The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb). Previous structural studies revealed that MarP contains a chymotrypsin fold and a disulphide bond that stabilizes the protease active site in the substrate-bound conformation. Here, we determined that MarP is located in the Mtb periplasm and showed that this localization is essential for function. Using the recombinant protease domain of MarP we identified its substrate specificity using two independent assays: Positional-Scanning Synthetic Combinatorial Library profiling and Multiplex Substrate Profiling by Mass Spectrometry. These methods revealed that MarP prefers bulky residues at P4, tryptophan or leucine at P2, arginine or hydrophobic residues at P1 and alanine or asparagine at P1 prime. Guided by these data, we designed fluorogenic peptide substrates and characterized the kinetic properties of MarP. Finally, we tested the impact of mutating MarP cysteine residues on the peptidolytic activity of recombinant MarP and its ability to complement phenotypes of Mtb ΔMarP. Taken together, our studies provide insight into the enzymatic properties of MarP, its substrate preference and the importance of its transmembrane helices and disulfide bond.
"In M. tuberculosis, the bd oxidase cannot sustain growth as the sole terminal oxidase because the aforementioned cytochrome bcc–aa 3 supercomplex is essential (Matsoso et al., 2005). However, a defect in the cytochrome c biosynthesis pathway causes increased expression of cydAB (Small et al., 2013); this suggests that, while CydAB activity does not yield enough energy to support growth, it might be sufficient to maintain a membrane potential when the haem–copper oxidase is downregulated or inhibited. Cytochrome bd oxidase has not yet been explored as a therapeutic target. "
[Show abstract][Hide abstract] ABSTRACT: The emergence and spread of drug-resistant pathogens and our inability to develop new antimicrobials to overcome resistance has inspired scientists to consider new targets for drug development. Cellular bioenergetics is an area showing promise for the development of new antimicrobials, particularly in the discovery of new anti-tuberculosis drugs where several new compounds have entered clinical trials. In this review, we have examined the bioenergetics of various bacterial pathogens, highlighting the versatility of electron donor and acceptor utilisation and the modularity of electron transport chain components in bacteria. In addition to re-examining classical concepts, we explore new literature that reveals the intricacies of pathogen energetics, for example, how Salmonella enterica and Campylobacter jejuni exploit host and microbiota to derive powerful electron donors and sinks; the strategies Mycobacterium tuberculosis and Pseudomonas aeruginosa use to persist in lung tissues; and the importance of sodium energetics and electron bifurcation in the chemiosmotic anaerobe Fusobacterium nucleatum. A combination of physiological, biochemical, and pharmacological data suggests that, in addition to the clinically-approved target F1Fo-ATP synthase, NADH dehydrogenase type II, succinate dehydrogenase, hydrogenase, cytochrome bd oxidase, and menaquinone biosynthesis pathways are particularly promising next-generation drug targets. The realisation of cellular energetics as a rich target space for the development of new antimicrobials will be dependent upon gaining increased understanding of the energetic processes utilised by pathogens in host environments and the ability to design bacterial-specific inhibitors of these processes.
Advances in Microbial Physiology 11/2014; DOI:10.1016/bs.ampbs.2014.08.001 · 3.25 Impact Factor
"The gene rv3671c, recently named marP for mycobacterial acid resistance protease , encodes a membrane-associated serine protease with the C-terminal protease domain located within the periplasm . The purified extracellular domain exhibits autoproteolytic activity and is capable of cleaving β-casein and select oligopeptides , demonstrating that it can function as a protease; however, no substrates have been identified that are known to be involved in its biologic function . While marP is important for pHIB homeostasis in Mtb, additional, marP-independent pathways of pHIB homeostasis are likely to exist. "
[Show abstract][Hide abstract] ABSTRACT: Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pHIB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pHIB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis.
PLoS ONE 08/2013; 8(7):e68942. DOI:10.1371/journal.pone.0068942 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Mycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis.
Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis.
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