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- All Subjects: neglected disease
- Creators: Bean, Heather
Description
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection.
ContributorsMaarsingh, Jason (Author) / Haydel, Shelley E (Thesis advisor) / Roland, Kenneth (Committee member) / Sandrin, Todd (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2019
Description
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022