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Transcriptional profiling of Mycobacterium tuberculosis Mmpl3 conditional mutant

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 shiftdown.

ORGANISM(S): Mycobacterium tuberculosis

PROVIDER: GSE89830 | GEO | 2017/01/25

SECONDARY ACCESSION(S): PRJNA353497

REPOSITORIES: GEO

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Project description:Background: Mtb's cell wall comprises peptidoglycan, arabinogalactan, and mycolic acids linked to capsule proteins and polysaccharides by noncovalent bonds. Cell division requires extensive remodeling by inserting cell wall-building subunits, which require multiple enzymes to ensure precision and accuracy during the addition and conjugation of biomolecules to the cell wall. Approximately 35% of division and cell wall cluster operon genes are involved in cell wall biosynthesis, 22% in cell division, and 43% are still unstudied. Results: Rv2166c was examined in M. smegmatis, a substitute for M. tuberculosis, using three strains: CRISPR-Cas12a-knockout ∆Ms_4236, complemented ∆Ms_4236::Rv2166c, and pALACE transformed Ms_Vec were grown at 370C in 7H9 or 7H10 to evaluate phenotypic, chemical stress, and antibiotic responses in normoxia and hypoxia. We also examined the whole transcriptome to identify genes associated with MSMEG_4236 deletion of the Rv2166c homolog under normoxia and hypoxia. Deletion and overexpression of Rv2166c affect mycobacterial biofilm formation, cell elongation, colony morphology, and sliding motility in normoxia but are alleviated in hypoxia. Overexpression showed resistance to nucleic acid-target antibiotics, but sensitivity to cell wall-target drugs, yet long-term expression arrests growth. According to transcriptome investigation, the deletion of MSMEG_4236 upregulated all division and cell wall cluster operon genes, several mycolic acids and arabinogalactan biosynthesis genes and downregulated numerous promoters outside of the dcw cluster operon through binding at AAAGTG[G/T] sequence motifs. Conclusion: This study shows that the mycobacterial Rv2166c represses the division and cell wall cluster operon as a transcriptional regulator. Thus, modulating this gene's expression affects many mycobacteria phenotypes and environmental stress resistance. Several AAAGTG[G/T] motifs containing promoters have been found, demonstrating that Rv2166c regulates genes other than its operon. These data suggest that the Rv2166c can be targeted as future antituberculosis medicines.

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Transcriptional profiling of Mycobacterium tuberculosis Mmpl3 conditional mutant

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