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ABSTRACTThe arginine deiminase system (ADS) has been identified in various bacteria and functions to supplement energy production and enhance biological adaptability. The current understanding of the regulatory mechanism of ADS and its effect on bacterial pathogenesis is still limited. Here, we found that the XRE family transcriptional regulator XtrSs negatively affected Streptococcus suis virulence and significantly repressed ADS transcription when the bacteria were incubated in blood. Electrophoretic mobility shift (EMSA) and lacZ fusion assays further showed that XtrSs directly bind to the promoter of ArgR, an acknowledged positive regulator of bacterial ADS, to repress ArgR transcription. Moreover, we provided compelling evidence that S. suis could utilize arginine via ADS to adapt to acid stress, while ΔxtrSs enhanced this acid resistance by upregulating the ADS operon. Moreover, whole ADS-knockout S. suis increased arginine and antimicrobial NO in the infected macrophage cells, decreased intracellular survival, and even caused significant attenuation of bacterial virulence in a mouse infection model, while ΔxtrSs consistently presented the opposite results. Our experiments identified a novel ADS regulatory mechanism in S. suis, whereby XtrSs regulated ADS to modulate NO content in macrophages, promoting S. suis intracellular survival. Meanwhile, our findings provide a new perspective on how Streptococci evade the host’s innate immune system.

Abstract Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Streptococcus suis serotype 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 ΔrbpA strain 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. suis and providing new insights into bacterial pathogenicity.

Streptococcus suis serotype 2 (SS2) is a noteworthy zoonotic pathogen that has been responsible for large economic losses in pig production and a great threat to human health. Pentraxin 3 (PTX3) is an essential regulator of the innate immune response to bacterial pathogens; however, its role during SS2 infection is not fully understood. In this study, we found that the SS2 strain HA9801 induced a significant inflammatory response in the mouse air pouch model; this response was amplified by the treatment of exogenous PTX3 simultaneously in terms of the results of inflammatory cell recruitment and proinflammatory cytokine IL-6 production. In addition, PTX3 facilitated the phagocytosis of macrophage Ana-1 against SS2 strain HA9801. The supplementation of exogenous PTX3 significantly reduced the bacterial loads in a dose-dependent manner in lungs, livers and bloods of SS2-infected mice compared to the samples with HA9801 infection alone; this finding indicated that PTX3 may facilitate the bacterial clearance through enhancing the host inflammatory response during SS2 infection. Both PTX3 and SS2 capsular polysaccharide (CPS2) were required for the robust inflammatory response, implying that the host PTX3 protein and SS2 surface CPS2 modulate the host innate immune response in concert. All of these results suggested that PTX3 is a potential novel biological agent for the SS2 infection; however, the recommended dose of PTX3 must be evaluated strictly to avoid inducing an excessive inflammatory response that can cause serious tissue injury and animal death.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements that play an essential role in multidrug tolerance and virulence of bacteria. So far, little is known about the TA systems in Streptococcus suis. In this study, the Xress-MNTss TA system, composed of the MNTss toxin in the periplasmic space and its interacting Xress antitoxin, was identified in S. suis. β-galactosidase activity and electrophoretic mobility shift assay (EMSA) revealed that Xress and the Xress-MNTss complex could bind directly to the Xress-MNTss promoter as well as downregulate streptomycin adenylyltransferase ZY05719_RS04610. Interestingly, the Xress deletion mutant was less pathogenic in vivo following a challenge in mice. Transmission electron microscopy and adhesion assays pointed to a significantly thinner capsule but greater biofilm-formation capacity in ΔXress than in the wild-type strain. These results indicate that Xress-MNTss, a new type II TA system, plays an important role in antibiotic resistance and pathogenicity in S. suis.

Infection with the epidemic virulent strain of Streptococcus suis serotype 2 (SS2) can cause septicemia in swine and humans, leading to pneumonia, meningitis and even cytokine storm of Streptococcal toxic shock-like syndrome. Despite some progress concerning the contribution of bacterial adhesion, biofilm, toxicity and stress response to the SS2 systemic infection, the precise mechanism underlying bacterial survival and growth within the host bloodstream remains elusive. Here, we reported the SS2 virulent strains with a more than 20 kb endoSS-related insertion region that showed significantly higher proliferative ability in swine serum than low-virulent strains. Further study identified a complete N-glycans degradation system encoded within this insertion region, and found that both GH92 and EndoSS contribute to bacterial virulence, but that only DndoSS was required for optimal growth of SS2 in host serum. The supplement of hydrolyzed high-mannose-containing glycoprotein by GH92 and EndoSS could completely restore the growth deficiency of endoSS deletion mutant in swine serum. EndoSS only hydrolyzed a part of the model glycoprotein RNase B with high-mannose N-linked glycoforms into a low molecular weight form, and the solo activity of GH92 could not show any changes comparing with the blank control in SDS-PAGE gel. However, complete hydrolyzation was observed under the co-incubation of EndoSS and GH92, suggesting GH92 may degrade the high-mannose arms of N-glycans to generate a substrate for EndoSS. In summary, these findings provide compelling evidences that EndoSS-related N-glycans degradation system may enable SS2 to adapt to host serum-specific availability of carbon sources from glycoforms, and be required for optimal colonization and full virulence during systemic infection.