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.