title: A novel ESX-1 locus reveals that surface-associated ESX-1 substrates mediate virulence in Mycobacterium marinum.
abstract: EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.
authors.affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Kennedy
authors.forename: Gwendolyn C
authors.initials: GC
authors.lastname: Hooley
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.forename: Felix
authors.initials: F
authors.lastname: Mba Medie
authors.forename: Patricia A DiGiuseppe
authors.initials: PA
authors.lastname: Champion
mesh_terms: D048248:Acanthamoeba castellanii; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages; D051379:Mice; D019910:Mycobacterium marinum / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D021381:Protein Transport; D014774:Virulence; D037521:Virulence Factors
chemical_list: D001426:Bacterial Proteins; D018398:Homeodomain Proteins; D037521:Virulence Factors