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-ß-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.

Immunomodulatory effect of mycobacterial outer membrane vesicles coated nanoparticles.

Tuberculosis (TB) is one of the most widely prevalent infectious diseases that cause significant mortality. Bacillus Calmette-Guérin (BCG), the current TB vaccine used in clinics, shows variable efficacy and has safety concerns for immunocompromised patients. There is a need to develop new and more effective TB vaccines. Outer membrane vesicles (OMVs) are vesicles released by Mycobacteria that contain several lipids and membrane proteins and act as a good source of antigens to prime immune response. However, the use of OMVs as vaccines has been hampered by their heterogeneous size and low stability. Here we report that mycobacterial OMVs can be stabilized by coating over uniform-sized 50 nm gold nanoparticles. The OMV-coated gold nanoparticles (OMV-AuNP) show enhanced uptake and activation of macrophages and dendritic cells. Proteinase K and TLR inhibitor studies demonstrated that the enhanced activation was attributed to proteins present on OMVs and was mediated primarily by TLR2 and TLR4. Mass spectrometry analysis revealed several potential membrane proteins that were common in both free OMVs and OMV-AuNP. Such strategies may open up new avenues and the utilization of novel antigens for developing TB vaccines.

Redefining genetic essentiality in Mycobacterium tuberculosis.

Sequencing transposon mutant libraries have been pivotal in annotating essential and non-essential genes in bacteria. This is particularly very helpful in the case of Mycobacterium tuberculosis with a large part of its genome without known function. It is not known whether there are any variations in the essentiality states as a function of optimal growth in the absence of any selection pressure. We here grow a high-density mutant library of M. tuberculosis through serial cultures and monitor the temporal fluctuations in insertion frequencies across all TA dinucleotides in the genome. Genes that cause morphological and physiological heterogeneity or enable metabolic bypass were found to gradually lose insertions, while genes comprising the toxin-antitoxin systems were found to get enriched with insertions during growth in nutrient replete conditions. High levels of fluctuations were observed in genes involved in cell wall and cell processes, intermediary metabolism, and genes involved in virulence, suggesting new modes of adaptation undertaken by the mutants. We also report the essentiality status of several newly annotated genetic features.