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

  1. Patricia A. Championa,4
  1. aDepartment of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556;
  2. bDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556;
  3. cDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824
  1. Edited by Lalita Ramakrishnan, University of Cambridge, Cambridge, United Kingdom, and approved November 2, 2017 (received for review June 6, 2017)

Significance

Mycobacteria use ESX systems to transport protein substrates across the cytoplasmic membrane. The ESX-1 system is required for mycobacterial pathogenesis in Mycobacterium tuberculosis (M. tb), the cause of tuberculosis (TB). Differences in the expression of genes encoding ESX substrates directly impacts M. tb transmission and virulence. Deletion of genes encoding ESX exporters results in reduced levels of ESX substrates in mycobacteria. Here, we define a fundamental mechanism of regulation of ESX-1 substrates in M. marinum, a pathogenic mycobacterial species and a model for M. tb. We demonstrate that the transcriptional regulation of genes encoding ESX-1 substrates is linked to the presence or absence of the ESX-1 exporter. These findings provide insight into how substrate levels are intricately controlled in mycobacteria.

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 Mycobacterium marinum. 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 whiB6 gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in M. marinum strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb1, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the whiB6 gene. Finally, we found that the loss of WhiB6 resulted in a virulent M. marinum strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in M. marinum are fine-tuned by negative feedback control, linking the expression of the whiB6 gene to the presence, not the functionality, of the ESX-1 membrane complex.

Footnotes

  • ?1R.E.B. and T.T.N. contributed equally to this work.

  • ?2Present address: Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, TX 77030-1503.

  • ?3Present address: Eli Lilly and Company, Indianapolis, IN 46285.

  • ?4To whom correspondence should be addressed. Email: pchampio{at}nd.edu.
  • Author contributions: R.E.B., T.T.N., M.M.C., R.B.A., and P.A.C. designed research; R.E.B., T.T.N., K.G.S., A.E.C., M.J.F., C.R.T., M.M.C., and R.B.A. performed research; M.J.F. and M.M.C. contributed new reagents/analytic tools; R.E.B., T.T.N., K.G.S., A.E.C., M.M.C., R.B.A., and P.A.C. analyzed data; and R.E.B., T.T.N., and P.A.C. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, http://www.danielhellerman.com/geo (accession no. GSE99632).

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1710167114/-/DCSupplemental.

Published under the PNAS license.

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