Type IV Secretion by Neisseria gonorrhoeae

描述(申请人提供)：淋病奈瑟菌可引起性传播疾病淋病以及盆腔炎、败血症、脑膜炎和关节炎。淋球菌产生令人难以置信的遗传多样性和抗原多样性，使它们能够避免人类的免疫反应。相当数量的遗传多样性是通过自然转化创造的。自然变形是淋球菌的一种生活方式。它们在构成上具有转化的能力，它们优先从其他淋球菌或其他奈瑟菌中摄取DNA，即它们摄取最有可能用于与基因组重组的DNA。我们在淋球菌染色体的基因组岛上发现了一个IV型分泌系统(T4SS)。T4SS将细菌的染色体DNA分泌到它们周围的环境中。分泌的DNA在附近其他淋球菌的转化中很活跃。我们证明了分泌的DNA是单链的，在5‘端被阻断，而且为了分泌它，它必须首先被一种松弛酶处理， 作用于染色体上的单一位置，也就是转移的起点。T4SS对宿主细胞的相互作用有影响。它还会影响细菌之间以及与细胞和表面的粘附性。这项建议的重点是如何调控T4SS的表达。与许多其他T4SS一样，淋球菌IV型分泌基因在正常培养条件下受到抑制。许多T4SS蛋白不能被免疫印迹检测到，免疫金电子显微镜在细胞亚群上发现了一个单一的斑点。这些结果表明，每个细胞可能有一个T4SS装置，或者培养物中只有几个细胞产生分泌系统。为了进一步研究分泌的机制，寻找额外的分泌底物，并阐明IV型分泌在感染过程中的作用，将确定控制IV型分泌的机制。这一建议的具体目的是：1)确定影响T4SS表达的调节因子和环境条件；2)确定TRAH调控区对T4SS转录、翻译和蛋白质定位的机制和作用；3)使用模型底物和全基因组测序来研究DNA分泌的机制和后果。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae causes the sexually-transmitted disease gonorrhea as well as pelvic inflammatory disease, sepsis, meningitis, and arthritis. Gonococci generate an incredible degree of genetic diversity and antigenic diversity allowing them to avoid the human immune response. A significant amount of the genetic diversity is created through natural transformation. Natural transformation is a way of life for gonococci. They are constitutively competent for transformation, and they preferentially take up DNA from other gonococci or other Neisseria, i.e., they take up DNA that is most likely to be useful for recombination with the genome. We discovered a type IV secretion system (T4SS) encoded in a genomic island in the gonococcal chromosome. The T4SS secretes chromosomal DNA from the bacteria into their surroundings. The secreted DNA is active in the transformation of other gonococci in the vicinity. We demonstrated that the secreted DNA is single- stranded, blocked at the 5' end, and that for it to be secreted it must first be processed by a relaxase that acts at a single site in the chromosome, the origin of transfer. The T4SS has effects on host cell interactions. It also affects adherence of the bacteria to each other and to cells and surfaces. This proposal is focused on how expression of the T4SS is regulated. As is the case for many other T4SSs, the gonococcal type IV secretion genes are repressed under normal culture conditions. Many T4SS proteins are not detectable by western blot and immune-gold electron microscopy identifies a single spot on a subpopulation of cells. These results suggest that each cell might have one T4SS apparatus or that only a few cells in the culture produce the secretion system. To further study the mechanism of secretion, to identify additional secretion substrates, and to elucidate the effects of the type IV secretion during infection, the mechanism controlling type IV secretion will be determined. The specific aims of this proposal are: 1) to identify regulators and environmental conditions that affect T4SS expression, 2) to determine the mechanisms and effects of the traH regulatory region on T4SS transcription, translation, and protein localization, and 3) to use model substrates and whole genome sequencing to examine the mechanism and consequences of DNA secretion.