Type IV secretion by Neisseria gonorrhoeae

描述(申请人提供)：淋病奈瑟氏菌引起的性传播疾病淋病，更严重的感染盆腔炎(PID)和播散性淋球菌感染(DGI)。我们在淋病奈瑟菌中发现了一个57kb的遗传岛，显示了致病岛的特征。在疾病分离株中存在几种形式的淋球菌基因岛(GGI)，大约80%的淋球菌菌株携带某种形式的GGI。我们鉴定了GGI中编码的一个肽聚糖水解酶(ATLA)，并发现它参与了一种来自细菌细胞壁的不寻常的细胞毒素的产生。这种由肽聚糖衍生的细胞毒素(PG-细胞毒素)与百日咳杆菌的气管细胞毒素相同，是淋球菌感染的重要毒力因子。PG细胞毒素可引起器官培养中纤毛输卵管细胞的死亡，诱发大鼠关节炎，并诱导培养细胞产生IL-1和IL-6。基因岛上ATLA的存在与引起DOT的菌株显著相关。 DNA测序表明，GGI编码一个可能的IV型分泌系统。IV型分泌系统包括质粒接合系统和毒力因子输出系统，一些IV型分泌系统执行这两个过程。GGI中假定的IV型分泌基因突变导致DNA分泌丧失。这些突变体还表现出对原代宫颈细胞的延迟黏附，并在与细胞结合时表现出不同寻常的微集落表型。ATLA突变导致与其他IV型分泌物突变相同的表型，并显示PG细胞毒素产生减少。因此，ATLA是IV型细胞分泌所必需的，并直接或间接参与PG细胞毒素的产生。这些结果表明，GGI编码一个活跃的IV型分泌系统，在淋病奈瑟菌与宿主的相互作用中起重要作用。 这项提案的目标包含在两个具体目标中。1)我们将进行旨在影响IV型细胞分泌的突变，并测试这些突变株的蛋白质、DNA和PG-细胞毒素的分泌。2)我们将测试分泌缺陷的突变株与毒力相关的表型，即上皮细胞的黏附和侵袭、细胞内存活和微集落形成。总之，这些研究旨在揭示这种新的分泌系统的分子机制，并识别以前未被认识到的在淋球菌感染中重要的毒力因子，这些毒力因子可能成为化疗或免疫保护的新靶点。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea, the more serious infections pelvic inflammatory disease (PID) and disseminated gonococcal infection (DGI). We identified a 57 kb genetic island present in N. gonorrhoeae that shows the characteristics of a pathogenicity island. Several forms of the gonococcal genetic island (GGI) are present among disease isolates, with approximately 80 percent of gonococcal strains carrying some form of the GGI. We identified a peptidoglycan hydrolase (atlA) encoded in the GGI and found that it is involved in the production of an unusual cytotoxin derived from the bacterial cell wall. This peptidoglycan-derived cytotoxin (PG-cytotoxin) is identical to the tracheal cytotoxin of Bordetella pertussis and is an important virulence factor in gonococcal infections. PG-cytotoxin causes the death of ciliated fallopian tube cells in organ culture, induces arthritis in rats, and induces IL-1 and IL-6 in cultured cells. The presence of atlA in the genetic island is significantly correlated with strains that cause DOT. DNA sequencing revealed that the GGI encodes a putative type IV secretion system. Type IV secretion systems include plasmid conjugation systems and virulence factor export systems and some type IV secretion systems carry out both processes. Mutations in the putative type IV secretion genes in the GGI result in loss of DNA secretion. These mutants also show delayed adherence to primary cervical cells and exhibit an unusual microcolony phenotype upon binding to cells. Mutations in atlA result in the same phenotypes as the other type IV secretion mutations as well as showing decreased PG-cytotoxin production. Thus AtlA is necessary for type IV secretion and is either directly or indirectly involved in PG-cytotoxin production. These results suggest that the GGI encodes an active type IV secretion system important in interaction of N. gonorrhoeae with the host. The goals of this proposal are contained in two specific aims. 1) We will make mutations designed to affect type IV secretion and test these mutants for secretion of proteins, DNA, and PG-cytotoxin. 2) We will test mutants with defects in secretion for phenotypes relevant to virulence, i.e., epithelial cell adherence and invasion, intracellular survival, and microcolony formation. Overall, these studies are designed to reveal the molecular mechanisms of this novel secretion system and identify previously unrecognized virulence factors important in gonococcal infection which may serve as new targets for chemotherapy or immunoprotection.