Iron acquisition mechanisms utilized by Neisseria gonorrhoeae

人类特有的病原体淋病奈瑟菌是性传播感染淋病的原因。这种疾病在男性和女性中都很常见，但在女性中发病率很高。在感染过程中，N.淋病病毒必须从人类宿主那里获得必需的营养素铁。这种病原体已经进化出从多种宿主特异性来源同化铁的能力，包括从铁结合糖蛋白、转铁蛋白和乳铁蛋白。铁从这些铁结合蛋白的运输是由一个完整的，外膜转运蛋白，这需要能量和TonB蛋白完成。由于TonB依赖性转运蛋白具有序列同源性，因此可以在测序的细菌基因组中鉴定编码它们的基因。N.淋球菌基因组表明，其他营养物质可以通过这些未表征的转运蛋白内化，并且这些营养物质，可能包括铁，可能对淋球菌在人类宿主中的存活至关重要。一些铁螯合物被N.淋病的能量和TonB的独立途径，表明淋球菌有多个，重叠和潜在的冗余铁收购系统，这强调了获得这种营养的重要性。本研究的长期目标是确定功能性的、依赖于TonB或不依赖于TonB的转运系统，表征其配体，探测其摄取机制，并确定体内转运功能干扰的生物学相关性。这些研究是重要的，因为这些途径的组分最终可以用作免疫预防的疫苗抗原或作为治疗淋球菌疾病的治疗剂的靶点。这项研究的总体目标是确定依赖TonB和不依赖TonB的运输过程如何影响N.淋病在体外和培养的宿主细胞内生长。本提案的具体目标是解决以下问题：1.淋球菌通过什么样的分子机制从其他细菌产生的低分子铁螯合物中获得铁？2.宿主细胞表达的TonB依赖性转运蛋白TdfF在上皮细胞内淋球菌存活中起什么作用？用于解决这些问题的方法包括定向和随机诱变，以确定TonB独立的铁螯合物利用途径的关键组成部分。控制TonB依赖性转运蛋白TdfF表达的机制将通过基因融合、RT-PCR和蛋白表达谱进行评估。最后，将使用细胞内存活测定和最先进的免疫荧光显微镜技术评价TdfF对培养细胞内生长和存活的贡献。

The human-specific pathogen, Neisseria gonorrhoeae is responsible for the sexually-transmitted infection, gonorrhea. This disease is common among both men and women, but causes significant morbidity amongst women. During the course of infection, N. gonorrhoeae must obtain a necessary nutrient, iron, from the human host. This pathogen has evolved the capacity to assimilate iron from a variety of host-specific sources, including from the iron-binding glycoproteins, transferrin and lactoferrin. Iron transport from these iron-binding proteins is accomplished by an integral, outer membrane transporter, which requires energy and the TonB protein. Because the TonB-dependent transporters share sequence homology, genes that encode them can be identified within sequenced bacterial genomes. The presence of genes in the N. gonorrhoeae genome suggests that other nutrients can be internalized via these uncharacterized transporters and that these nutrients, potentially including iron, may be crucial for gonococcal survival within the human host. Some iron chelates are internalized by N. gonorrhoeae in an energy- and TonB-independent pathway, suggesting that the gonococcus has multiple, overlapping and potentially redundant iron acquisition systems, which emphasizes the importance of obtaining this nutrient. The long-term goal of this research is to identify functional, TonBdependent or TonB-independent transport systems, to characterize their ligands, to probe their mechanism of uptake, and to determine the biological relevance of interference with transporter function in vivo. These studies are significant because components of these pathways could ultimately be used as vaccine antigens for immunoprophylaxis or as targets for therapeutics to treat gonococcal disease. The overall goal of the proposed study is to determine how TonB-dependent and TonB-independent transport processes contribute to the ability of N. gonorrhoeae to grow in vitro and within cultured host cells. The specific aims of this proposal address the following questions: 1. By what molecular mechanism does the gonococcus acquire iron from low molecular iron-chelates produced by other bacteria? 2. What function does the host-cell expressed TonBdependent transporter, TdfF, serve in intra-epithelial cell gonococcal survival? The methods to be employed to address these questions include directed and random mutagenesis to identify key components of the TonBindependent iron-chelate utilization pathway. The mechanisms that control expression of the TonB-dependent transporter, TdfF will be evaluated by genetic fusions, RT-PCR and protein expression profiling. Finally, the contribution that TdfF provides to growth and survival within cultured cells will be evaluated using intracellular survival assays and state-of-the-art immunofluorescence microscopy techniques.