Source data for the graphs in Figs 1-9 and S1-S8.

Vibrio cholerae cytolysin (VCC) is a potent exotoxin secreted by Vibrio cholerae, the etiological agent of the severe diarrheal disease cholera. VCC is a membrane-damaging pore-forming toxin by nature, and is well known for its ability to cause host cell death. Using wild type V. cholerae and VCC-deleted mutant variant of the bacteria, we show that VCC plays an important role in the inflammatory responses during infection in mice. This observation supports that VCC can function as a pathogen-associated molecular pattern (PAMP). Toll-like receptors (TLRs) are the key initiators of inflammation. Upon ligand recognition, TLR1 and TLR6 generally form heterodimers with TLR2 for triggering pro-inflammatory signals. In the present study, we show that VCC engages novel TLR1/4 heterodimer assembly, and elicits pro-inflammatory responses in both dendritic cells (DCs) and macrophages. Along with TLR1/4, VCC-induced pro-inflammatory response in macrophages also involves TLR2. It has been shown earlier that VCC is implicated in the V. cholerae-mediated killing of the immune cells following biofilm formation. Here we show that TLRs play an important role in VCC-mediated killing of DCs and macrophages following V. cholerae infection. Interestingly, we find that TLR1/4 signalling is specifically crucial for the VCC-induced inflammatory and death responses in DCs, as well as in mice. Additionally, we observe that similar to DCs and macrophages, TLR1/4-MyD88 play an important role in VCC-mediated inflammatory responses in another crucial immune cell type, neutrophils. Taken together, our study shows novel TLR heterodimer formation, differential recognition of the same ligand by different TLR combination in cell type-dependent manner, and their implications in the context of V. cholerae and VCC-induced immune cell death and mortality.霍乱弧菌溶细胞素（Vibrio cholerae cytolysin，VCC）是由引发重症腹泻性疾病霍乱的病原菌霍乱弧菌（Vibrio cholerae）分泌的一种强效外毒素。VCC本质上是一类破坏细胞膜的成孔毒素，因其可诱导宿主细胞死亡而被广泛熟知。本研究利用野生型霍乱弧菌与敲除VCC的细菌突变株，证实VCC在小鼠感染过程中的炎症反应中发挥关键作用。该发现表明，VCC可作为病原体相关分子模式（pathogen-associated molecular pattern，PAMP）行使功能。 Toll样受体（Toll-like receptors，TLRs）是炎症反应的核心启动因子。当识别配体后，TLR1与TLR6通常会与TLR2形成异二聚体，以触发促炎信号通路。在本研究中，我们证实VCC可介导全新的TLR1/4异二聚体组装，并在树突状细胞（dendritic cells，DCs）与巨噬细胞中诱导促炎反应。在巨噬细胞中，VCC诱导的促炎反应除依赖TLR1/4外，还涉及TLR2的参与。 此前已有研究表明，VCC参与了霍乱弧菌在生物膜形成后对免疫细胞的杀伤过程。本研究证实，在霍乱弧菌感染后，TLRs在VCC介导的DCs与巨噬细胞杀伤过程中发挥重要作用。值得注意的是，我们发现TLR1/4信号通路对于DCs乃至小鼠体内VCC诱导的炎症与细胞死亡反应均具有特异性关键作用。此外，我们观察到，与DCs和巨噬细胞类似，TLR1/4-髓系分化初级应答基因88（MyD88）信号轴在另一种关键免疫细胞——中性粒细胞（neutrophils）的VCC介导的炎症反应中同样扮演重要角色。 综上，本研究揭示了全新的TLR异二聚体组装形式，以及不同TLR组合以细胞类型依赖的方式对同一配体的差异性识别，并阐明了其在霍乱弧菌与VCC诱导的免疫细胞死亡及宿主致死效应中的生物学意义。