Genetic screens to identify mycobacterial porins

描述（由申请人提供）：分枝杆菌是一个主要寄生于许多生态位的微生物家族。这些成员中有几个是非常重要的人类病原体，包括结核病的病原体（M. tuberculosis，M. africanum和M. bovis），以及M. avium和M.主要感染免疫功能低下的患者。分枝杆菌的一个标志性特征是富含脂质的细胞包膜，这使得这些生物体高度不可渗透，并使它们对许多杀菌化合物具有内在抗性。为了促进亲水性溶质的摄取，分枝杆菌，如革兰氏阴性生物，利用孔蛋白，孔蛋白是水填充的通道，其允许亲水性化合物如营养素和抗生素通过细胞包膜的外层。因此，孔蛋白在细胞生理学中起着关键作用，作为守门人控制各种环境化合物进入细胞，在病原体的情况下，抗生素。因此，分枝杆菌孔蛋白的鉴定和表征不仅将增强我们对其生理学的了解，而且还将有助于设计更有效的治疗方法。已经从全细胞提取物中对几种分枝杆菌孔蛋白进行了物理表征，已经鉴定出了耻垢病，并对主要的孔蛋白基因MspA进行了结构表征。然而，对M.结核没有M的同源物在M.结核病基因组此外，多次尝试从M.结核或M.牛的鉴定尚未成功。我们的长期目标是研究孔蛋白在缓慢生长的分枝杆菌中的功能。本研究的近期目标是研究M.并建立各种遗传筛选方法以鉴定M.结核对分枝杆菌生理学这方面的研究可以帮助设计更有效的治疗各种分枝杆菌感染的疗法。这将是一个重要的贡献，以减少公共卫生负担的分枝杆菌疾病在美国。

DESCRIPTION (provided by applicant): Mycobacteria are a family of primarily saprophytic organisms that inhabit many ecological niches. Several of these members are highly significant human pathogens, including the causative agents of tuberculosis (M. tuberculosis, M. africanum, and M. bovis), and the opportunistic agents such as M. avium and M. intracellulare that infect primarily immunocompromised patients. A hallmark characteristic of mycobacteria is the lipid-rich cell envelope which makes these organisms highly impermeable and rendering them intrinsically resistant to many bacteriocidal compounds. To facilitate uptake of hydrophilic solutes, mycobacteria, like Gram-negative organisms, utilize porins which are water-filled channels that allow passage of hydrophilic compounds such as nutrients and antibiotics through the outer layer of the cell envelope. Thus, porins play a critical role in cell physiology, serving as gatekeepers that control access of various environmental compounds to the cell, and in the case of pathogens, antibiotics. Therefore, the identification and characterization of mycobacterial porins will not only enhance our knowledge of their physiology, but will also aid in the design of more effective therapeutics. Several mycobacterial porins have been physically characterized from whole cell extracts, the porin genes of M. smegmatis have been identified and the major porin gene, MspA, structurally characterized. However, very little is known about the porins of M. tuberculosis. No homologs to the M. smegmatis porins have been found in the M. tuberculosis genome. In addition, multiple attempts to chemically isolate sufficient amounts of porins from M. tuberculosis or M. bovis for identification have not been successful. Our long-term goal is study porin function in the slow growing mycobacteria. The immediate aims of this proposal are to study the physiology of the porins of M. smegmatis and to develop various genetic screens to identify porin genes of M. tuberculosis. Research to characterize this aspect of mycobacterial physiology could aid in the design of more effective therapeutics for the treatment of various mycobacterial infections. This would be an important contribution to reduce the public health burden of mycobacterial disease in the United States.