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.
Highlights
Tuberculosis remains a significant global health concern with estimates indicating that one-third of the world’s population is currently latently infected by the causative organism, Mycobacterium tuberculosis [1]
The genetic programs required by M. tuberculosis for establishment, maintenance, and/or reactivation from persistent infection within the host remain poorly defined, but are thought to include stress-adaptation systems such as extracytoplasmic function (ECF) sigma factors and two-component signal transduction systems. mprAB is one of 11 complete two-component system encoded within the genome of M. tuberculosis [2]
MprAB is required for in vivo growth of the tubercle bacillus during persistent stages of infection [7], and its expression is up-regulated within an artificial granuloma model system [8] and under various conditions in vitro likely to be experienced by M. tuberculosis during residence within the granuloma [4,6,9]
Summary
Tuberculosis remains a significant global health concern with estimates indicating that one-third of the world’s population is currently latently infected by the causative organism, Mycobacterium tuberculosis [1]. MprAB is one of 11 complete two-component system encoded within the genome of M. tuberculosis [2] This system directly regulates expression of numerous stress-responsive determinants in M. tuberculosis including ECF sigma factors sigE and sigB, alpha crystallin gene acr, and serine protease pepD [3,4,5,6]. PepD mutants of M. tuberculosis display a pleiotrophic phenotype; they are unaltered in survival following exposure to SDS [12], and they exhibit similar in vivo growth kinetics within tissues of infected mice compared to their wild-type counterparts [11] These mutants do display an increased time to death in mice and are associated with reduced tissue pathology [11]. These phenotypes, coupled with the observation that pepD deletion results in upregulation of numerous stressresponsive determinants in M. tuberculosis under physiological conditions including sigE [12], underscores the complex regulation and multifaceted activity of this protein
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