Williams J M, Chen G C, Zhu L, Rest R F
Department of Microbiology and Immunology, MCP-Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, PA 19129, USA.
Mol Microbiol. 1998 Jan;27(1):171-86. doi: 10.1046/j.1365-2958.1998.00670.x.
Neisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two-hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteria-host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified five different OIPs, designated OIP1-OIP5, two of which are homologous to human proteins--thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two-hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.
淋病奈瑟菌不透明相关（Opa）蛋白是一类外膜蛋白，参与淋球菌对人细胞的黏附和侵袭。我们想要确定Opa在感染过程中的其他作用，并利用酵母双杂交系统来鉴定与Opa蛋白相互作用的人上皮细胞蛋白。尽管该系统已成功用于鉴定多种类型的相互作用蛋白，但尚未用于筛选人细胞cDNA文库以寻找原核外膜蛋白的结合伴侣。因此，我们也对探索酵母双杂交系统在鉴定细菌与宿主相互作用方面的通用性感兴趣。以F62SF菌株的OpaP作为诱饵，我们筛选了HeLa细胞cDNA文库以寻找与Opa相互作用的蛋白（OIPs）。我们鉴定出了五种不同的OIPs，命名为OIP1 - OIP5，其中两种与人蛋白同源——甲状腺激素受体相互作用蛋白（TRIP6）和丙酮酸激酶同工酶M2（PK）。在本文所述的研究中，我们更深入地研究了Opa蛋白与PK之间的相互作用。通过独立于酵母双杂交系统的体外和体内试验证实了Opa与PK的相互作用。在体外结合试验中，表达来自淋球菌菌株FA1090的六种不同Opa蛋白的大肠杆菌比Opa阴性的大肠杆菌结合更多的PK。使用抗PK抗体和荧光显微镜，我们表明人上皮细胞PK与细胞内Opa + 淋球菌以及表达Opa蛋白的大肠杆菌共定位。使用不能在丙酮酸或乳酸上生长的淋病奈瑟菌突变体，似乎细胞内丙酮酸对于淋球菌的生长和存活至关重要。这些结果提示了细菌致病机制中的一种新机制，即需要与宿主代谢酶（PK）进行直接分子相互作用以获取必需的细胞内碳源和生长底物（丙酮酸）。这些结果表明酵母双杂交系统是鉴定细菌与宿主蛋白之间生物学相关相互作用的有价值工具，为进一步研究细菌致病新机制提供了有价值的线索。
