HC2091 kills Mycobacterium tuberculosis by targeting the MmpL3 mycolic acid transporter
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ABSTRACT:
PROVIDER: PRJNA420343 | ENA |
REPOSITORIES: ENA
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Project description:Mechanisms governing Mycobacterium tuberculosis acid-fastness and its capacity to induce long-term infections remain unknown. Serine/Threonine phosphorylation represents an emerging theme allowing mycobacteria to adapt their cell envelope structure/composition in response to environmental changes. We addressed whether phosphorylation of KasB, a mycolic acid biosynthetic enzyme, modulates M. tuberculosis pathogenicity. Phosphorylation of KasB occurred at Thr334 and Thr336 in vitro and in mycobacteria. A mutant strain bearing an kasB_T334D/T336D allele, mimicking constitutive KasB phosphorylation, was generated by specialized linkage transduction. This resulted in shortened mycolic acids and the lack of trans-cyclopropanation. Structural/modeling analyses revealed Thr334 and Thr336 in the vicinity of the catalytic triad, implying that phosphorylation of these residues impaired KasB activity. Importantly, the phosphomimetic strain lost acid-fast staining and was more attenuated than a kasB deletion mutant in immunocompetent and immunodeficient mice. The absence of lung pathology and mortality infers this mutant to represent a valuable vaccine candidate. This work emphasizes the critical role of Ser/Thr kinase-dependent signaling in controlling mycolic acid elongation, acid-fastness, virulence and has important clinical implications for diagnosis of latent infections. Transcriptome of kasB null, phosphoablative, and phosphomimetic mutants compared to parental. Triplicate 10ml cultures of M. tuberculosis CDC1551 and kasB null, phosphoablative, and phosphomimetic mutants were grown to OD 1.0 and harvested for transcriptional profiling.
2014-04-01 | E-GEOD-47640 | biostudies-arrayexpress
Project description:The spread of antibiotic resistance is a major challenge for treatment of Mycobacterium tuberculosis infection. In addition, efficacy of drugs is often limited by the restricted permeability of the mycomembrane. Frontline antibiotics inhibit mycomembrane biosynthesis leading to rapid cell death. Inspired by this mechanism we exploit β-lactones as putative mycolic acid mimics to block serine hydrolases involved in their biosynthesis. Among a collection of β-lactones we found one hit with potent anti-mycobacterial and bactericidal activity. Chemical proteomics using an alkynylated probe identified Pks13 and Ag85 serine hydrolases as major targets. Validation via enzyme assays and customized 13C metabolite profiling showed that both targets are functionally impaired by the β-lactone. Co-administration with front-line antibiotics enhanced the potency against M. tuberculosis by more than 100-fold demonstrating a therapeutic potential of targeting mycomembrane biosynthesis serine hydrolases.
2022-01-19 | PXD007699 | Pride
Project description:Mycobacterium tuberculosis has a complex cell envelope that is remodelled throughout infection to respond and survive the hostile and variable intracellular conditions within the host. Despite the importance of cell wall homeostasis in pathogenicity, little is known about the environmental signals and regulatory networks controlling cell wall biogenesis in mycobacteria. The mycolic acid desaturase regulator (MadR) is a transcriptional repressor responsible for regulation of the essential aerobic desaturases desA1 and desA2 that are differentially regulated throughout infection along with mycolate modification genes and thus, likely involved in mycolic acid remodelling. Here we generated a madR null mutant in M. smegmatis that exhibited traits of an impaired cell wall with increased permeability, susceptibility to rifampicin and cell surface disruption as a consequence of desA1/desA2 dysregulation. Analysis of mycolic acids revealed the presence of a highly desaturated mycolate in the null mutant that exists in relative trace amounts in the wildtype, but increases in abundance upon cell surface disruption as a result of relieved repression on the desA1/desA2 promoters. Transcriptomic profiling confirmed MadR as a cell surface disruption responsive regulator of desA1/desA2 and further implicating it in the control of bespoke β-oxidation pathways and transport evolutionarily diversified subnetworks associated with virulence. In vitro characterisation of MadR using electromobility shift assays and analysis of binding affinities is suggestive of a unique acyl-CoA pool sensing mechanism, whereby MadR is able to bind a range of acyl-CoA but MadR repression of desA1/desA2 promoters is only relieved upon binding of saturated acyl-CoA of chain length C16-C24. We propose this acyl effector ligand mechanism as distinct to other regulators of mycolic acid biosynthesis or fatty acid desaturases and places MadR as the key regulatory checkpoint that coordinates mycolic acid remodelling in response to host derived cell surface perturbation
2022-01-01 | GSE171759 | GEO
Project description:Mechanisms governing Mycobacterium tuberculosis acid-fastness and its capacity to induce long-term infections remain unknown. Serine/Threonine phosphorylation represents an emerging theme allowing mycobacteria to adapt their cell envelope structure/composition in response to environmental changes. We addressed whether phosphorylation of KasB, a mycolic acid biosynthetic enzyme, modulates M. tuberculosis pathogenicity. Phosphorylation of KasB occurred at Thr334 and Thr336 in vitro and in mycobacteria. A mutant strain bearing an kasB_T334D/T336D allele, mimicking constitutive KasB phosphorylation, was generated by specialized linkage transduction. This resulted in shortened mycolic acids and the lack of trans-cyclopropanation. Structural/modeling analyses revealed Thr334 and Thr336 in the vicinity of the catalytic triad, implying that phosphorylation of these residues impaired KasB activity. Importantly, the phosphomimetic strain lost acid-fast staining and was more attenuated than a kasB deletion mutant in immunocompetent and immunodeficient mice. The absence of lung pathology and mortality infers this mutant to represent a valuable vaccine candidate. This work emphasizes the critical role of Ser/Thr kinase-dependent signaling in controlling mycolic acid elongation, acid-fastness, virulence and has important clinical implications for diagnosis of latent infections.
2014-04-01 | GSE47640 | GEO
Project description:The mycobacterial cell wall is a distinctive thick layer that protects the tubercle bacillus from general antibiotics and the host’s immune system. Mycolic acids, which are long-chain α-alkyl-β-hydroxy fatty acids, are the major constituents of this protective layer, and their synthesis has been shown to be critical for the survival of M. tuberculosis. This model captures the mycolic acid pathway in M. tuberculosis with 197 metabolites participating in 219 reactions catalysed by 28 proteins. The model helps in the rational identification of potential anti-tubercular drug targets.
2023-11-24 | BIOMD0000001046 | BioModels
Project description:Glycolipids and lipids are prominent components of bacterial cell wall that play critical roles not only in maintaining the biofilm formation but also in resistance to environmental stress. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are the key glycolipid components in the cell wall of Mycobacterium tuberculosis. However, whether or not PatA regulates the lipids synthesis and affects drugs resistance and biofilm formation in mycobacterium is unclear, and the mechanisms involved remain to be explored. Here we show that PatA can directly regulate the synthesis of glycolipids and lipids to maintain the drugs resistance and biofilm formation in Mycobacterium smegmatis. Interestingly, the INH resistance of patA-deleted mutant is significantly enhanced although its biofilm formation is reduced. This is due to PatA negative regulation the synthesis of mycolic acids through a novel mycolic acid synthesis pathway other than the FAS pathway, which could efficiently counteract the inhibition of isoniazid on mycolic acids synthesis in mycobacterium. Furthermore, PatA is highly conserved including amino acid sequences and physiological functions in mycobacterium. Therefore, PatA regulates the synthesis of glycolipids and lipids to affect drugs resistance and biofilm formation in mycobacterium. Our findings provide a novel mycolic acids synthetic pathway and novel insights into the molecular mechanism of glycolipids and lipids metabolism regulation and their correlation with bacterial physiological phenotypes.
2023-05-22 | MTBLS6758 | MetaboLights
Project description:Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer
| EGAS00001006334 | EGA
Project description: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.
2017-01-25 | GSE89830 | GEO
Project description:A functional polymorphism responsible for TOLLIP deficiency is associated with increased tuberculosis risk. However, TOLLIP’s role in TB pathogenesis is unknown. Tollip-/- mice developed severe Mycobacterium tuberculosis (Mtb) disease, characterized by macrophage and T cell inflammation from multiple cell populations alongside foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) selectively accumulated lipid and underwent necrosis in an autonomous manner. Transcriptional and protein analysis analysis of Mtb-infected, Tollip-/- AM demonstrated increased EIF2 signaling, a key regulator of the integrated stress response (ISR) to variable environmental conditions. The ISR, inflammation, and lipid accumulation were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM led to ISR activation and Mtb replication. Correspondingly, ISRIB, a small-molecule ISR inhibitor, selectively reduced Mtb intracellular carriage in AM and improved Mtb control, overcoming the inflammatory phenotypeinflammation. Targeting the ISR broadly improves Mtb control and immunopathology, offering a promising target for host-directed tuberculosis therapy.
2023-09-27 | GSE243818 | GEO
Project description:Transcriptional profile of Mycobacterium tuberculosis in in vitro acid-nitrosative multistress, comparing untreated control cells and bacteria under multi-stress.
2013-11-05 | GSE34623 | GEO