Type IV secretion by Neisseria gonorrhoeae

描述（由申请人提供）：淋病奈瑟菌是性传播疾病淋病、盆腔炎和播散性淋球菌感染的病原体。淋球菌通过产生高度的遗传多样性，导致抗原变异和避免宿主免疫反应而在人群中存活。遗传多样性是基因组内重组和基因组间重组的结果。基因组间重组是由与其他淋球菌DNA的自然转化引起的。自然转化是如此频繁，以至于淋球菌种群结构被认为是泛型的。我们发现并正在努力表征淋病奈瑟菌的IV型分泌系统（T4SS）。我们已经证明T4SS分泌染色体DNA，并且分泌的DNA在群体中其他淋球菌的转化中是活跃的。T4SS是在80%的淋病奈瑟菌菌株中存在的一个基因岛上编码的。T4SS基因的突变导致DNA分泌到培养基中的损失，菌株为自然转化提供DNA的能力急剧下降，并改变了与宿主细胞的相互作用。在原代宫颈细胞感染中，T4SS突变体附着延迟。在感染后的后期，T4SS突变体以异常大的聚集体结合到宿主细胞上。此外，对转化的宫颈细胞系的研究表明，编码T4SS的基因岛的突变体被删除后，侵袭性降低。本课题主要研究淋球菌T4SS的功能，探讨其DNA分泌机制及分泌系统对宿主细胞相互作用的影响。这项建议的目标包含在三个具体目标中。我们将产生针对组成分泌装置的特定蛋白质的抗体，并使用这些抗体来确定蛋白质在淋球菌细胞室和整个淋球菌细胞中的位置。我们将研究DNA分泌的机制，使用遗传方法来鉴定和表征结合和切割DNA分泌的蛋白质。我们将利用显微镜、侵袭和细胞内生长试验以及宿主细胞信号测量来表征T4SS对宿主细胞感染的影响。我们假设分泌的dna -蛋白复合物通过改变细菌粘附或分散直接影响感染，或者通过影响宿主细胞信号传导导致影响细菌粘附、侵袭或细胞内存活的宿主蛋白的不同表达间接起作用。预计这些研究不仅将使我们更好地理解IV型分泌和基因通过转化的水平转移，而且还将确定可能成为药物或免疫治疗靶点的潜在表面分子。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae is the causative agent of the sexually-transmitted disease gonorrhea as well as pelvic inflammatory disease, and disseminated gonococcal infection. Gonococci survive in the human population by generating a high degree of genetic diversity resulting in antigenic variation and the avoidance of the host immune response. The genetic diversity results from both intragenomic and intergenomic recombination. Intergenomic recombination results from natural transformation with DNA from other gonococci. Natural transformation is so frequent that the gonococcal population structure is considered panmictic. We discovered and are working to characterize a type IV secretion system (T4SS) in N. gonorrhoeae. We have shown that the T4SS acts to secrete chromosomal DNA, and that the secreted DNA is active in the transformation of other gonococci in the population. The T4SS is encoded on a genetic island present in eighty per cent of N. gonorrhoeae strains. Mutations in the T4SS genes resulted in loss of DNA secretion into the medium, drastic reduction in the ability of a strain to donate DNA for natural transformation, and altered interactions with host cells. In infection of primary cervical cells, T4SS mutants were delayed in attachment. At later times post-infection, the T4SS mutants were bound to the host cells as unusually large aggregates. Furthermore, studies with a transformed cervical cell line showed decreased invasion by a mutant deleted for the genetic island that encodes the T4SS. This proposal is focused on determining the function of the gonococcal T4SS - examining the mechanism of DNA secretion and how the secretion system affects host cell interactions. The goals of this proposal are contained in three specific aims. We will generate antibodies to specific proteins that make up the secretion apparatus and use these antibodies to determine the locations of the proteins in gonococcal cell compartments and on whole gonococcal cells. We will examine the mechanism of DNA secretion using genetic methods to identify and characterize proteins that act to bind and cleave DNA for secretion. We will characterize the effects of the T4SS on host cell infection using microscopy, invasion and intracellular growth assays, and measures of host cell signaling. We hypothesize that a secreted DNA-protein complex affects infection directly, by altering bacterial adherence or dispersion, or acts indirectly by affecting host cell signaling leading to different expression of host proteins affecting bacterial adherence, invasion, or intracellular survival. It is anticipated that these studies will not only lead to a better understanding of type IV secretion and the horizontal transfer of genes via transformation, but that they will also identify potential surface molecules that may be targets for drugs or immunotherapy. NARRATIVE: This project will characterize the mechanism of secretion of DNA and proteins by a secretion apparatus found in the bacterium Neisseria gonorrhoeae. These studies will lead to a better understanding of how genes are transferred between bacteria and how N. gonorrhoeae infects human cells.