Abstract
Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology.
Highlights
From the ‡Department of Microbiology and Immunology, §Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina, 27599; ¶Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655; ʈHoward Hughes Medical Institute, Chevy Chase, Maryland, 20815
By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria
Proteomic Analysis of Differentially Abundant Proteins in the Cell Wall of H37Rv versus ⌬secA2 Mutant M. tuberculosis—Triplicate cultures of M. tuberculosis H37Rv and ⌬secA2 mutant were grown to mid-log phase, at which time cells were sterilized by gamma irradiation and lysed with a French pressure cell to generate whole cell lysates (WCL)
Summary
From the ‡Department of Microbiology and Immunology, §Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina, 27599; ¶Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655; ʈHoward Hughes Medical Institute, Chevy Chase, Maryland, 20815. In M. tuberculosis, SecA2 is required for virulence in both mice and macrophage models of infection, making the identification of SecA2-dependent exported proteins important for understanding M. tuberculosis pathogenesis [5,6,7]. The other type of system, which is the case in mycobacteria, does not include an accessory SecY and the repertoire of exported proteins is more diverse (5, 14 –17) In these SecA2-only or multisubstrate SecA2 systems, SecA2 appears to work with the SecYEG channel for protein translocation [9, 17,18,19,20]. Studies conducted in mycobacteria suggest that the proteins exported by its SecA2-only pathway have a tendency to fold in the cytoplasm prior to export, which distinguishes them from proteins that remain unfolded and are exclusively exported by SecA1. This folding feature of SecA2-exported proteins may dictate the need for SecA2 in their export [21]
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