IDENTIFICATION OF GONOCOCCAL PH-REGULATED PROTEINS

淋病奈瑟菌产生保守而独特的热休克蛋白， 或应激蛋白，以应对高温、营养匮乏和 厌氧症。因为淋球菌暴露在广泛的pH值变化中 它在体内感染的各种生态位，可能会产生pH调节的应激 蛋白质也是。这项提议的目标是识别蛋白质 淋病奈瑟菌对公认的pH胁迫的反应 用淋病患者的恢复期血清。实验方案 被选中是因为它们被成功地用来描述压力 其他致病细菌中的蛋白质。此外，这些协议是 相当直截了当，使本科生能够参与。标准的一条- 而二维电泳分析将被用来检测 胞质和外膜蛋白的诱导和抑制 在体外对pH变化的反应。然后，分离的外膜免疫印迹 将探索生长在不同pH值下的生物体的膜蛋白 用淋病患者的免疫血清。免疫印迹结果 实验将表明，pH诱导的外膜蛋白是否 在淋球菌感染期间表达，以及它们是否具有免疫显性。 最后，淋球菌pH调节蛋白被患者免疫识别 血清将被提纯，印迹在聚偏二氟乙烯膜上， 对N端20-30个氨基酸进行了测序。这些序列将是 与来自其他生物的胁迫蛋白质氨基酸序列进行比较。 这些数据将使淋球菌pH调节蛋白的分类成为可能 作为高度保守的应激蛋白家族的成员，或作为 淋球菌。独特的暴露在表面的应激蛋白将是极好的 淋病疫苗的候选者。这项研究将有助于 对淋球菌应激蛋白在发病机制中作用的认识 和宿主的免疫反应。

Neisseria gonorrhoeae produces conserved and unique heat shock proteins, or stress proteins, in response to heat, nutrient deprivation, and anaerobiasis. As the gonococcus is exposed to wide variations in pH in the various niches it infects in vivo, it may produce pH-regulated stress proteins as well. The objective of this proposal is to identify proteins synthesized by N. gonorrhoeae in response to pH stress that are recognized by convalescent serum from patients with gonorrhea. Experimental protocols were chosen as they have been used successfully to describe stress proteins in other pathogenic bacteria. Furthermore, these protocols are quite straightforward, enabling undergraduate participation. Standard one- and two-dimensional electrophoretic analyses will be used to detect induction and repression of cytoplasmic and outer membrane proteins in response to pH changes in vitro. Then, immunoblots of separated outer membrane proteins from organisms grown at different pHs will be probed with immune serum from patients with gonorrhea. Results of immunoblotting experiments will indicate whether pH-induced outer membrane proteins are expressed during gonococcal infection, and if they are immunodominant. Finally, gonococcal pH-regulated proteins recognized by patient immune serum will be purified, blotted onto polyvinylidene difluoride membranes, and the N-terminal 20-30 amino acids sequenced. These sequences will be compared to stress protein amino acid sequences from other organisms. These data will enable classification of gonococcal pH-regulated proteins as members of highly conserved stress protein families, or as unique to the gonococcus. Unique surface-exposed stress proteins would be excellent candidates for a gonococcal vaccine. This research will contribute toward an understanding of the role of gonococcal stress proteins in pathogenesis and the host immune response.