A Systems Biology Approach to Identify Adjunctive Drug Targets in Two Major Mycobacterial Pathogens

This thesis explores novel drug targets to accelerate therapy for infections caused by the important human pathogens Mycobacterium avium (Mav) and Mycobacterium tuberculosis (Mtb). Infections with these bacterial species are notoriously difficult to treat - requiring months to years of intensive antibiotic therapy. Decreasing this length may help reduce expenditures necessary for monitoring therapy, improve patient outcomes, and reduce the significant morbidity and mortality caused by Mav and especially the global pathogen Mtb. Bacterial antibiotic persistence has been defined as the ability of bacteria to survive in high concentrations of antibiotics without genetic mutation (ie antibiotic resistance). Thus, infections caused by Mav and Mtb are highly persistent. A major focus of this work is the discovery of mechanisms underlying this persistence phenomenon in hopes that this knowledge can be exploited to improve available therapies. A major portion of the work is carried out using high-throughput genomic screens involving techniques such as transposon mutagenesis and transposon sequencing (Tn-seq). Statistical methods are developed and implemented to analyze this dataset with a focus on non-parametric methods. Novel discoveries include identification of the essential genes of Mav as well as particular genes that assist in bacterial survival during antibiotic exposure. Mechanisms underlying antibiotic persistence are discussed and explored in follow-up experiments guided by the high-throughput data. The mechanisms of action of antibiotics beyond well-established drug-target binding are also discussed. The results presented are relevant to the understanding of antibiotic persistence and may be informative to efforts to develop new drugs for these difficult-to-treat pathogens

Alkaline residues and the environment: A review of impacts, management practices and opportunities

Around two billion tonnes of alkaline residues are produced globally each year by industries such as steel production, alumina refining and coal-fired power generation, with a total production estimate of 90 billion tonnes since industrialization. These wastes are frequently stored in waste piles or landfills, and can be an environmental hazard if allowed to generate dust, or if rainwater infiltrates the waste. This review will focus on the environmental impacts associated with alkaline residues, with emphasis on the leachates produced by rainwater ingress. Many alkaline industrial wastes can produce leachates that are enriched with trace metals that form oxyanions (e.g. As, Cr, Mo, Se, V), which can be very mobile in alkaline water. The management options for the residues and their leachates are also discussed, distinguishing active and passive treatment options. Potential reuses of these materials, in construction materials, as agricultural amendments, and in environmental applications are identified. The mechanisms of carbon sequestration by alkaline residues are assessed, and the potential for enhancing its rate as a climate change off-setting measure for the industry is evaluated. The potential for recovery of metals critical to e-technologies, such as vanadium, cobalt, lithium and rare earths, from alkaline residues is considered. Finally research needs are identified, including the need to better understand the biogeochemistry of highly alkaline systems in order to develop predictable passive remediation and metal recovery technologies

Gene Enrichment Analysis Reveals Major Regulators of Mycobacterium tuberculosis Gene Expression in Two Models of Antibiotic Tolerance

The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure