WhiB6 regulation of ESX-1 gene expression is controlled by a negative feedback loop inMycobacterium marinum

Abstract

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the gene. Finally, we found that the loss of WhiB6 resulted in a virulent strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in are fine-tuned by negative feedback control, linking the expression of the gene to the presence, not the functionality, of the ESX-1 membrane complex.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112

“…The induced genes annotated as transcriptional regulators were csoR , rv1129c , rv1460 , rv2017 , rv2250c , rv3334 , sigB , whiB3 and whiB6 (Table ). These transcription factors regulate copper homoeostasis (CsoR) (Marcus et al , ), iron–sulphur cluster biogenesis (Rv1460) (Willemse et al , ), cholesterol catabolism (Rv1129c/PrpR) (Masiewicz et al , ), the enduring hypoxic response (Rv3334) (Rustad et al , ), multiple stress responses (SigB) (Lee et al , ), redox stress and complex lipid biosynthesis (WhiB3) (Mehta and Singh, ) and virulence factor expression (WhiB6) (Bosserman et al , ).…”

Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria

“…NanoUHPLC-MS/MS was performed essentially as described(Bosserman et al 2017; 462 Bosserman et al 2019). 1µg of each digest was analyzed in technical triplicate and 463 biological duplicate on an Orbitrap instrument running a TOP15 data-dependent 464 acquisition (Q-Exactive Thermo San Jose, CA).…”

WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates in pathogenic mycobacteria

“…WhiB proteins are actinobacteria-specific regulators that contain iron-sulfur clusters and are thought to act as redox-sensing transcription factors that can cause both gene activation and repression [37]. WhiB6 was suggested to be involved in the regulation of EsxA secretion [38], and subsequent studies have confirmed this suggestion [39–41]. To determine whether Whib6 had an effect on the expression levels of esx-1 associated genes, we overexpressed this protein in the ESX-1-deficient M. marinum eccCb 1 transposon mutant strain.…”