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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.

ABSTRACTType VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenic Escherichia coli(APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucial clpVclusters of these two T6SS loci and their vgrGgenes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines cultured in vitro, decreased pathogenicity in duck and mouse infection models in vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.

In this differential RNA-seq study, Wu et al. identified 29 small RNAs expressed by the pig pathogen Streptococcus suis. The authors then went on to show that S. suisstrains lacking some of these small RNAs were attenuated in a zebrafish infection model or were more sensitive to killing by pig blood. The study thus provides further evidence that small, regulatory RNAs play roles in bacterial virulence.

Streptococcus suisis one of the most important pathogens affecting the swine industry and is also an emerging zoonotic agent for humans. Two-component signaling systems (TCSs) play important roles in the adaptation of pathogenic bacteria to host environments. In this study, we identified a novel TCS, named TCS09HKRR, which facilitated Streptococcus suisserotype 2 (SS2) resistance to clearance by the host immune system and contributed to bacterial pathogenicity.

ABSTRACTStreptococcus suishas received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located in S. suisserotype 2 (SS2), designated VraSRSS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that the yvqFSSgene, located upstream of vraSRSS, shared the same promoter with the TCSS genes, which was directly regulated by VraSRSS, as shown in electrophoretic mobility shift assays. Notably, YvqFSSand VraSRSSconstitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSRSSinactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSRSSexpression observed in host blood, strongly suggested that VraSRSSis an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSRSSstrains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSRSSindirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused by vraSRSSdeletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSRSS. Taken together, our data suggest that the novel TCSS, VraSRSS, plays critical roles for multidrug resistance and full virulence in SS2.