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

Williams, Emily A.; Medie, Felix Mba; Bosserman, Rachel E.; Johnson, Benjamin K.; Reyna, Cristal; Ferrell, Micah J.; Champion, Matthew M.; Abramovitch, Robert B.; Champion, Patricia A.

doi:10.1128/iai.00653-16

Cited by 23 publications

(49 citation statements)

References 81 publications

(144 reference statements)

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“…They found no differences between the wild-type and espA mutant strains in Nile red or ethidium bromide (EtBr) uptake or in sensitivity to SDS, which are common measures of permeability (134). We observed no differences in EtBr uptake between ESX-1-deficient and wild-type M. marinum strains (135). Differences in strain background could account for the divergent conclusions.…”

Section: Esx Systems and Envelope Permeabilitycontrasting

confidence: 43%

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

Bosserman

Champion

2017

J Bacteriol

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Mycobacterial 6-kDa early secreted antigenic target (ESAT-6) system (ESX) exporters transport proteins across the cytoplasmic membrane. Many proteins transported by ESX systems are then translocated across the mycobacterial cell envelope and secreted from the cell. Although the mechanism underlying protein transport across the mycolate outer membrane remains elusive, the ESX systems are closely connected with and localize to the cell envelope. Links between ESX-associated proteins, cell wall synthesis, and the maintenance of cell envelope integrity have been reported. Genes encoding the ESX systems and those required for biosynthesis of the mycobacterial envelope are coregulated. Here, we review the interplay between ESX systems and the mycobacterial cell envelope.

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Section: Esx Systems and Envelope Permeabilitycontrasting

confidence: 43%

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

Bosserman

Champion

2017

J Bacteriol

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“…Indeed, our previous findings suggest that NTA may promote the transport of EsxA, an ESX-1 substrate, across the mycobacterial cell envelope. 61 …”

Section: Resultsmentioning

confidence: 99%

“…61 M. tb Erdman (Gift of Jeffery S. Cox) was maintained in 7H9 with the addition of 10% oleic albumin dextrose catalase (OADC, BD Biosciences), 0.5% Tween-80, and 0.5% glycerol and grown at 37 °C as previously described. 35 …”

Section: Methodsmentioning

confidence: 99%

“…35,61 In brief, M. marinum and M. tb were grown under conditions permissive for ESX-1-mediated protein secretion. Following growth, bacterial cells were harvested by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…Desalted digests of enriched and depleted fractions were analyzed by bottom-up LC−MS/MS essentially, as described in Williams et al 61 In brief, peptides corresponding to ~1 μ g of digest were loaded onto C 18 BEH column 100 μ m × 100 mm (Waters, Billerica, MA) and separated over a 90−120 min gradient from 5 to 35% aqueous (A) to organic (B) phase (A = H 2 O + 0.1% formic acid; B = acetonitrile + 0.1% formic acid) running at 800 nL/min. The column outlet was directly connected to an electrospray emitter, and MS and MS/MS data were acquired on an LTQ Velos Orbitrap FTMS instrument (Thermo, San Jose, CA) (three technical replicates, first biological replicate) or Q-Exactive HF FTMS instrument (three technical replicates, second biological replicate).…”

Section: Methodsmentioning

confidence: 99%

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Quantitative N-Terminal Footprinting of Pathogenic Mycobacteria Reveals Differential Protein Acetylation

Thompson

Champion

2018

J. Proteome Res.

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N-terminal acetylation (NTA) is a post-transcriptional modification of proteins that is conserved from bacteria to humans. In bacteria, the enzymes that mediate protein NTA also promote antimicrobial resistance. In pathogenic mycobacteria, which cause human tuberculosis and other chronic infections, NTA has been linked to pathogenesis and stress response, yet the fundamental biology underlying NTA of mycobacterial proteins remains unclear. We enriched, defined, and quantified the NT-acetylated populations of both cell-associated and secreted proteins from both the human pathogen, Mycobacterium tuberculosis, and the nontuberculous opportunistic pathogen, Mycobacterium marinum. We used a parallel N-terminal enrichment strategy from proteolytic digests coupled to charge-based selection and stable isotope ratio mass spectrometry. We show that NTA of the mycobacterial proteome is abundant, diverse, and primarily on Thr residues, which is unique compared with other bacteria. We isolated both the acetylated and unacetylated forms of 256 proteins, indicating that NTA of mycobacterial proteins is homeostatic. We identified 16 mycobacterial proteins with differential levels of NTA on the cytoplasmic and secreted forms, linking protein modification and localization. Our findings reveal novel biology underlying the NTA of mycobacterial proteins, which may provide a basis to understand NTA in mycobacterial physiology, pathogenesis, and antimicrobial resistance.

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Measuring Efflux and Permeability in Mycobacteria

Rodrigues

Aínsa

Viveiros

2021

Methods in Molecular Biology

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Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps, must be determined. In this chapter, we will describe a method that allows: i) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (ii) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.

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

Cited by 23 publications

References 81 publications

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

Quantitative N-Terminal Footprinting of Pathogenic Mycobacteria Reveals Differential Protein Acetylation

Measuring Efflux and Permeability in Mycobacteria

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