Genetic screens to identify mycobacterial porins

描述(申请人提供)：分枝杆菌是一类主要生活在许多生态环境中的腐生生物。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(结核分枝杆菌、非洲分枝杆菌和牛分枝杆菌)，以及主要感染免疫受损患者的机会性病原体，如禽型分枝杆菌和细胞内分枝杆菌。分枝杆菌的一个特征是富含脂质的细胞膜，这使得这些生物高度不透气，并使它们对许多杀菌化合物具有内在的抵抗力。为了促进亲水性溶质的吸收，分枝杆菌像革兰氏阴性生物体一样，利用孔道，孔道是充满水的通道，允许营养物质和抗生素等亲水化合物通过细胞膜的外层。因此，孔蛋白在细胞生理学中扮演着关键角色，起着把关人的作用，控制着各种环境化合物进入细胞的途径，对于病原体来说，就是抗生素。因此，对分枝杆菌孔蛋白的鉴定和鉴定不仅将提高我们对其生理学的认识，还将有助于设计更有效的治疗方法。已从全细胞提取液中鉴定了几种分枝杆菌孔蛋白，鉴定了耻垢分枝杆菌的孔蛋白基因，并对主要的孔蛋白基因mspA进行了结构特征分析。然而，人们对结核分枝杆菌的致病因子知之甚少。在结核分枝杆菌基因组中还没有发现与耻垢分枝杆菌孔蛋白同源的基因。此外，从结核分枝杆菌或牛分枝杆菌中化学分离足够数量的孔蛋白以进行鉴定的多次尝试都没有成功。我们的长期目标是研究孔蛋白在生长缓慢的分枝杆菌中的作用。这项建议的直接目的是研究耻垢分枝杆菌孔蛋白的生理学，并开发各种基因筛查来识别结核分枝杆菌的孔蛋白基因。对分枝杆菌生理学这一方面的研究有助于设计更有效的治疗各种分枝杆菌感染的疗法。这将是对减轻美国分枝杆菌疾病的公共卫生负担的重要贡献。

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.