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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, continues to be the world’s deadliest human bacterial pathogen. Current treatments are notoriously limited, lengthy, and becoming increasingly ineffective due to drug-resistant mutant strains. WhiB7, a putative transcriptional regulator, is an essential component of intrinsic antibiotic resistance in Mtb. Unique to Actinobacteria, multiple paralogous WhiB-like proteins have diverse roles in physiology, but little is known about their mode of action or regulation. To investigate WhiB7, a combination of in vitro run-off, two-hybrid assays, protein pull-down experiments, and genetic approaches was used. WhiB7 was characterized as an auto-regulatory, redox-sensitive transcriptional activator, providing the first biochemical proof that a WhiB-like protein directly promotes transcription. WhiB7’s antibiotic resistance function was dependent on three regions: an iron-sulfur cluster binding region likely required for stability; a middle region for binding to the vegetative sigma factor SigA; and a C-terminal DNA-binding region. Mutations disrupting any one of the regions led to an inability of WhiB7 to activate resistance. These experimental constraints were combined with protein modelling techniques to visualize the WhiB7:SigA:DNA complex which may serve as a platform for the design of inhibitors. Additionally, a GFP reporter was constructed to monitor whiB7 induction, and was used to screen our custom library of almost 600 bioactive compounds including the majority of clinical antibiotics. Expression was induced by compounds having diverse structures and targets, which did not correlate with drug susceptibility of the whiB7 mutant. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced intracellular condition reflected by the reduced:oxidized mycothiol ratio. Amino acid metabolism also contributed to WhiB7-mediated intrinsic resistance. To gain insights into whether other genetic loci contribute to WhiB7-mediated antibiotic resistance, a transposon library of Mycobacterium smegmatis was screened for WhiB7-like drug susceptibility or resistance. These studies revealed a putative aspartate aminotransferase (MSMEG_4060) that contributed to whiB7 repression and a pair of adjacent hypothetical genes (MSMEG_3637/3638) that contributed to whiB7 induction. Continued characterization of WhiB7 may serve as a paradigm for other WhiB-like proteins and lead to novel and desperately needed therapies for tuberculosis.
Master's Student Supervision (2010 - 2018)
Intrinsic resistance of the intracellular pathogen Mycobacterium tuberculosis is one of the main reasonsthat the disease tuberculosis is difficult to treat and why it remains as one of the world’s most prevalentand dangerous infectious diseases. The intrinsic resistance regulator WhiB7 controls a regulon thatcontains many genes predicted to have physiological functions including aspartate aminotransferases,aspB and aspC. Multi-drug susceptibility was observed in an aspC mutant and an aspB constitutiveexpression strain. The expression of aspC was positively regulated by WhiB7 while expression of aspBdownregulated whiB7 expression. The fitness of Mycobacterium smegmatis was affected negatively byoxaloacetate and positively by α-ketoglutarate, substrates of aspartate aminotransferase, which thenaltered the growth inhibition by antibiotics. Recombinant AspB and AspC both catalyze measurabletransamination of aspartate and α-ketoglutarate. AspC plays an important role in mycobacteriaphysiology as deletion of this gene caused many growth deficiencies. Furthermore, the physiological roleof AspC extended beyond amino acid intermediary metabolism to redox homeostasis and oxidativestress detoxification. These results revealed a link between intrinsic antibiotic resistance andmetabolism mediated through AspB and AspC. Since antibiotic resistance in mycobacterium is a complexfunction of its physiology, it is important to screen for tuberculosis drugs under growth conditions thatresemble those found in vivo.
Mycobacterium tuberculosis, the causative agent of tuberculosis, is an adept pathogen partly due to its intrinsic antibiotic resistance systems which make an infection difficult to treat with antibiotics. Parts of these intrinsic antibiotic resistance systems are regulated by the product of the gene whiB7. A yeast two-hybrid experiment suggested that WhiB7 might interact with the anti-sigma factor RsbW, indirectly implying involvement with the alternate sigma factor SigF. Co-expression of WhiB7 and RsbW in Escherichia coli followed by pull-down experiments did not support this hypothesis. In order to understand mycobacterial instrinsic antibiotic resistance, a new screening method was designed and implemented to identify pairs of compounds that inhibited mycobacteria by synergistic interactions. Mycobacterium bovis BCG(lux) cells were exposed to pairwise compound combinations and assessed for viability. A computer program was created to identify synergistic combination leads from the screen. The synergistic interactions might provide us with a greater understanding of the intrinsic antibtiotic resistance systems in mycobacteria. A sample of the leads from the screen tested further confirmed that 38% of domperidone combinations and 25% of rifampicin combinations were synergistic.The data from the screen has also identified new potential anti-mycobacterial compounds, specifically the avermectins. The avermectins, as a family, have never been reported to demonstrate antibiotic activity, but they have been used to treat people and other mammals for parasitic infections. The antibiotic activity of the avermectins was investigated in this work and found to be specific to mycobacteria. The potential of this compound family as a possible future treatment for tuberculosis therapy warrants further study.
During an infection, Mycobacterium tuberculosis resides within a phagosome of a macrophage (MΦ), an environment thought to be hypoxic, nitrosative, oxidative and carbohydrate poor. Previous research evaluating which M. tuberculosis genes are important for surviving the harsh MΦ phagosome environment suggested that the fumarate reductase (FRD) enzyme might be an important factor not only for intra-MΦ survival but for M. tuberculosis virulence as well. FRD is used in anaerobic respiration (when oxygen is limiting) and helps maintain a balanced cellular redox state by using fumarate as a terminal electron acceptor. We engineered an M. tuberculosis FRD knock out that demonstrated a decrease in viability in hypoxic conditions, confirming the role of FRD in surviving hypoxia. The M. tuberculosis FRD knock out also showed a statistically significant decrease in intracellular colony forming units 4-days post MΦ infection. This suggested that FRD is also an M. tuberculosis virulence factor. As such, targeting and inhibiting FRD with a drug may be a novel means for treating tuberculosis by preventing M. tuberculosis from performing anaerobic respiration. Putative FRD inhibitors were tested for mycobactericidal activity. Three out of 7 putative FRD inhibitors tested showed mycobactericidal activity in hypoxic conditions, which supported the hypothesis. Additionally, we engineered an Mycobacterium smegmatis strain to express M. tuberculosis FRD. This changed its colony morphology and rendered it more susceptible to a variety of antimicrobials, suggesting that FRD may also affect cell envelope composition. These findings further implicate FRD as a target of interest for novel anti-mycobacterial drug options. In summary, this research gives hope to alleviate the ever growing resistance problem seen in tuberculosis infections by opening up a new route for drug development and discovery.