Pleiotropic consequences of gene knockouts in the phthiocerol dimycocerosate and phenolic glycolipid biosynthetic gene cluster of the opportunistic human pathogen<i>Mycobacterium marinum</i>

Mohandas, Poornima; Budell, William C.; Mueller, Emily L.; Au, Andrew; Bythrow, Glennon V.; Quadri, Luis E. N.

doi:10.1093/femsle/fnw016

Cited by 35 publications

(38 citation statements)

References 39 publications

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“…PDIM directly contributes to the impermeable nature of the envelope (24). The loss of PDIM in both M. tuberculosis and M. marinum results in increased envelope fluidity and permeability (24,27,42). The observed permeabilization of the suppressor strains may be driven by the reduced levels of PDIM and PGL.…”

Section: Discussionmentioning

confidence: 99%

“…These findings demonstrate that the insertion in the ppsC gene was polar on the expression of the downstream genes. The loss of PDIMs and PGLs has previously been shown to increase the permeability of the mycobacterial cell wall, including the cell walls of both M. tuberculosis and M. marinum (24,27). To test if the suppressor strains bearing the ppsC frameshift allele had altered cell wall impermeability, we conducted an ethidium bromide (EtBr) uptake assay.…”

Section: Spontaneous Mutation and Suppression In M Marinummentioning

confidence: 99%

“…To test if the suppressor strains bearing the ppsC frameshift allele had altered cell wall impermeability, we conducted an ethidium bromide (EtBr) uptake assay. Previously, this assay was used to demonstrate that deletions in genes in the PDIM/PGL biosynthetic locus in M. marinum led to a 2 to 2.5% increase in EtBr uptake due to increased permeability (27). The data from the uptake assay are shown in Fig.…”

Section: Spontaneous Mutation and Suppression In M Marinummentioning

confidence: 99%

“…PDIM and PGLs are abundant and complex cell wall-associated lipids that play roles in cell impermeability and virulence in pathogenic mycobacteria (23)(24)(25)(26)(27). The spontaneous and frequent loss of PDIM has been widely reported in the literature during routine laboratory manipulation of Mycobacterium tuberculosis, the causative agent of human tuberculosis (20)(21)(22).…”

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confidence: 99%

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A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

et al. 2017

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Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and wholegenome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

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Section: Discussionmentioning

confidence: 99%

Section: Spontaneous Mutation and Suppression In M Marinummentioning

confidence: 99%

Section: Spontaneous Mutation and Suppression In M Marinummentioning

confidence: 99%

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confidence: 99%

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A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

et al. 2017

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“…The biofilm formation assay was carried out using the MBEC™ device (Innovotech Inc.) and a protocol comparable to one we recently reported (Mohandas et al, 2016). Briefly, Mk cells from actively growing cultures were harvested by centrifugation and resuspended to an OD 600 of 0.1 in Middlebrook 7H9 lacking ADN supplementation (Chavadi, Stirrett, et al, 2011) and Tween-80.…”

Section: Biofilm Formationmentioning

confidence: 99%

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth

et al. 2020

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Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis‐like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long‐term, multidrug courses with adverse side effects. The emergence of drug‐resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next‐generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

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Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility

et al. 2019

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Edited by Peter BrzezinskiPhthiocerol dimycocerosates and phenolic glycolipids (PGL) are considered as major virulence elements of Mycobacterium tuberculosis, in particular because of their involvement in cell wall impermeability and drug resistance. The biosynthesis of these waxy lipids involves multiple enzymes, including thioesterase A (TesA). We observed that purified recombinant M. tuberculosis TesA is able to dimerize in the presence of palmitoyl-CoA and our 3D structure model of TesA with this acyl-CoA suggests hydrophobic interaction requirement for dimerization. Furthermore, we identified that methyl arachidonyl fluorophosphonate, which inhibits TesA by covalently modifying the catalytic serine, also displays a synergistic antimicrobial activity with vancomycin further warranting the development of TesA inhibitors as valuable antituberculous drug candidates.

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Pleiotropic consequences of gene knockouts in the phthiocerol dimycocerosate and phenolic glycolipid biosynthetic gene cluster of the opportunistic human pathogenMycobacterium marinum

Cited by 35 publications

References 39 publications

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility

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