Flanking N- and C-terminal domains of PrsA in Streptococcus suis type 2 are crucial for inducing cell death independent of TLR2 recognition.

Streptococcus suis type 2 (SS2), a major emerging/re-emerging zoonotic pathogen found in humans and pigs, can cause severe clinical infections, and pose public health issues. Our previous studies recognized peptidyl-prolyl isomerase (PrsA) as a critical virulence factor promoting SS2 pathogenicity. PrsA contributed to cell death and operated as a pro-inflammatory effector. However, the molecular pathways through which PrsA contributes to cell death are poorly understood. Here in this study, we prepared the recombinant PrsA protein and found that pyroptosis and necroptosis were involved in cell death stimulated by PrsA. Specific pyroptosis and necroptosis signalling inhibitors could significantly alleviate the fatal effect. Cleaved caspase-1 and IL-1ß in pyroptosis with phosphorylated MLKL proteins in necroptosis pathways, respectively, were activated after PrsA stimulation. Truncated protein fragments of enzymatic PPIase domain (PPI), N-terminal (NP), and C-terminal (PC) domains fused with PPIase, were expressed and purified. PrsA flanking N- or C-terminal but not enzymatic PPIase domain was found to be critical for PrsA function in inducing cell death and inflammation. Additionally, PrsA protein could be anchored on the cell surface to interact with host cells. However, Toll-like receptor 2 (TLR2) was not implicated in cell death and recognition of PrsA. PAMPs of PrsA could not promote TLR2 activation, and no rescued phenotypes of death were shown in cells blocking of TLR2 receptor or signal-transducing adaptor of MyD88. Overall, these data, for the first time, advanced our perspective on PrsA function and elucidated that PrsA-induced cell death requires its flanking N- or C-terminal domain but is dispensable for recognizing TLR2. Further efforts are still needed to explore the precise molecular mechanisms of PrsA-inducing cell death and, therefore, contribution to SS2 pathogenicity.

Deletion of lacD gene affected stress tolerance and virulence of Streptococcus suis serotype 2.

Streptococcus suis type 2 (S. suis type 2, SS2), an infectious pathogen which is zoonotic and can induce severely public health concern. Our previous research identified a newly differential secreted effector of tagatose-bisphosphate aldolase (LacD) mediated by VirD4 factor within the putative type IV secretion system of SS2, whereas the functional basis and roles in virulence of LacD remain elusive. Here in this study, the LacD was found enzymatic and can be activated to express under oxidative stress. Gene mutant and its complemental strain (ΔlacD and cΔlacD) were constructed to analyze the phenotypes, virulence and transcriptomic profiles as compared with the parental strain. The lacD gene deletion showed no effect on growth capability and cells morphology of SS2. However, reduced tolerance to oxidative and heat stress conditions, increased antimicrobial susceptibility to ciprofloxacin and kanamycin were found in ΔlacD strain. Further, the LacD deficiency led to weakened invasion and attenuated virulence since an easier phagocytosed and more prone to be cleared of SS2 in macrophages were shown in ΔlacD mutant. Distinctive transcriptional profiling in ΔlacD strain and typical down-regulated genes with significant mRNA changes including alcohol dehydrogenase, GTPase, integrative and conjugative elements, and iron ABC transporters which were mainly involved in cell division, stress response, antimicrobial susceptibility and virulence regulation, were examined and confirmed by RNA sequencing and real time qPCR. In summary, the results demonstrated for the first time that LacD was a pluripotent protein mediated the metabolic, stress and virulent effect of SS2.

Development of a recombinase polymerase amplification assay for rapid detection of Streptococcus suis type 2 in nasopharyngeal swab samples.

Streptococcus suis serotype 2 (SS2), an emerging zoonotic pathogen, may induce severe infections and symptoms manifested as septicemia, meningitis and even death both in human and pigs. The aim of this article was to develop a new methodology as real-time recombinase polymerase amplification (RT-RPA) assay targeting cps2J gene for the detection of SS2 (or SS1/2). The sensitivity and reproducibility of RT-RPA results were evaluated and compared with a real-time quantitative PCR (RT-qPCR). The established RT-RPA reaction could be completed in 20 minutes with distinguishable specificity against the predominant S. suis infection serotypes of 3, 4, 5, 7, 9, 14, and 31. Lower detection limit for RT-RPA was 102 genomic DNA copies per reaction. The specimen performance of RT-RPA was tested in nasopharyngeal swab samples with the sensitivity and specificity as 97.5% and 100%, respectively. Thus, this RT-RPA method is a rapid and potential molecular diagnostic tool for SS2 detection.