Maintenance of cell wall remodeling and vesicle production are connected inMycobacterium tuberculosis

Abstract

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced byMycobacterium tuberculosis(Mtb) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related genevirR(virRmut) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process inMtb. We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in thevirRmutant. Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

Extracellular vesicles in mycobacteria: new findings in biogenesis, host-pathogen interactions, and diagnostics

“…Recent studies have shown that a cell wall imbalance in Mtb leads to an increase in MEV release ( 16 , 19 ). For instance, the exposure of Mtb to sublethal concentrations of isoniazid (INH), a first-line antitubercular drug that targets mycolic acid synthesis, triggers MEV production ( 19 ).…”

“…However, no lipidomic analyses were performed in this study. In an independent study focused on the functional analysis of virR , the first gene known to have a role in vesiculogenesis in Mtb , it was shown the absence of apolar lipids in MEVs derived from WT, a mutant in virR ( virR mut ) and a virR complemented strain when analyzed by thin-layer chromatography analysis or mass spectrometry (MS), suggesting that MEVs from Mtb do originate at the cell membrane under normal growing conditions ( 16 ). Acknowledging that some of the stresses analyzed in closely related species to Mtb may be physiologically relevant ( 33 ), we still lack confirmation that MEVs can include mycomembrane lipids and that their release is triggered by dying Mtb cells.…”

“…Nevertheless, the mechanism by which virR mut , defective in a LytR single domain protein with no catalytic domain, could generate a hypervesiculation phenotype was not known. A recent study investigating the mechanistic role of VirR in Mtb vesiculogenesis indicated that this protein may indirectly control cell permeability via PG remodeling ( 16 ). In this study, a detailed ultrastructural, transcriptional, proteomic, and lipidomic analysis of virR mut revealed profound cell wall alterations at the PG level.…”

“…In this study, a detailed ultrastructural, transcriptional, proteomic, and lipidomic analysis of virR mut revealed profound cell wall alterations at the PG level. This mutant possesses an enlarged PG layer with a higher number of pores, but smaller than WT and complemented strains, as shown by cryo-electron microscopy (cryo-EM) analysis and high-resolution atomic force microscopy (AFM) of whole cells and purified PG, respectively ( 16 ). This phenotype is linked to the observed enhanced permeability of virR mut and the mutant in the homolog gene cei ( c ell e nvelope i ntegrity, Rv2700) ( 16 , 41 ), indicating that PG defects derived from the absence of VirR lead to enhanced permeability and vesiculation.…”

“…General biogenesis mechanisms indicate that bacterial EVs can be produced naturally or induced by distinct stressors, and several studies support the notion that vesiculogenesis is genetically regulated ( 5 , 12 – 14 ). Our group reported virR ( v esiculogenesis and i mmune r esponse r egulator) as the first gene implicated in the biogenesis of EVs in Mtb ( 15 , 16 ). A transposon mutant in virR overproduces MEVs, which provokes augmentation of cytokine responses in mouse and human macrophages.…”