Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

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...

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Autores principales:	M. Sloan Siegrist, Magnus Steigedal, Rushdy Ahmad, Alka Mehra, Marte S. Dragset, Brian M. Schuster, Jennifer A. Philips, Steven A. Carr, Eric J. Rubin
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d46b5f6c7ca24dd09d0a99f0dd19d43b
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spelling	oai:doaj.org-article:d46b5f6c7ca24dd09d0a99f0dd19d43b2021-11-15T15:47:38ZMycobacterial Esx-3 Requires Multiple Components for Iron Acquisition10.1128/mBio.01073-142150-7511https://doaj.org/article/d46b5f6c7ca24dd09d0a99f0dd19d43b2014-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01073-14https://doaj.org/toc/2150-7511ABSTRACT 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.M. Sloan SiegristMagnus SteigedalRushdy AhmadAlka MehraMarte S. DragsetBrian M. SchusterJennifer A. PhilipsSteven A. CarrEric J. RubinAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 3 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 M. Sloan Siegrist Magnus Steigedal Rushdy Ahmad Alka Mehra Marte S. Dragset Brian M. Schuster Jennifer A. Philips Steven A. Carr Eric J. Rubin Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition
description	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.
format	article
author	M. Sloan Siegrist Magnus Steigedal Rushdy Ahmad Alka Mehra Marte S. Dragset Brian M. Schuster Jennifer A. Philips Steven A. Carr Eric J. Rubin
author_facet	M. Sloan Siegrist Magnus Steigedal Rushdy Ahmad Alka Mehra Marte S. Dragset Brian M. Schuster Jennifer A. Philips Steven A. Carr Eric J. Rubin
author_sort	M. Sloan Siegrist
title	Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition
title_short	Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition
title_full	Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition
title_fullStr	Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition
title_full_unstemmed	Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition
title_sort	mycobacterial esx-3 requires multiple components for iron acquisition
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/d46b5f6c7ca24dd09d0a99f0dd19d43b
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Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

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