Prophage-associated virulence factors in pathogenic Neisseria species

描述（由申请人提供）： 这项研究的长期目标是确定毒力因子，这些毒力因子可能用于设计淋病奈瑟菌（淋球菌，GC）和脑膜炎奈瑟菌（脑膜炎球菌，MC）的有效疫苗和治疗药物，淋病奈瑟菌（淋球菌，GC）引起淋病和盆腔炎，脑膜炎奈瑟菌（脑膜炎球菌，MC）引起败血症和脑膜炎。R 03应用的目的是研究脑膜炎球菌病相关（MDA）前噬菌体在GC和MC发病机制中的作用。在GC和MC基因组的多个位点发现的MDA原噬菌体编码一种潜在的毒素，该毒素是霍乱弧菌CTX原噬菌体带状隐带毒素（Zot）的同系物，其改变上皮和内皮紧密连接。因此，GC和MC中的MDA原噬菌体Zot蛋白具有参与炎症、脓毒症和/或血脑屏障侵袭的潜力。MDA噬菌体DNA插入（和切除）宿主染色体以建立溶原性和可能的切除以起始复制可以由ISNgo 2/3元件的转座酶介导，ISNgo 2/3元件是噬菌体基因组的一部分。确定噬菌体DNA运动的机制对于理解噬菌体在GC和MC发病机制中的作用至关重要。具体目标是：1.确定脑膜炎球菌和淋球菌Zot蛋白的活性，以及2.定义ISNgo 2/3转座酶Irg在指导MDAphi DNA插入和/或切除以及控制MC和GC中zot表达中的作用。实验方法将包括测定上皮和脑微血管内皮细胞膜上来自MC和GC的Zot的闭锁小带毒素活性;定量逆转录酶PCR以测量不同生长条件下Zot和irg的表达;以及定量PCR以检测MDAphi的移动和复制。 这项工作与公共卫生有关，因为美国没有针对淋病奈瑟菌或脑膜炎奈瑟菌最流行血清群（B）的有效疫苗。美国淋球菌病的年发病率为130/100，000，而脑膜炎球菌病的发病率为1/100，000。尽管使用了化疗药物，但10-40%的侵袭性脑膜炎球菌感染是致命的，10-15%的幸存者有严重的后遗症，包括智力迟钝和耳聋。

DESCRIPTION (provided by applicant): The long-term goal of this research is to identify virulence factors that may be targeted for the design of effective vaccines and therapeutics for Neisseria gonorrhoeae (gonococci, GC), which causes gonorrhoeae and pelvic inflammatory disease, and Neisseria meningitidis (meningococci, MC), which causes septicemia and meningitis. The objective of this R03 application is to examine the role of the meningococcal disease-associated (MDA) prophage in the pathogenesis of GC and MC. The MDA prophage, which is found at multiple loci on the GC and MC genomes, encodes a potential toxin that is a homologue of the Vibrio cholera CTX prophage zonular occulens toxin (Zot) that alters epithelial and endothelial tight junctions. Thus, the MDA prophage Zot proteins in GC and MC have the potential to be involved in inflammation, sepsis, and/or invasion of the blood-brain barrier. The insertion (and excision) of the MDA phage DNA into the host chromosome to establish lysogeny and possible excision to initiation replication may be mediated by the transposase of the ISNgo2/3 elements, which are part of the phage genome. Defining the mechanism for the movement of the phage DNA is essential to understanding the role of the phage in GC and MC pathogenesis. The specific aims are to: 1. Determine the activities of the meningococcal and gonococcal Zot proteins, and 2. Define the role of the ISNgo2/3 transposase, Irg, in directing insertion and/or excision of MDAphi DNA and controlling zot expression in MC and GC. Experimental approaches will include assays for zonular occludens toxin activity for Zot from MC and GC on epithelial and brain microvascular endothelial cell membranes; quantitative reverse transcriptase PCR to measure expression of zot and irg under different growth conditions; and quantitative PCR to detect movement and replication of MDAphi. This work is relevant to public health because there is no effective vaccine for Neisseria gonnorrhoeae or for the most prevalent serogroup (B) of Neisseria meningitidis in the U.S. The annual U.S. incidence of gonococcal disease is 130 in 100,000, while the incidence of meningococcal disease is 1 in 100,000. Despite the use of chemotherapeutics, 10-40% of invasive meningococcal infections are fatal and 10-15% of survivors have serious sequelae, including mental retardation and deafness.