Investigation of iron acquisition genes in Sodalis glossinidius using new tools.

描述（由申请人提供）：舌蝇是一种兼性细胞内细菌，是采采蝇的次级共生体，采采蝇携带引起非洲昏睡病的锥虫。培养这种细菌的纯培养技术直到最近才发表，因此，用于Sodalis分子遗传操作的优化程序和技术还没有完全开发出来。此外，使这种兼性内共生细菌在昆虫体内生存和繁殖的生理过程，包括细胞内环境，才刚刚开始被研究。研究这些过程将有助于PI研究的长期科学目标，即阐明病原体和非病原体在真核细胞内生活的重要生理过程。该项目的科学目标是开发和完善分子遗传工具，以测试假设，这些假设解决了两种索达利斯假定的铁获取系统对昆虫细胞和采采蝇内索达利斯生长的贡献。预计这些系统会编码一种蛋白质，促进血红素作为铁源（HemS）的有效利用，以及不舍菌蛋白铁载体系统。在Specific Aim 1中，基于内含子的诱变程序将在Sodalis中进行优化，以在hem中产生定向突变。通过移除含有消色肌动蛋白基因的质粒，将消色肌动蛋白基因从Sodalis中去除。在特定目标2中，将检查Sodalis hemS和achromoactin突变体在采采蝇和昆虫培养中生长的能力，以确定哪些Sodalis铁获取基因对生长重要。在Specific Aim 3中，假定的Sodalis hemS和achromobactin基因的功能将通过使用Sodalis基因来补充携带类似基因突变的痢疾志贺氏菌和大肠杆菌菌株来验证。在Specific Aim 4中，当Sodalis在采采蝇体内时，将检测Sodalis hem和achromobactin基因的表达模式。公共卫生相关性：研究采采蝇与索达利斯的共生关系将对公共卫生具有重要意义，因为索达利斯在采采蝇中的存在可能会增加引起非洲昏睡病的锥虫的采采媒介传播；因此，增加对索达菌和采采蝇细胞内共生性质的了解可能会提供信息，以帮助控制采采蝇传播的疾病。此外，许多重要的人类病原体可以作为内共生体生存，并且需要高亲和力的铁转运来致病，本提案所述工作获得的数据将提供与此类致病性共生相关的信息。

DESCRIPTION (provided by applicant): Sodalis glossinidlus is a facultative intracellular bacterium that is a secondary symbiont of the tsetse fly, which carries the trypanosome that causes African sleeping sickness. Pure culture techniques for growing the bacterium have only recently been published and, thus, procedures and techniques optimized for molecular genetic manipulation of Sodalis have not fully been developed. Furthermore, the physiological processes that enable this facultative endosymbiotic bacterium to survive and multiply within the insect, including the intracellular environment, are just beginning to be examined. Investigating these processes will contribute to the long-term scientific goals of the PI's research, which are to elucidate the physiological processes that are important for pathogens and non-pathogens to live within eukaryotic cells. The scientific goals of this project are to develop and refine molecular genetic tools for the purpose of testing hypotheses which address the contribution of two Sodalis putative iron acquisition systems to growth of Sodalis within insect cells and the tsetse fly. These systems are predicted to encode a protein that facilitates efficient utilization of heme as an iron source (HemS) and the achomobactin siderophore system. In Specific Aim 1, intron-based mutagenesis procedures will be optimized in Sodalis to generate directed mutations in hemS. The achromobactin genes will be eliminated from Sodalis by removing the plasmid that contains these genes. In specific aim 2, the ability of the Sodalis hemS and achromobactin mutants to grow in tsetse flies and in insect culture will be examined to determine which Sodalis iron acquisition genes are important for growth. In Specific Aim 3, the function of the putative Sodalis hemS and achromobactin genes will be verified by using the Sodalis genes to complement Shigella dysenteriae and E. coli strains that carry mutations in similar genes. In Specific Aim 4, the expression pattern of the Sodalis hemS and achromobactin genes will be examined when Sodalis is in the tsetse fly. PUBLIC HEALTH RELEVANCE: Studying the tsetse fly-Sodalis symbiosis will have significance to public health because the presence of Sodalis in the tsetse fly may increase tsetse-mediated transmission of the trypanosome that causes African sleeping sickness; thus, an increased understanding of the nature of the intracellular symbiosis of Sodalis and tsetse may yield information to help control tsetse-borne disease. Furthermore, many important human pathogens can live as endosymbionts and require high affinity iron transport for pathogenesis, and the data obtained from the work described in this proposal will provide information relevant to such pathogenic symbioses.