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以及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而在人群中存活，从而导致抗原变异和避免宿主免疫应答。基因组内和基因组间的重组导致了遗传多样性。基因组间重组是由来自其他淋球菌的DNA自然转化引起的。自然转化是如此频繁，淋球菌的人口结构被认为是panmictic。我们发现并正在研究N.淋病我们已经证明T4 SS分泌染色体DNA，并且分泌的DNA在群体中的其他淋球菌的转化中是活跃的。T4 SS编码在一个遗传岛上，存在于80%的N。淋病菌株。T4 SS基因的突变导致DNA分泌到培养基中的损失，菌株捐赠DNA用于自然转化的能力急剧降低，并改变了与宿主细胞的相互作用。在感染原代宫颈细胞时，T4 SS突变体的附着延迟。在感染后的稍后时间，T4 SS突变体作为异常大的聚集体结合到宿主细胞。此外，对转化的宫颈细胞系的研究显示，缺失编码T4 SS的遗传岛的突变体的侵袭性降低。该提案的重点是确定淋球菌T4 SS的功能-检查DNA分泌的机制以及分泌系统如何影响宿主细胞相互作用。该提案的目标包含在三个具体目标中。我们将产生针对组成分泌器的特定蛋白质的抗体，并使用这些抗体来确定淋球菌细胞隔室和整个淋球菌细胞中蛋白质的位置。我们将研究DNA分泌的机制，使用遗传学方法来识别和表征蛋白质，其作用是结合和切割DNA进行分泌。我们将使用显微镜、侵袭和细胞内生长测定以及宿主细胞信号传导的测量来表征T4 SS对宿主细胞感染的影响。我们假设分泌的DNA-蛋白质复合物通过改变细菌粘附或分散直接影响感染，或通过影响宿主细胞信号传导间接作用，导致宿主蛋白质的不同表达影响细菌粘附、侵袭或细胞内存活。预计这些研究不仅将导致更好地理解IV型分泌和通过转化的基因水平转移，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。 说明：该项目将描述淋病奈瑟氏菌分泌器分泌DNA和蛋白质的机制。这些研究将使我们更好地了解基因是如何在细菌之间转移的，以及N。淋病感染人体细胞。

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.