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.

中文翻译：

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结核分枝杆菌外膜的蛋白质组学分析对小分子摄取的潜在影响

对当前抗分枝杆菌药物的耐药性增加以及对相对疏水性化学实体的潜在偏向突出表明迫切需要了解当前的抗结核药物如何进入结核杆菌。虽然内膜蛋白已得到充分研究，但小分子如何穿过不可穿透的外膜仍然未知。在这里，我们采用基于质谱的蛋白质组学来证明辛基-β- d-吡喃葡萄糖苷选择性提取结核分枝杆菌的外膜蛋白。富集营养充足和营养匮乏条件下差异表达的蛋白质，以鉴定参与营养吸收的蛋白质。我们使用免疫荧光证明了七种新蛋白质的细胞表面定位，并表明蛋白质 LpqY 和 ProX 的过度表达会导致对链霉素的超敏反应，而 SubI、SpmT 和 Rv2041 的过度表达表现出更高的膜通透性（通过 EtBr 累积测定进行评估）。此外，质子核磁共振代谢组学表明六种外膜蛋白在甘油摄取中的作用。这项研究确定了几种参与亲水性小分子渗透的外膜蛋白，它们是增强抗结核药物的吸收和功效的潜在靶标。