The characterization of Mycobacterium tuberculosis amidase-like proteins
Date
2024
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Abstract
Tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis, is a significant global threat to human health. The emergence of drug resistant M. tuberculosis necessitates the identification of new drug targets for the development of new, shorter regimens. The peptidoglycan (PG) core of the M. tuberculosis cell wall is a potential source of drug targets because it is unique to bacteria and plays a vital role in a multitude of cellular processes and host-mediated immune responses. PG is constantly remodelled by PG synthases and hydrolases in response to external stimuli. This research focuses on N-acetylmuramyl-Lalanine amidases (amidases), PG hydrolases that are implicated in bacterial growth, cell division, virulence and antibiotic tolerance. More specifically, this PhD aims to characterize the M. tuberculosis Ami1 (Rv3717) homologue and to highlight its potential as a drug target. Genotypic characterization of a previously generated M. tuberculosis mutant strain lacking ami1 (H37Δami1S) was conducted prior phenotypic assessments. The deletion of ami1 had no significant effect on growth rate and cell division in standard 7H9 media. In contrast, the growth rate of H37Δami1S was significantly reduced when grown in Sauton’s minimal media with (1%) or without glycerol as a carbon source. We then surmised that Ami1 possibly plays a role in intracellular survival, where host-derived carbon sources support bacterial growth. The survival of H37Δami1S was reduced in IFN-γ stimulated U937 macrophages.
H37Δami1S displayed increased susceptibility to rifampicin when assessed by broth microdilution. This observation was credited to a weakened, more permeable cell wall. Consistent with this, H37Δami1S exhibited an increased ethidium bromide uptake. Subsequently, we hypothesized that H37Δami1S may display alterations in antibiotic tolerance/persistence. In a 7-day time-course experiment, H37Δami1S displayed increased susceptibility to vancomycin, ethionamide and isoniazid as evidenced by declining bacterial survival. We interrogated the isoniazid-associated phenotype further, by assessing the transcription of all three amidase-encoding genes. Only ami1 was induced following exposure to isoniazid whereas the expression of ami3 and ami4 remained at basal levels. The regulation of the ami1 gene was explored further through bioinformatics, which revealed two putative transcriptional regulators predicted to bind upstream of ami1, namely Rv1423 and Rv1776c. Protein homology modelling detected HTH DNA binding domains in both proteins. These proteins were then cloned for recombinant expression in the pET29a+ system for purification. Rv1776c was successfully expressed and purified. Electrophoretic mobility shift assays yielded preliminary data that suggested that Rv1776c binds the promoter region of the ami1 gene. Attempts to optimize binding were unsuccessful. To further evaluate the role of Rv1776c and Rv1423 in regulating ami1 gene expression, we over-expressed the regulators, using the tetracycline operator, and assessed effect on cell wall stability, via an ethidium bromide diffusion assay. Over-expression of Rv1776c was not achieved despite increasing concentrations of anhydrotetracycline, suggesting possible downstream regulation of Rv1776c; however, over-expression of Rv1423 was achieved. An increase in ethidium bromide uptake was observed in strains over-expressing Rv1776c and Rv1423. Increasing anhydrotetracycline concentrations in both strains resulted in marginal decreases in the transcription of ami1. Overall, this study has demonstrated that Ami1 plays a vital role in how M. tuberculosis utilizes a carbon source during normal growth and survival in vitro. Moreover, the transcription of ami1 is specifically and directly responsive to isoniazid exposure, possibly via two transcriptional repressors. This work therefore supports further characterization and development of Ami1 as novel drug target in M. tuberculosis.
Description
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2023
Keywords
Tuberculosis (TB), Mycobacterium tuberculosis, Peptidoglycan