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Title: Mycobacterium tuberculosis aconitase-IRE interactions : integrating iron, oxidative and nitrosative stress responses to regulate cell wall biosynthesis
Author: Bancroft, Peter
ISNI:       0000 0004 8504 7739
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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With over one million deaths per year caused by tuberculosis and the rise in drug-resistant infections, the need to improve our understanding of Mycobacterium tuberculosis biology has never been greater, particularly the bacterium's cell wall processes and responses to the stresses encountered during infection. Aconitase is a bi-functional protein that has been characterised in both eukaryotes and prokaryotes. Under iron-replete conditions, aconitase possesses a [4Fe-4S] cluster that enables it to act as a Krebs cycle enzyme, catalysing the interconversion of citrate and isocitrate via the intermediate cis-aconitate. Conditions such as oxidative stress, nitrosative stress and iron starvation can result in the disassembly of the iron-sulfur cluster. Apo-aconitase instead functions as an RNA-binding protein, specifically able to bind iron-responsive elements (IREs) in certain mRNA molecules. This RNA-binding activity serves as a mechanism of post-transcriptional regulation of gene expression under stressful conditions such as oxidative damage and iron starvation. An IRE-like sequence was identified in the cwlM gene of M. tuberculosis, a gene encoding an essential protein involved in peptidoglycan biosynthesis. This project confirms that the aconitase of M. tuberculosis (AcnA) is a bi-functional protein with mutually exclusive enzyme and RNA-binding activities. Dismantling of the iron-sulfur cluster and AcnA-RNA binding occur under stressful conditions, and apo-AcnA is able to bind the IRE in cwlM mRNA. The effect of this binding was studied using a mutant strain of M. smegmatis defective for IRE structure, and revealed that the IRE is important for growth and recovery of stressed mycobacteria, with the mutant strain unable to recover from a combination of oxidative and nitrosative stress. Induction of cwlM expression under stress conditions was also shown to be detrimental to growth and survival of bacteria. Mycobacteria with wild type cwlMMtb exhibited a decrease in the abundance of CwlM in response to oxidative stress and iron deprivation, while the mutant with a disrupted IRE showed no decrease in CwlM abundance. Transcription of cwlM was shown to be up-regulated under nitrosative stress, as was CwlM at the protein level. These findings indicate that regulation of cwlM mRNA translation by AcnA has the effect of reducing CwlM expression and improving the recovery of mycobacteria under stress, and that disruption of the IRE in the cwlM mRNA results in dysregulation of CwlM levels with deleterious effects for survival.
Supervisor: Greeb, Jeff Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available