PMID:21445360

Citation	White, MJ, Savaryn, JP, Bretl, DJ, He, H, Penoske, RM, Terhune, SS and Zahrt, TC (2011) The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein. PLoS ONE 6:e18175
Abstract	Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.
Links	PubMed PMC3062566 Online version:10.1371/journal.pone.0018175
Keywords	Antigens, Bacterial/immunology; Bacterial Outer Membrane Proteins/metabolism; Chromatography, Liquid; Epitopes/immunology; Immunoprecipitation; Mycobacterium tuberculosis/enzymology; Mycobacterium tuberculosis/immunology; Serine Proteases/metabolism; Substrate Specificity; Tandem Mass Spectrometry; Two-Hybrid System Techniques
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MYCTU:O53896		GO:0006508: proteolysis	ECO:0000314:		P		Proteins that interact with PepD were selected by co-immunoprecipitation carried out with with M. smegmatis ΔpepD (Figure 2C). These were then identified using LC MS/MS, and co-immunoprecipitation and LC MS/MS were repeated with M. tuberculosis Δpep, indicating several proteins which interact with PepD (Tables S3-7).The 35-kDa antigen was the top scoring protein for probable interaction with PepD(Table 1). Specifically with the 35-kDa antigenFigure 3D shows the presence of a lower molecular weight protein that is immunoreactive with the anti-FLAG monoclonal antibody in the wild-type M. tuberculosis pepD strain. This indicates that PepD interacts with/ cleaves the 35-kDa antigen	complete
MYCTU:MPRB		GO:0005886: two-component signal transduction system (phosphorelay)	ECO:0000314:		P		Figure 1 Schematic depicting the differential centrifugation protocol used to fractionate PepD in M. tuberculosis.	complete

References

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