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GONOCOCCAL PIA--ROLE IN INVASION OF FALLOPIAN TUBE CELL

淋球菌 PIA——侵入输卵管细胞的作用

基本信息

批准号：
2886956
负责人：
GARY L GORBY
金额：
$ 8.36万
依托单位：
CREIGHTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-09-30 至 2001-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2886956
关键词：
Escherichia coli G protein Neisseria gonorrhoeae calmodulin disease /disorder model fallopian tubes gene expression human tissue membrane proteins site directed mutagenesis tissue /cell culture

项目摘要

Neisseria gonorrhoeae (GC) is able to attach to and invade non-ciliated epithelial cells of the Fallopian tube organ culture model (FTOC). The mechanism of this interaction remains to be defined. GC Por or PI, the focus of this proposal, is an outer membrane porin protein that comes in two forms, PIA or PIB. It does not vary within a single GC strain. It can transfer spontaneously from GC to lipid bilayers and human cells. It is able to bind calmodulin, but the impact of this on pathogenesis is unknown. Two surface-exposed regions of PIA have amino acid sequences compatible with a basic amphiphilic a-helical conformation, a motif that calmodulin recognizes in binding to a large variety of proteins. This motif also allows peptides and some transmembrane receptors to activate G proteins. PIA has been cloned and expressed in E. coli. In the FTOC model, these E. coli fail to attach, which has prevented assessment of PIA's invasion-triggering properties. GC opacity-associated proteins (Opa) and lipooligosaccharides (LOS), have been implicated as attachment and invasion factors in some cell culture models. Opa and LOS undergo rapid phase and antigenic variation. One type of LOS defined by the 3F11 monoclonal antibody binds to the asialoglycoprotein receptor in liver cell lines. It is not known if the 3F11 epitope mediates attachment to or invasion of fallopian tube epithelium. Opas and the 3F11 LOS have been cloned and expressed in E. coli. Opa production facilitates attachment of E. coli in the human FTOC model, but observed bacterial transport through the cells has been much less than that seen with GC. Thus, we hypothesize that following intimate Opa- and/or LOS-mediated attachment, PI insertion into host cells triggers significant GC invasion via calmodulin- and/or G protein-mediated pathways. By providing for bacterial attachment and eliminating phase and antigenic variation via subcloning of Opa and/or LOS into PIA-bearing E. coli, "GC surrogates" will be constructed to study invasion-triggering by PIA. Variants which express Opa, 3F11 LOS, and PIA in various combinations will be tested in the FTOC model for invasiveness as measured by computerized image analysis in conjunction with digital confocal microscopy. Invasive subclones of E. coli and GC will be tested in the FTOC model with G protein and calmodulin activators or inhibitors to determine if these pathways are connected to invasion through GC PIA. Site-directed mutagenesis of putative basic amphiphilic alpha-helical PIA regions with a-helix breaking proline residues should ablate calmodulin-binding properties and invasiveness. Morphometric comparisons of these mutants to E. coli expressing wild-type PIA in the FTOC model will confirm the pathogenic role of these regions.

淋病奈瑟菌(GC)能够附着和入侵非纤毛虫输卵管上皮细胞器官培养模型(FTOC)。这个这种相互作用的机制还有待确定。GC POR或PI，这项提议的焦点是一种外膜孔蛋白，它进入两种形式，PIA或PIB。它在单个GC菌株内不会发生变化。它可以自发地从GC转移到脂质双分子层和人体细胞。它是能够结合钙调蛋白，但这对发病机制的影响是未知。PIA的两个表面暴露区域具有氨基酸序列与基本的两亲性α-螺旋构象兼容，该基序钙调蛋白可识别多种蛋白质的结合。这 Motif还允许多肽和一些跨膜受体激活 G蛋白。Pia基因已被克隆并在大肠杆菌中表达。在FTOC中模型中，这些大肠杆菌未能附着，这阻碍了对 PIA的入侵触发特性。 GC不透明相关蛋白(OpA)和脂寡糖(LOS)，有在某些细胞培养中被认为是附着和侵袭因素模特们。OPA和LOS经历快速时相和抗原性变化。一由3F11单抗定义的LOS类型与肝细胞系的去唾液酸糖蛋白受体。目前尚不清楚 3F11表位介导输卵管附着或侵袭上皮组织。OPAs和3F11LOS被克隆并在E. Coli.OPA的产生促进了大肠杆菌在人FTOC中的附着模型，但观察到的细菌通过细胞的传输低于GC。因此，我们假设下列亲密的OPA和/或LOS调节附着，PI插入宿主细胞触发显著的GC侵袭通过钙调素和/或G蛋白介导的途径。通过规定细菌的附着和消除阶段与抗原变异 OpA和/或LOS亚克隆到含PIA的大肠杆菌“GC替代物”中将被构建来研究PIA触发入侵的机制。其中的变种各种组合的Express Opa、3F11 LOS和PIA将在用计算机图像测量侵入性的FTOC模型结合数字共聚焦显微镜进行分析。侵入性大肠杆菌和GC的亚克隆将在FTOC模型中与G 蛋白质和钙调蛋白激活剂或抑制物来确定这些途径通过GC-PIA与侵袭有关。站点定向可能的碱性两亲性α-螺旋PIA区的突变 A-螺旋断裂的脯氨酸残基应该会破坏钙调蛋白结合财产和侵入性。这些突变体的形态计量学比较在FTOC模型中表达野生型PIA的大肠杆菌将证实这些区域的致病作用。