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

描述（由申请人提供）：Sodalis glossinidlus是一种兼性细胞内细菌，是采采蝇的次级共生体，其携带引起非洲昏睡病的锥虫。用于生长细菌的纯培养技术最近才被公布，因此，用于Sodalis的分子遗传操作的优化的程序和技术尚未完全开发。此外，使这种兼性内共生细菌能够在昆虫体内生存和繁殖的生理过程，包括细胞内环境，才刚刚开始被检查。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存的重要生理过程。该项目的科学目标是开发和完善分子遗传工具，以测试两种Sodalis假定的铁获取系统对昆虫细胞和采采蝇中Sodalis生长的贡献的假设。这些系统被预测编码的蛋白质，有利于有效利用血红素作为铁源（HemS）和achomobactin铁载体系统。在特定目标1中，将在Sodalis中优化基于内含子的诱变程序，以在hemS中产生定向突变。通过去除含有这些基因的质粒，将从Sodalis中消除无色菌素基因。在具体目标2中，将检查Sodalis hemS和无色菌素突变体在采采蝇和昆虫培养物中生长的能力，以确定哪些Sodalis铁获取基因对生长是重要的。在具体目标3中，将通过使用Sodalis基因补充志贺菌和大肠杆菌来验证推定的Sodalis hemS和无色菌素基因的功能。携带相似基因突变的大肠杆菌菌株。在具体目标4中，当Sodalis在采采蝇中时，将检查Sodalis hemS和无色菌素基因的表达模式。公共卫生相关性：研究采采蝇Sodalis共生将有意义的公共卫生，因为存在的Sodalis在采采蝇可能会增加采采介导的锥虫，导致非洲昏睡病的传播;因此，增加了解胞内的Sodalis和采采蝇共生的性质可能会产生信息，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活，并需要高亲和力的铁运输的发病机制，从本提案中描述的工作获得的数据将提供相关的信息，这种致病性共生。

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.