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ABSTRACTStreptococcus suisserotype 2 (SS2), an emerging zoonotic pathogen, causes swine diseases and human cases of streptococcal toxic shock syndrome. RNA-binding proteins (RBPs) can modulate gene expression through post-transcriptional regulation. In this study, we identified an RBP harbouring an S1 domain, named RbpA, which facilitated SS2 adhesion to host epithelial cells and contributed to bacterial pathogenicity. Comparative proteomic analysis identified 145 proteins that were expressed differentially between ΔrbpAstrain and wild-type strain, including several virulence-associated factors, such as the extracellular protein factor (EF), SrtF pilus, IgA1 protease, SBP2 pilus, and peptidoglycan-binding LysM’ proteins. The mechanisms underlying the regulatory effects of RbpA on their encoding genes were explored, and it was found that RbpA regulates gene expression through diverse mechanisms, including post-transcriptional regulation, and thus acts as a global regulator. These results partly reveal the pathogenic mechanism mediated by RbpA, improving our understanding of the regulatory systems of S. suisand providing new insights into bacterial pathogenicity.

ABSTRACTStreptococcus suiscan cause severe infections in pigs and humans. The tonsils of pigs are major niches for S. suis, and different serotypes of S. suiscan be found in the same tonsil. Pig tonsil colonization by S. suisis believed to be an important source of infection for humans and pigs. However, how S. suiscompetes for a stable tonsil niche is unknown. Here, we found that S. suisstrain WUSS351, isolated from a healthy pig tonsil, is virulent and multidrug-resistant. The ABC transporter system SstFEG, conferring resistance to bacitracin, was reported to confer a competitive survival advantage in vivo. In addition, strain WUSS351 has several antimicrobial systems, including a novel type VII secretion system (T7SS), lantibiotic bacteriocin, and lactococcin972-like bacteriocin Lcn351. Bacterial competition experiments demonstrated T7SS-mediated cell contact-dependent antagonism of S. suis. Antibacterial activity analysis and 16S rRNA gene sequencing of the culture-independent and culture-dependent pig tonsillar microbiome revealed that Lcn351 mainly targets S. suis, one of the core microbiomes in pig tonsils. Taken together, our results revealed the mechanism of the stable persistence of S. suisin the tonsil niche, which might have important implications for S. suisepidemiology, potentially influencing strain prevalence and disease progression.

ABSTRACTThe Streptococcus suisserotype 2 (SS2) is a significant zoonotic pathogen that is responsible for various swine diseases, even causing cytokine storms of Streptococcal toxic shock-like syndromes amongst human. Cell wall anchoring proteins with a C-terminal LPxTG are considered to play vital roles during SS2 infection; however, their exporting mechanism across cytoplasmic membranes has remained vague. This study found that YSIRK-G/S was involved in the exportation of LPxTG-anchoring virulence factors MRP and SspA in virulent SS2 strain ZY05719. The whole-genome analysis indicated that diverse LPxTG proteins fused with an N-terminal YSIRK-G/S motif are encoded in strain ZY05719. Two novel LPxTG proteins SspB and YzpA were verified to be exported via a putative transport system that was dependent on the YSIRK-G/S directed translocation, and portrayed vital functions during the infection of SS2 strain ZY05719. Instead of exhibiting an inactivation of C5a peptidase in SspB, another LPxTG protein with an N-terminal YSIRK-G/S motif from Streptococcus agalactiaewas depicted to cleave the C5a component of the host complement. The consequent domain-architecture retrieval determined more than 10,000 SspB/YzpA like proteins that are extensively distributed in the Gram-positive bacteria, and most of them harbor diverse glycosyl hydrolase or peptidase domains within their middle regions, thus presenting their capability to interact with host cells. The said findings provide compelling evidence that LPxTG proteins with an N-terminal YSIRK-G/S motif are polymorphic effectors secreted by Gram-positive bacteria, which can be further proposed to define as cell wall anchoring effectors in a new subset.

ABSTRACTStreptococcus suisis a bacterium that can cause infections in pigs and humans. Although oxidative stress is common occurrence during bacterial growth and infection, the regulation networks of S. suisunder oxidative stress remain poorly understood. To address this, we utilized RNA-Seq to reveal the transcriptional landscape of S. suisin response to H2O2stress. We identified novel genes responsible for S. suisresistance to oxidative stress, including those involved in DNA repair or protection, and essential for the biosynthesis of amino acids and nucleic acids. In addition, we found that a novel aquaporin, Aagp, belonging to atypical aquaglyceroporins and widely distributed in diverse S. suisserotypes, plays a crucial role during H2O2stress. By performing oxidative stress assays and measuring the intracellular H2O2concentrations of the wild-type strain and Aagpmutants during H2O2stress, we found that Aagp facilitated H2O2efflux. Additionally, we found that Aagp might be involved in glycerol transport, as shown by the growth inhibition and H2O2production in the presence of glycerol. Mice infection experiments indicated that Aagp contributed to S. suisvirulence. This study contributes to understanding the mechanism of S. suisoxidative stress response, S. suispathogenesis, and the function of aquaporins in prokaryotes.

ABSTRACTStreptococcus suisis a major porcine bacterial pathogen and emerging zoonotic agent. S. suis5ʹ-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitroand it is an important virulence factor. Here, we show that S. suis5ʹ-nucleotidase not only enables producing 2ʹ-deoxyadenosine from 2ʹ-deoxyadenosine monophosphate by the enzymatic assay and reversed-phase high performance liquid chromatography (RP-HPLC) analysis in vitro, but also synthesizes both 2ʹ-deoxyadenosine and adenosine in mouse blood in vivoby RP-HPLC and liquid chromatography with tandem mass spectrometry analyses. Cellular cytotoxicity assay and Western blot analysis indicated that the production of 2ʹ-deoxyadenosine by 5ʹ-nucleotidase triggered the death of mouse macrophages RAW 264.7 in a caspase-3-dependent way. The in vivoinfection experiment showed that 2ʹ-deoxyadenosine synthesized by 5ʹ-nucleotidase caused monocytopenia in mouse blood. The in vivotranscriptome analysis in mouse blood showed the inhibitory effect of 5ʹ-nucleotidase on neutrophil functions and immune responses probably mediated through the generation of adenosine. Taken together, these findings indicate that S. suissynthesizes 2ʹ-deoxyadenosine and adenosine by 5ʹ-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suispathogenesis.

ABSTRACTMuramidase-released protein (MRP) of Streptococcus suisserotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1(Δmrp-d1), and Δmrp domain 2(Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242–596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239–598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848–1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.