Tuberculosis remains a major global health challenge and the increasing strength and prevalence of drug resistance threaten to undo much of the good progress made. As one of the primary, frontline anti-tuberculous drugs, growing resistance to rifampicin in particular is concerning. Sub-lethal drug exposure and the development of adaptive phenotypic drug resistance, represent an important avenue by which genetic resistance and treatment failure or relapse may occur. Proteins and general metabolites are molecular classes that are highly dynamic, responsive and essential to understanding the state of an organism, while mass spectrometry-based proteomics and metabolomics are powerful tools by which these can be examined. For mycobacteria in particular, the lipidome and cell wall are compartments of major importance with respect to virulence, adaptation, host-pathogen interactions and persistence. As such, we sought to determine the effect of sub-lethal rifampicin exposure upon the model organism Mycobacterium smegmatis over time and determine what phenotypic adaptations might be observed and explained by alterations in the proteome and lipidome, with special focus on the cell wall sub-proteome. From these data we formed several new hypotheses with respect to virulence and mechanisms of both drug resistance and sensing, which were investigated further. Finally, we examined the effect of sub-lethal rifampicin exposure, and consequent proteomic alterations, upon the M. smegmatis lipidome and propose a model by which mycobacteria respond to sub-lethal challenge with rifampicin: Upon initial insult, drug-susceptible mycobacterial growth slows and stress response networks, including the SOS response, are temporarily activated. For both susceptible and resistant bacteria, cell wall remodelling begins early through dysregulation of cell wall and lipid synthesis enzymes — such as MtrAB, Mur proteins and PimB — resulting in ultimate accumulation of lipids with composition such as to impede passive diffusion of rifampicin into the cell. Some of this lipid accumulation, namely with PIMs, may take place rapidly and so ultimately reveal extremely large increases in abundance, which possibly necessitates downregulation of enzymes such as PimB by ~4 hours post treatment. In concert with ongoing lipid dysregulation, the cell wall proteome is altered as ABC transporter proteins are generally downregulated as an additional mechanism by which to control cell wall permeability through altered cell wall composition — through removal of cell wall penetrating transport proteins — and by limiting controlled influx of exogenous compounds. Bacterial efforts to resume normal growth and adapt to rifamipicin stress involves the dysregulation of numerous virulence factors, such as PknG, which results in impaired virulence. Transcriptional and translational machinery are also gradually upregulated so as to compensate for intracellular rifampicin's inhibition of RpoB, with transcriptional activity regulated separately to that of translational machinery. Ultimately, the combination of increased transcription, translation, and cell wall impermeability allows mycobacteria to overcome rifampicin challenge and resume normal growth. In M. smegmatis specifically, all this is accompanied by the gradual upregulation of the chromosomal resistance factor Arr which, at a later timepoint, modifies extracellular rifampicin to alleviate drug pressure.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/35546 |
| Date | 20 January 2022 |
| Creators | Giddey, Alexander D |
| Contributors | Blackburn, Jonathan |
| Publisher | Faculty of Health Sciences, Division of Chemical and Systems Biology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, Doctoral, PhD |
| Format | application/pdf |
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