Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

<i>Trueperella pyogenes</i> (<i>T. pyogenes</i>) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of <i>T. pyogenes</i>. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1β, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO. <b>Abbreviations</b>: <i>T. pyogenes: Trueperella pyogenes</i>; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-β-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin–Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1β: interleukin-1β; IFN-γ: interferon-γ; TGF-β: transforming growth factor-β; ELISA: enzyme-linked immunosorbent assay

*化脓隐秘杆菌*（*Trueperella pyogenes*，简称*T. pyogenes*）是一种重要的条件致病菌。化脓素（Pyolysin，PLO）是其致病过程中的核心毒力因子。然而，目前关于PLO的结构-功能关系及其毒力机制的研究仍有待完善。本研究成功制备了重组PLO（recombinant PLO，rPLO）及3株rPLO突变体。其中，将第238位天冬氨酸（aspartic acid，Asp）替换为精氨酸（arginine，Arg）的突变体rPLO D238R，在含胆固醇脂质体上的寡聚化活性以及绵羊红细胞（sheep red blood cells，sRBCs）膜上的成孔活性均出现显著受损。利用所制备的突变体开展的后续研究证实，PLO的成孔活性是其诱导小鼠产生过度炎症应答的关键介导因素，可通过上调白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）及白细胞介素6（IL-6）的表达水平实现炎症激活。与之相对，另一株细胞膜结合能力受损的突变体rPLO P499F，其诱发的炎症应答依赖于病原体相关分子模式（pathogen-associated molecular pattern，PAMP）活性：该突变体可显著上调巨噬细胞及小鼠体内白细胞介素10（IL-10）的表达，而野生型rPLO则无此效应。研究结果表明，PLO分子的结构域1在维持成孔活性中发挥关键作用。进一步明确，PLO诱发炎症的核心效应由其成孔活性介导，而非PAMP活性。本研究为PLO的结构、功能及毒力相关研究领域提供了新的实验依据与理论参考。**缩写说明**：*T. pyogenes*：化脓隐秘杆菌（Trueperella pyogenes）；PLO：化脓素（Pyolysin）；rPLO：重组PLO（recombinant PLO）；PAMP：病原体相关分子模式（pathogen-associated molecular pattern）；CDCs：胆固醇依赖性溶素（cholesterol-dependent cytolysins）；PLY：肺炎球菌溶素（pneumolysin）；NLRP3：NLR家族pyrin结构域包含蛋白3（NLR family pyrin domain containing protein 3）；PRRs：模式识别受体（pattern recognition receptors）；Asp：天冬氨酸（aspartic acid）；TLR4：Toll样受体4（Toll-like receptor 4）；Arg：精氨酸（arginine）；Asn：天冬酰胺（asparagine）；IPTG：异丙基-β-D-硫代半乳糖苷（Isopropyl-β-d-thiogalactoside）；PBS：磷酸盐缓冲液（phosphate-buffered saline）；sRBCs：绵羊红细胞（sheep red blood cells）；TEM：透射电子显微镜（Transmission electron microscopy）；RBCM：红细胞膜（red blood cell membrane）；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate–polyacrylamide gel electrophoresis）；NC膜：硝酸纤维素膜（nitrocellulose membrane）；SDS-AGE：十二烷基硫酸钠琼脂糖凝胶电泳（dodecyl sulfate agarose gel electrophoresis）；MDBK细胞：马-达二氏牛肾细胞（Madin–Darby bovine kidney cells）；RPMI-1640培养基：RPMI-1640培养基（Roswell Park Memorial Institute-1640 medium）；FBS：胎牛血清（fetal bovine serum）；BMDMs：骨髓源巨噬细胞（bone marrow-derived macrophages）；TNF-α：肿瘤坏死因子α（tumor necrosis factor α）；IL-1β：白细胞介素1β（interleukin-1β）；IFN-γ：干扰素γ（interferon-γ）；TGF-β：转化生长因子β（transforming growth factor-β）；ELISA：酶联免疫吸附实验（enzyme-linked immunosorbent assay）