Copper transport in Mycobacterium tuberculosis

描述(申请人提供)：结核分枝杆菌在杀菌环境如巨噬细胞的吞噬小体中生存能力的一个重要组成部分是由其不寻常的外膜(OM)建立的有效的通透性屏障。我们发现，缺少OM通道蛋白Rv1698的Mtb突变体对铜更敏感。铜是一种必不可少的微量营养素，但过量的铜是有毒的。突变株rv1698的胞内铜浓度提高了100倍。我们的实验表明，Rv1698是铜跨OM外流和维持低细胞内铜水平所必需的。由于针对浓度梯度的外排需要能量，所以大肠杆菌外排系统的OM组件连接到内膜泵，该泵为运输过程提供底物特异性和能量。然而，目前还不清楚结核分枝杆菌是如何排出废物分子、药物和其他有毒溶质的。Rv1698是分枝杆菌外排系统中的第一个OM组分，因此，这是一个很好的机会，不仅可以研究Mtb如何控制必要的、但有毒的氧化还原活性金属，如铜，而且还可以控制Mtb的总体外排过程。当用干扰素-3刺激巨噬细胞时，含结核分枝杆菌吞噬小体内的铜增加到足以抑制结核分枝杆菌生长的浓度，这表明铜的动态平衡对结核分枝杆菌的毒力至关重要。然而，对于Mtb来说，这在很大程度上是一个未开发的领域。因此，我们建议对新的铜外排通道MctB和相互作用蛋白进行表征，以确定铜稳态缺失的成分，并评估它们在结核分枝杆菌毒力中的作用。公共卫生相关性：对分枝杆菌的科学兴趣不仅是因为结核分枝杆菌在医学上的至高无上的重要性，也是因为它不同寻常的外膜，这是它在杀菌环境中生存能力的关键组成部分。我们发现，一种新的结核分枝杆菌外膜通道蛋白是铜外流所必需的。这种蛋白是表面可及的，是分枝杆菌外排系统的第一个外膜成分。

DESCRIPTION (provided by applicant): A crucial component in the ability of Mtb to survive in bactericidal environments such as the phagosome of macrophages is the efficient permeability barrier established by its unusual outer membrane (OM). We have discovered that an Mtb mutant lacking the OM channel protein Rv1698 was more susceptible to copper. Copper is an essential micronutrient, but excess copper is toxic. The intracellular copper concentration of the rv1698 mutant was 100-fold increased. Our experiments demonstrated that Rv1698 is required for copper efflux across the OM and for maintaining low intracellular copper levels. Because efflux against a concentration gradient requires energy, the OM component of efflux systems in E. coli is connected to an inner membrane pump which contributes substrate specificity and energy to the transport process. However, it is unknown how Mtb extrudes waste molecules, drugs, and other toxic solutes. Rv1698 is the first OM component of any efflux system in mycobacteria and, hence, represents a great opportunity not only to examine how Mtb controls essential, but toxic redox-active metals such as copper, but also efflux processes in Mtb in general. Copper inside Mtb-containing phagosomes is increased upon stimulation of macrophages with interferon-3 to concentrations that are sufficient to inhibit growth of Mtb indicating that copper homeostasis is critical for virulence of Mtb. However, this is a largely unexplored field for Mtb. We, therefore, propose to characterize the novel copper efflux channel MctB and interacting proteins, to identify missing components of copper homeostasis and to assess their role in virulence of Mtb. PUBLIC HEALTH RELEVANCE: Scientific interest in mycobacteria has not only been sparked by the paramount medical importance of Mycobacterium tuberculosis but also by its unusual outer membrane which is a crucial component in its ability to survive in bactericidal environments. We have discovered that a novel outer membrane channel protein of M. tuberculosis is required for efflux of copper. This protein is surface-accessible and represents the first outer membrane component of any efflux system in mycobacteria.