Proteomic Analysis of the Mycobacterium tuberculosis Outer Membrane for Potential Implications in Uptake of Small Molecules

Increased
resistance to current antimycobacterial agents and a
potential bias toward relatively hydrophobic chemical entities highlight
an urgent need to understand how current anti-TB drugs enter the tubercle
bacilli. While inner membrane proteins are well-studied, how small
molecules cross the impenetrable outer membrane remains unknown. Here,
we employed mass spectrometry-based proteomics to show that octyl-β-d-glucopyranoside selectively extracts the outer membrane proteins
of Mycobacterium tuberculosis. Differentially
expressed proteins between nutrient-replete and nutrient-depleted
conditions were enriched to identify proteins involved in nutrient
uptake. We demonstrate cell surface localization of seven new proteins
using immunofluorescence and show that overexpression of the proteins
LpqY and ProX leads to hypersensitivity toward streptomycin, while
overexpression of SubI, SpmT, and Rv2041 exhibited higher membrane
permeability, assessed through an EtBr accumulation assay. Further,
proton NMR metabolomics suggests the role of six outer membrane proteins
in glycerol uptake. This study identifies several outer membrane proteins
that are involved in the permeation of small hydrophilic molecules
and are potential targets for enhancing the uptake and efficacy of
anti-TB drugs.

当前临床常用抗分枝杆菌药物的耐药性日益严峻，且结核分枝杆菌对相对疏水的化学实体存在潜在偏好，这凸显了阐明现有抗结核药物（anti-TB drugs）如何侵入结核分枝杆菌（Mycobacterium tuberculosis）的迫切需求。尽管内膜蛋白已得到充分研究，但小分子如何穿透难以渗透的外膜仍未可知。本研究采用基于质谱的蛋白质组学（mass spectrometry-based proteomics）技术，证实辛基-β-D-吡喃葡萄糖苷（octyl-β-d-glucopyranoside）可选择性提取结核分枝杆菌的外膜蛋白。通过对营养充足与营养匮乏条件下的差异表达蛋白进行富集分析，我们成功鉴定出参与营养摄取的相关蛋白。我们借助免疫荧光法验证了7种新蛋白的细胞表面定位，并发现过表达LpqY与ProX蛋白会使菌株对链霉素产生超敏反应；而过表达SubI、SpmT及Rv2041则可提升菌株的膜通透性，该结果通过溴化乙锭（EtBr）积累实验得以验证。此外，质子核磁共振代谢组学分析表明，6种外膜蛋白参与甘油的摄取过程。本研究鉴定出多种参与亲水性小分子渗透的外膜蛋白，它们可作为提升抗结核药物摄取效率与临床疗效的潜在靶点。