Abstract
MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate ≤1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic downshift.
Highlights
Conditional mutant confirming the essentiality of MmpL3 for growth in axenic media and its role in TMM export, and demonstrate its essentiality for intracellular growth
After constructing an M. tuberculosis mmpL3 conditional knockdown (cKD) mutant, we confirmed that MmpL3 is essential for growth in axenic culture and for transport of TMM17
We proved its essentiality for growth and survival of M. tuberculosis during human macrophage infection and showed that MmpL3 depletion leads to a complex change in the global transcriptional profile
Summary
Generation and characterization in vitro of mmpL3 conditional mutant in M. tuberculosis. Rv0516c was recently renamed osmosensory protein A (OprA) for its involvement in an osmosensory signaling pathway including PknD and SigF, which regulates peptidoglycan architecture[35], suggesting that cells depleted in MmpL3 might experience osmotic stress, probably due to a change in surface permeability as a result of a different lipid composition Consistent with this hypothesis, argC, the first gene of the arginine biosynthetic operon was found to be induced 3.9-fold: L-arginine is considered to be an osmoprotectant and induction of its biosynthetic operon was reported under conditions of osmotic stress[35]. Several other repressed genes were involved in energy production such as sdaA, involved in gluconeogenesis from serine[43], atpB, encoding a component of ATP synthase, nuoB, D and H encoding different subunits of NADH dehydrogenase, or the entire mce[1] operon, hypothesized to encode a lipid re-uptake system that enables mycolic acid recycling[44], suggesting a decrease in the metabolic activity of the cells
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