Essentiality of mmpL3 and impact of its silencing on Mycobacterium tuberculosis gene expression

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.

Figure 1

(A) Serial dilutions of log-phase cultures of the mmpL3 conditional mutant TB416 and its parental strain TB38 strains were spotted on Middlebrook 7H10 plates with or without 500 ng/ml ATc; (B) Growth curves of standing cultures of the mmpL3 conditional mutant in the presence of different ATc concentrations. Circles: ATc 500 ng/ml; squares: ATc 100 ng/ml; triangles: no ATc. The experiment was repeated three times giving comparable results. The figure shows one representative experiment.

Figure 2. Killing of the mmpL3 conditional mutant in the presence of ATc.

The mutant was grown in roller bottles in Middlebrook 7H9 containing 500 ng/ml ATc. When the culture reached mid-log phase, bacteria were diluted in fresh media containing ATc (T0). Samples were collected at different time points and plated on Middlebrook 7H10 to determine the number of colony forming units (CFU). The line represents the optical density of the culture, while bars represent the CFU/ml. The experiment was repeated four times and comparable results were obtained. The figure shows one representative experiment.

Figure 3. Accumulation of TMMs in the MmpL3-depleted strain.

(A) Lipids were extracted from M. tuberculosis TB416, grown in the absence and presence of ATc (ATc−/+), with CHCl₃/CH₃OH (1:2–1x; 2:1–2x), then separated by TLC in the solvent CHCl₃/CH₃OH/H₂O (20:4:0.5) and detected with 10% (w/v) CuSO4 in 8% (v/v) H₃PO₄ for the complete lipid profiles. From the same cultures, delipidated cells, as well as lipid fractions were saponified with 15% TBAH. Free fatty/mycolic acids were derivatized to methylesters, analysed by TLC in n-hexane:ethyl acetate (95:5, 3x) and detected with a CuSO₄ detection system. (B) Mycolic acids bound to cell wall. (C) Fatty and mycolic acids from extractable lipids. Samples are in duplicates. H37Rv was used as a control. TDM: trehalose dimycolate; TMM: trehalose monomycolate; PE: phosphatidylethanolamine; CL: cardiolipin; FAME: fatty acid methyl ester; α, methoxy, keto: respectively alpha-, methoxy- and keto-mycolic acids (MAME, mycolic acid methyl ester).

Figure 4. Growth of the mmpL3 cKD mutant and its parental strain TB38 in THP-1-derived macrophages.

Infection was performed at an MOI of 1:20 (bacteria/macrophage). The results are expressed as CFU/well. ATc (200 ng/ml) was added to the cell culture medium, when appropriate. Two separate infections were performed in duplicate. Triangles: mmpL3 mutant plus ATc; circles: parental strain TB38 plus ATc; squares: mmpL3 mutant with no ATc. While colonies were detectable until 4 days after infection starting from day 5 no colonies were detected from cell cultures infected with the mutant strain and treated with ATc (detection limit 20 cfu/well).