Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci.
Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci.
复制标题
DOI：
10.1128/msphere.00331-18
复制
发表时间：
2018-10-10
期刊：
影响因子：
4.8
通讯作者：
Christodoulides M
中科院分区：
文献类型：
--
作者：
Almonacid-Mendoza HL;Humbert MV;Dijokaite A;Cleary DW;Soo Y;Hung MC;Orr CM;Machelett MM;Tews I;Christodoulides M
淋病奈瑟菌（淋球菌 [Ng]）是性传播疾病淋病的病原体，世界卫生组织将其列为研究和开发新控制措施（包括疫苗）的高度优先病原体。在这项研究中，我们证明了淋病奈瑟菌粘附素复合物蛋白 (Ng-ACP) 在 50 种淋球菌菌株中是保守的和表达的，并且重组蛋白在小鼠体内诱导产生抗体，从而在体外杀死细菌。我们通过 X 射线晶体学确定了 Ng-ACP 的结构，并研究了脑膜炎奈瑟菌 ACP 和来自其他细菌的 MliC/PliC 蛋白的结构保守性，这些蛋白充当人类先天防御分子溶菌酶的抑制剂。这些发现很重要，表明 Ng-ACP 可以为解决淋球菌感染提供潜在的双重靶点。淋病奈瑟菌（淋球菌[Ng]）是性传播疾病淋病的病原体，目前尚无有效的疫苗。在这项研究中，介绍了 Ng-粘附素复合蛋白 (Ng-ACP) 的结构、生物学特性和疫苗潜力。重组 Ng-ACP (rNg-ACP) 蛋白的晶体结构在 1.65 Å 处得到解析。在计算机中检查了不同奈瑟菌属物种和淋球菌分离株（https://pubmlst.org/neisseria/ 数据库）中 Ng-ACP 的多样性和保守性，并通过蛋白质印迹检查了疾病控制和预防中心/食品和药物管理局 (CDCP/FDA) AR 分离株库中 50 种淋球菌菌株的蛋白质表达。使用不同佐剂将小鼠抗血清提高到等位基因 10（菌株 P9-17）编码的 rNg-ACP 蛋白，并通过酶联免疫吸附测定 (ELISA)、蛋白质印迹和人血清杀菌测定进行检查。测试了兔 rNg-ACP 抗血清在体外阻止 Ng-ACP 抑制人溶菌酶活性的能力。 Ng-ACP 在结构上与脑膜炎奈瑟菌 ACP 和 MliC/PliC 溶菌酶抑制剂同源。淋球菌主要表达等位基因 10 和等位基因 6 编码的 Ng-ACP（分别占分离株的 81% 和 15%）。鼠抗血清对同源 P9-17 菌株和异源（等位基因 6）FA1090 菌株具有杀菌作用（效价为 64 至 512，P<0.05）。兔抗 rNg-ACP 血清可阻止 Ng-ACP 抑制人溶菌酶，效率约为 100%。所有 50 个淋球菌分离株均表达 Ng-ACP 蛋白，但相对表达水平存在微小差异。 rNg-ACP 是一种潜在的候选疫苗，可诱导产生以下抗体：(i) 杀菌；(ii) 防止淋球菌抑制先天防御分子的裂解活性。重要性 淋病奈瑟菌（淋球菌 [Ng]）是性传播疾病淋病的病原体，世界卫生组织将其列为研究和开发新控制措施（包括疫苗）的高度优先病原体。在这项研究中，我们证明了淋病奈瑟菌粘附素复合物蛋白 (Ng-ACP) 在 50 种淋球菌菌株中是保守的和表达的，并且重组蛋白在小鼠体内诱导产生抗体，从而在体外杀死细菌。我们通过 X 射线晶体学确定了 Ng-ACP 的结构，并研究了脑膜炎奈瑟菌 ACP 和来自其他细菌的 MliC/PliC 蛋白的结构保守性，这些蛋白充当人类先天防御分子溶菌酶的抑制剂。这些发现很重要，表明 Ng-ACP 可以为解决淋球菌感染提供潜在的双重靶点。
Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and the organism is listed by the World Health Organization as a high-priority pathogen for research and development of new control measures, including vaccines. In this study, we demonstrated that the N. gonorrhoeae adhesin complex protein (Ng-ACP) was conserved and expressed by 50 gonococcal strains and that recombinant proteins induced antibodies in mice that killed the bacteria in vitro. We determined the structure of Ng-ACP by X-ray crystallography and investigated structural conservation with Neisseria meningitidis ACP and MliC/PliC proteins from other bacteria which act as inhibitors of the human innate defense molecule lysozyme. These findings are important and suggest that Ng-ACP could provide a potential dual target for tackling gonococcal infections. Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and no effective vaccine exists currently. In this study, the structure, biological properties, and vaccine potential of the Ng-adhesin complex protein (Ng-ACP) are presented. The crystal structure of recombinant Ng-ACP (rNg-ACP) protein was solved at 1.65 Å. Diversity and conservation of Ng-ACP were examined in different Neisseria species and gonococcal isolates (https://pubmlst.org/neisseria/ database) in silico, and protein expression among 50 gonococcal strains in the Centers for Disease Control and Prevention/Food and Drug Administration (CDCP/FDA) AR Isolate Bank was examined by Western blotting. Murine antisera were raised to allele 10 (strain P9-17)-encoded rNg-ACP protein with different adjuvants and examined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and a human serum bactericidal assay. Rabbit antiserum to rNg-ACP was tested for its ability to prevent Ng-ACP from inhibiting human lysozyme activity in vitro. Ng-ACP is structurally homologous to Neisseria meningitidis ACP and MliC/PliC lysozyme inhibitors. Gonococci expressed predominantly allele 10- and allele 6-encoded Ng-ACP (81% and 15% of isolates, respectively). Murine antisera were bactericidal (titers of 64 to 512, P < 0.05) for the homologous P9-17 strain and heterologous (allele 6) FA1090 strain. Rabbit anti-rNg-ACP serum prevented Ng-ACP from inhibiting human lysozyme with ∼100% efficiency. Ng-ACP protein was expressed by all 50 gonococcal isolates examined with minor differences in the relative levels of expression. rNg-ACP is a potential vaccine candidate that induces antibodies that (i) are bactericidal and (ii) prevent the gonococcus from inhibiting the lytic activity of an innate defense molecule. IMPORTANCE Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and the organism is listed by the World Health Organization as a high-priority pathogen for research and development of new control measures, including vaccines. In this study, we demonstrated that the N. gonorrhoeae adhesin complex protein (Ng-ACP) was conserved and expressed by 50 gonococcal strains and that recombinant proteins induced antibodies in mice that killed the bacteria in vitro. We determined the structure of Ng-ACP by X-ray crystallography and investigated structural conservation with Neisseria meningitidis ACP and MliC/PliC proteins from other bacteria which act as inhibitors of the human innate defense molecule lysozyme. These findings are important and suggest that Ng-ACP could provide a potential dual target for tackling gonococcal infections.