Dynamin‐like proteins mediate extracellular vesicle secretion in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) secretes extracellular vesicles (EVs) containing a variety of proteins, lipoproteins, and lipoglycans. While emerging evidence suggests that EVs contribute to tuberculosis pathogenesis, the factors and molecular mechanisms involved in mycobacterial EV production have not been identified. In this study, we use a genetic approach to identify Mtb proteins that mediate vesicle release in response to iron limitation and antibiotic exposure. We uncover a critical role for the isoniazid‐induced, dynamin‐like proteins, IniA and IniC, in mycobacterial EV biogenesis. Further characterization of a Mtb iniA mutant shows that the production of EVs enables intracellular Mtb to export bacterial components into the extracellular environment to communicate with host cells and potentially modulate the immune response. The findings advance our understanding of the biogenesis and functions of mycobacterial EVs and provide an avenue for targeting vesicle production in vivo.

Mycobacterium abscessus extracellular vesicles increase mycobacterial resistance to clarithromycin in vitro

“…To further investigate the role of EVs in mycobacterial resistance to CLR treatment, we purified EVs from untreated or CLR‐treated M. abscessus broth culture using density gradient ultracentrifugation as described previously [14]. Similar to Figure 2A, NanoSight analysis shows a comparable size distribution profile for EVs from untreated or CLR‐treated M. abscessus broth culture (Figure 3C).…”

“…Finally, mycobacterial EVs were resuspended in 500 μL 1X PBS and stored at −80°C. Alternatively, mycobacterial EVs were further purified using density gradient ultracentrifugation with Optiprep solution (Sigma–Aldrich) as described previously [14]. The concentration of purified mycobacterial EVs was determined via a Pierce Micro BCA assay (Cat.…”

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

“…Consequently, we tested whether the enhanced vesiculation described in virR mut (Rath et al, 2013) could be linked to cell lysis events. To do this, we analyzed the presence of cytoplasmic IdeR, a transcription factor which is not present in EVs and is not detected in secreted proteins preparations, in culture supernatants of Mtb strains, assuming that its detection could be linked to a loss of envelope integrity (Gupta et al, 2023). We first confirmed that the virR mut strain manifested an enhanced vesiculation by measuring EVs using nanoparticle tracking analysis (NTA) (Fig S2A).…”

“…In addition, it was shown that the Pst/SenX3-RegX3 signal transduction system regulates MEVs production independent from VirR (White et al, 2018), suggesting an alternative mechanism of vesicle production. We have recently demonstrated that both iron starvation and deletion of VirR in Mtb, two hypervesiculating conditions, trigger the induction of the isoniazid-induced gene operon iniBAC (Gupta et al, 2023). We determined that both IniA and IniC are dynamin-like proteins (DLP) presumably assisting MEV release through the fusion of the cell membrane (Gupta et al, 2023;Wang et al, 2019).…”

“…We have recently demonstrated that both iron starvation and deletion of VirR in Mtb, two hypervesiculating conditions, trigger the induction of the isoniazid-induced gene operon iniBAC (Gupta et al, 2023). We determined that both IniA and IniC are dynamin-like proteins (DLP) presumably assisting MEV release through the fusion of the cell membrane (Gupta et al, 2023;Wang et al, 2019). Understanding how defects in VirR lead to hypervesiculation through DLPs is important to provide mechanistic explanation for the vesiculation process in mycobacteria.…”