title: Mycobacterial Esx-3 requires multiple components for iron acquisition.
abstract: ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.
authors.forename: M Sloan
authors.initials: MS
authors.lastname: Siegrist
authors.forename: Magnus
authors.initials: M
authors.lastname: Steigedal
authors.forename: Rushdy
authors.initials: R
authors.lastname: Ahmad
authors.forename: Alka
authors.initials: A
authors.lastname: Mehra
authors.forename: Marte S
authors.initials: MS
authors.lastname: Dragset
authors.forename: Brian M
authors.initials: BM
authors.lastname: Schuster
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Philips
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
mesh_terms: D058947:Bacterial Secretion Systems / Q000235:genetics; D023061:Gene Order; D056426:Genetic Loci; D007501:Iron / Q000378:metabolism*; D009161:Mycobacterium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010080:Oxazoles / Q000378:metabolism
chemical_list: D058947:Bacterial Secretion Systems; D010080:Oxazoles; C018608:mycobactins; D007501:Iron