Genetic screens to identify mycobacterial porins

描述（由申请人提供）：分枝杆菌是一个主要腐生生物家族，栖息于许多生态位。其中一些成员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），以及主要感染免疫功能低下患者的机会性病原体，如鸟分枝杆菌和胞内分枝杆菌。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渗透，并使它们对许多杀菌化合物具有内在抵抗力。为了促进对亲水溶质的吸收，分枝杆菌和革兰氏阴性菌一样，利用孔蛋白，这是一种充满水的通道，允许亲水化合物（如营养物质和抗生素）通过细胞膜的外层。因此，孔蛋白在细胞生理学中起着至关重要的作用，作为看门人，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提高我们对其生理学的认识，而且将有助于设计更有效的治疗方法。利用全细胞提取物对几种分枝杆菌的孔蛋白进行了物理表征，对耻毛分枝杆菌的孔蛋白基因进行了鉴定，并对主要的孔蛋白基因MspA进行了结构表征。然而，人们对结核分枝杆菌的孔蛋白知之甚少。在结核分枝杆菌基因组中未发现耻毛分枝杆菌的同源物。此外，多次尝试从结核分枝杆菌或牛分枝杆菌中化学分离出足够数量的孔蛋白进行鉴定都没有成功。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。本研究的直接目的是研究耻垢分枝杆菌的孔蛋白生理学和开发各种基因筛选来鉴定结核分枝杆菌的孔蛋白基因。研究分枝杆菌生理学的这方面特征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，以减少分枝杆菌疾病在美国的公共卫生负担。

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.