Streptococcus suis (S. suis) ranks among the five most important porcine pathogens worldwide and occasionally threatens human health, especially in people that come into close contact with pigs or pork products. Streptococcus suis serotype 2 (SS2) is considered to be the most pathogenic and prevalent capsular type. As a first line of immune defense against SS2 infection, neutrophils can eliminate the invader not only by phagocytosis but also by neutrophil extracellular traps (NETs)-mediated killing. SS2 can resist phagocytosis through polysaccharide capsule (CPS), but how this strain evades the effects of NETs remains to be determined. The present study demonstrated that the epidemic strain 05ZY, the highly pathogenic strain P1/7 and the intermediately pathogenic strain A7 could induce the formation of NETs. Furthermore, SS2 strains could successfully resist NETs-mediated killing, and the CPS structure contributed to this resistance by escaping the trapping. Therefore, the CPS structure not only contributed to the SS2 strains' resistance to phagocytosis-mediated killing but also played an essential role in evading NETs trapping and further killing in vitro. This study strengthens our understanding of how S. suis can evade innate immune surveillance and elimination.

Streptococcus suis 2 is an important swine pathogen and an emergent zoonotic pathogen. Excessive inflammation caused by S. suis is responsible for the high levels of early mortality observed in septic shock-like syndrome cases. However, the mechanisms through which S. suis 2 (SS2) causes excessive inflammation remain unclear. Thus, this study aimed to identify novel pro-inflammatory mediators that play important roles in the development of therapies against SS2 infection. In this study, the novel pro-inflammatory protein HP0459, which was encoded by the SSUSC84_0459 gene, was discovered. The stimulation of RAW 264.7 macrophages with recombinant HP0459 protein induced the expression of pro-inflammatory cytokines (IL-1β, MCP-1 and TNF-α). Compared with the wild-type (WT) strain, the isogenic knockout of HP0459 in SS2 led to reduced production of pro-inflammatory cytokines in RAW264.7 macrophages and in vivo. The pro-inflammatory activity of HP0459 was significantly reduced by an antibody against Toll-like receptor 2 (TLR2) in RAW264.7 macrophages and was lower in TLR2-deficient (TLR2-/-) macrophages than in WT macrophages. Furthermore, specific inhibitors of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathways significantly decreased the HP0459-induced pro-inflammatory cytokine production, and a western blot assay showed that HP0459 stimulation induced the activation of the ERK1/2 pathway. Taken together, our data indicate that HP0459 is a novel pro-inflammatory mediator of SS2 and induces TLR2-dependent pro-inflammatory activity in RAW264.7 macrophages through the ERK1/2 pathway.

Streptococcus suis 2 (SS2) has evolved into a highly invasive pathogen responsible for two large-scale outbreaks of streptococcal toxic shock-like syndrome (STSLS) in China. Excessive inflammation stimulated by SS2 is considered a hallmark of STSLS, even it also plays important roles in other clinical symptoms of SS2-related disease, including meningitis, septicemia, and sudden death. However, the mechanism of SS2-caused excessive inflammation remains poorly understood. Here, a novel pro-inflammatory protein was identified (HP1330), which could induce robust expression of pro-inflammatory cytokines (TNF-α, MCP-1, and IL-1β) in RAW264.7 macrophages. To evaluate the role of HP1330 in SS2 virulence, an hp1330-deletion mutant (Δhp1330) was constructed. In vitro, hp1330 disruption led to a decreased pro-inflammatory ability of SS2 in RAW 264.7 macrophages. In vivo, Δhp1330 showed reduced lethality, pro-inflammatory activity, and bacterial loads in mice. To further elucidate the mechanism of HP1330-induced pro-inflammatory cytokine production, antibody blocking and gene-deletion experiments with macrophages were performed. The results revealed that the pro-inflammatory activity of HP1330 depended on the recognition of toll-like receptor 2 (TLR2). Furthermore, a specific inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathways could significantly decrease HP1330-induced pro-inflammatory cytokine production, and western blot analysis showed that HP1330 could induce activation of the ERK1/2 pathway. Taken together, our findings demonstrate that HP1330 contributes to SS2 virulence by inducing TLR2- and ERK1/2-dependent pro-inflammatory cytokine production and influencing in vivo bacterial loads, implying that HP1330 may be associated with STSLS caused by SS2.

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Protein-protein interactions between bacteria and their hosts are responsible for all types of infection processes. The investigation of the bacteria-host crosstalk can provide a comprehensive understanding of the pathogenesis of bacterial disease. Despite scattered efforts in this field, a systematic identification of interactions between host and bacterial proteins remains unavailable. Here, we develop ACSP (affinity chromatography-based surface proteomics), which combines affinity chromatography and shotgun proteomics (LC-MS/MS), to investigate the interactions on a large-scale. Using ACSP, the potential surface interacting proteins (SIPs) of Streptococcus suis serotype 2 (SS2) were captured by the chromatographic resin, which was immobilized with the native surface molecules of Hep-2 cells. And then 40 potential SIPs were identified from the preys by LC-MS/MS, including 3 SIPs that have been previously reported in the literature. We selected 8 important SIPs and confirmed their ability to adhere to Hep-2 cells. Additionally, 3 newly identified SIPs, or their polyclonal antibodies, were found to significantly inhibit the adherence of SS2 to Hep-2 cells, indicating their essential role in the interaction between SS2 and Hep-2 cells. Using this example, we show that ACSP represents a new valuable tool for investigating the bacteria-host interactions.