken together, these findings suggest that surface exposed AgI/II likely play a key role in the first steps of the pathogenesis of the S. suis serotype 9 infection.

The protective protein Sao (surface antigen one) is not a critical virulence factor for Streptococcus suis serotype 2.

Surface antigen one (Sao) is protein that was shown to protect pigs in vaccination trials against disease caused by Streptococcus suis. Here, we used an isogenic Δsao mutant to investigate the insofar unknown role of Sao in S. suis virulence. The Δsao mutant and its wild-type parent strain (sequence type 1, European strain) did not present significant differences on levels of adhesion/invasion of swine respiratory epithelial cells. Moreover, the Δsao mutant resisted phagocytosis by macrophages similarly to the parent strain and it was still virulent in a mouse model of infection. Our findings suggest a minor role of Sao in S. suis serotype 2 virulence.

Interactions of Streptococcus suis serotype 9 with host cells and role of the capsular polysaccharide: Comparison with serotypes 2 and 14.

Streptococcus suis is an important porcine bacterial pathogen and a zoonotic agent responsible for sudden death, septic shock and meningitis, of which serotype 2 is the most widespread, with serotype 14 also causing infections in humans in South-East Asia. Knowledge of its pathogenesis and virulence are almost exclusively based on these two serotypes. Though serotype 9 is responsible for the greatest number of porcine cases in Spain, the Netherlands and Germany, very little information is currently available regarding this serotype. Of the different virulence factors, the capsular polysaccharide (CPS) is required for S. suis virulence as it promotes resistance to phagocytosis and killing and masks surface components responsible for host cell activation. However, these roles have been described for serotypes 2 and 14, whose CPSs are structurally and compositionally similar, both containing sialic acid. Consequently, we evaluated herein the interactions of serotype 9 with host cells and the role of its CPS, which greatly differs from those of serotypes 2 and 14. Results demonstrated that serotype 9 adhesion to but not invasion of respiratory epithelial cells was greater than that of serotypes 2 and 14. Furthermore serotype 9 was more internalized by macrophages but equally resistant to whole blood killing. Though recognition of serotypes 2, 9 and 14 by DCs required MyD88-dependent signaling, in vitro pro-inflammatory mediator production induced by serotype 9 was much lower. In vivo, however, serotype 9 causes an exacerbated inflammatory response, which combined with persistent bacterial presence, is probably responsible for host death during the systemic infection. Though presence of the serotype 9 CPS masks surface components less efficiently than those of serotypes 2 and 14, the serotype 9 CPS remains critical for virulence as it is required for survival in blood and development of clinical disease, and this regardless of its unique composition and structure.

Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus.

The capsular polysaccharide (CPS) represents a key virulence factor for most encapsulated streptococci. Streptococcus suis and Group B Streptococcus (GBS) are both well-encapsulated pathogens of clinical importance in veterinary and/or human medicine and responsible for invasive systemic diseases. S. suis and GBS are the only Gram-positive bacteria which express a sialylated CPS at their surface. An important difference between these two sialylated CPSs is the linkage between the side-chain terminal galactose and sialic acid, being α-2,6 for S. suis but α-2,3 for GBS. It is still unclear how sialic acid may affect CPS production and, consequently, the pathogenesis of the disease caused by these two bacterial pathogens. Here, we investigated the role of sialic acid and the putative effect of sialic acid linkage modification in CPS synthesis using inter-species allelic exchange mutagenesis. To this aim, a new molecular biogenetic approach to express CPS with modified sialic acid linkage was developed. We showed that sialic acid (and its α-2,6 linkage) is crucial for S. suis CPS synthesis, whereas for GBS, CPS synthesis may occur in presence of an α-2,6 sialyltransferase or in absence of sialic acid moiety. To evaluate the effect of the CPS composition/structure on sialyltransferase activity, two distinct capsular serotypes within each bacterial species were compared (S. suis serotypes 2 and 14 and GBS serotypes III and V). It was demonstrated that the observed differences in sialyltransferase activity and specificity between S. suis and GBS were serotype unrestricted. This is the first time that a study investigates the interspecies exchange of capsular sialyltransferase genes in Gram-positive bacteria. The obtained mutants represent novel tools that could be used to further investigate the immunomodulatory properties of sialylated CPSs. Finally, in spite of common CPS structural characteristics and similarities in the cps loci, sialic acid exerts differential control of CPS expression by S. suis and GBS.

Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28.

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.