Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.

Systems biology approaches have allowed for comprehensive understanding of complicated biological processes. Here, we’ve developed a global phenotypic profiling method by improving upon transposon mutagenesis methods for identifying genes required for bacterial growth in various conditions. By using the massively parallel power of Illumina sequencing, we precisely redefined the genes required for the growth of Mycobacterium Tuberculosis (Mtb) in vitro. This adapted technique provided more informative data with both increased dynamic range and resolution. As a result, we quantitatively assessed the fitness of individual mutants, as well as identified sub-genic essentiality. Mtb is well adapted to its nutrient-limiting intracellular niche. One important and novel adaptation is its ability to consume cholesterol for both energy and carbon. A combination of this genome-wide phenotypic analysis and global metabolite profiling was used to define the dedicated cholesterol catabolic pathway, as well as important transcriptional and metabolic adaptations required for the consumption of this carbon source. We identified the methylcitrate cycle (MCC) and an unexpected gluconeogenic route as essential pathways. Furthermore, we found that the cholesterol-dependent transcriptional induction of these metabolic enzymes was also essential for growth on this substrate, a function mediated by the Rv1129c regulatory protein. Using a combination of genetic and chemical methods to inhibit these pathways, we show that cholesterol represents a significant source of carbon during intracellular growth in macrophages. Finally, we have begun to define the mechanism by which lipids, such as cholesterol, are imported into the cell by investigating the assembly of the ABC-like lipid transporter, Mce1. The subunits of this system are localized to the cell wall and data is provided to support a novel mechanism for Mce-dependent import of lipids, such as cholesterol. In sum, this global analysis of host cholesterol utilization during infection provides insight into each step of this complicated process; import into the bacterial cell, the degradation of the molecule into primary metabolites, and the transformation of these metabolites into carbon and energy.

Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRb deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3b sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

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Thesis (MPH. (Epidemiology)) -- University of Limpopo, 2022 / Background: Tuberculosis (TB) is a fatal disease globally, if not managed well, with a million or more people dying by the disease annually in low and middle-income countries (LMIC). Around two billion people are thought to be asymptomatically (latently) infected with Mycobacterium tuberculosis, putting them at risk of acquiring active tuberculosis. Tests that identify immunoreactivity to mycobacterial antigens rather than live bacteria, as well as mathematical modelling, are used to estimate the prevalence of latent tuberculosis infection. According to reports, tuberculosis (TB) was the cause of 1.3 million fatalities among HIV-negative people in 2016, surpassing the global number of HIV/acquired immune deficiency syndrome (AIDS) deaths. In addition, TB was a factor in 374,000 HIV-related deaths. Despite the effectiveness of chemotherapy over the last seven decades, tuberculosis remains the world's leading infectious killer. In 2016, 10.4 million new cases were reported, a number that has remained constant since the dawn of the twenty-first century, confounding public health specialists tasked with designing and implementing measures to lessen the global burden of tuberculosis disease. As a result, the current study aims to look into the epidemiology of tuberculosis in Lesotho in order to help policymakers make decisions on TB control in the country. Methodology:. In the current investigation, a cross-sectional, retrospective descriptive study design was used, as well as a probability sampling strategy. The National TB-Database from the Ministry of Health in Lesotho was used as the source of data for this quantitative investigation, which was analyzed using STATA statistical software version 12 for Windows (STATA Corporation, College Station, Texas). A Chi-Squared test was used to compare categorical variables, while a t-test was used to examine continuous variables. A statistically significant P-value of 0.05 was used. Results: A total of 18 836 TB patient records were recovered, with 45 percent of the TB patients being females. The average age of the TB patients was 35.9 years, with a standard deviation of 12.7%, and the ages ranged from one year to 84 years. There vi was a statistically significant difference between the age groups (p value 0.001), with 33.1 percent of TB patents being in the age group 25–34 years, followed by 29 percent, 15.4 percent, 11.2 percent, and 5.5 percent in the age groups 35–44 years, 45–55 years, 15–24 years, and 55–64 years 65 years.. There has been a fluctuating treatment outcome of TB from 63.5% for cured patients in 2012 to 57.2% in 2013 and this rose to 60.4% in 2014 then eventually reached 76.7% in 2019. The TB treatment success rate in Lesotho also showed a similar trend as the cure rate. The overall TB death rates in the current study was found to be increasing on an annual basis from 7.4% in 2012 to 9.2% in 2018 then dropped to 8.5% in 2019. The TB patients who have not been evaluated for treatment outcomes have been decreasing annually from 4.4% in 2012 to 0.8% in 2019. The proportion of TB patients with known HIV status increased from 22.3% in 2015 to 90.5% in 2019 and similarly to the proportion of TB patients with HIV status positive increased from 15.1% in 2015 to 60.4% in 2019. The proportion of TB patients with HIV status positive increased with increasing age group all age groups. Conclusion: TB is still a concern in Lesotho, where treatment target goals have not yet been fulfilled, the findings of this study underline the importance of addressing the underlying socio-economic causes of TB. The most important goal in TB control is to detect 70% and cure at least 85% of sputum smear positive cases. If these goals are met, the prevalence, incidence, transmission, and medication resistance to tuberculosis (TB) could all decrease. Despite the National Tuberculosis Control Programme's attempts to enhance TB patients' access to treatment and adherence to therapy, the percentage of patients who have good treatment outcomes remains low. Despite having an 84 percent detection rate and using the DOTS technique, the available data did not identify the types of tuberculosis, therefore we were unable to forecast multidrug-resistant tuberculosis (MDR-TB).

Tuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective against MDR-TB. In order to discover compounds that target under-exploited pathways that may be essential only in vivo, the proposed screening platform will use a novel approach to drug discovery by blocking essential protein-protein interactions (PPI). In Mtb, PPI can be monitored by mycobacterial protein fragment complementation (M-PFC). This project will re-engineer the M-PFC assay to include the red fluorescent mCherry reporter for increased efficiency and sensitivity in high-throughput screening applications. To optimize the mCherry assay, we have developed fluorescent M-PFC reporter strains to monitor distinct PPI required for Mtb virulence: homodimerization of the dormancy regulator DosR. A drug screen will then identify novel compounds that inhibit this essential PPI. The screen will involve positional-scanning combinatorial synthetic libraries, which are made up of chemical compounds with varying side chains. This work will develop novel tools for TB drug discovery that could identify new treatments for the emerging world threat of MDR-TB.

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