Studies on the stress responses of M. tuberculosis : tmRNA and α-crystallins

• Main Author: Al Hashmi, Salim M.
• Published: University of Surrey 2016
• Subjects: 616.99
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690422

Mycobacterium tuberculosis remains a major human health problem killing millions of people around the world. Therefore, the need for an in depth understanding of its pathogenicity is very important to enable rational development of new control strategies.  Not all M. tuberculosis infected individuals progress to active disease at the time of primary infection as some carry an asymptomatic persistent infection that may reactivate later in life to cause disease. Despite a great deal of M. tuberculosis research, the ability of M. tuberculosis to cause long-term persistent infection in immune-competent hosts is poorly understood. When M. tuberculosis is inside the body it faces many environmental stress conditions including hypoxia, starvation and oxidative stress that damage proteins, DNA and other molecules. Gene expression studies have identified many bacterial genes that are differentially regulated when M. tuberculosis is subjected to such environmental stresses. This thesis focusses on three of these genes, the ssrA gene, which encodes the tmRNA molecule involved in ribosome recycling and degradation of denatured proteins, and two genes encoding -crystallin molecular chaperones (acr1 and acr2). This study was designed to achieve two aims: (1) to examine the role of tmRNA in translational control and protein homeostasis in stressed mycobacteria; and (2) to understand the roles of Acr1 and Acr2 in the stress response of M. tuberculosis and to reveal the degree of redundancy between them. In this thesis it was shown that ssrA/tmRNA is essential for bacterial viability as it was not possible to delete the gene unless a second fully functional copy was introduced elsewhere in the genome. The results suggested that the protease tagging function of tmRNA is essential alongside its role in ribosome recycling. A recombinant His-tagged tmRNA was expressed in the mycobacteria in an attempt to identify if tmRNA is directly involved in the translation of stress proteins. Expression of the His-tagged tmRNA was detrimental to the cell and appeared to preclude successful tagging of tmRNA substrate polypeptides. Thus there was insufficient evidence to support the hypothesis. Ultrastructural localisation of Acr1 and Acr2 by immuno-electron microscopy and Western blotting of subcellular fractions of mycobacteria showed that Acr1 and Acr2 were localised in different parts of the cell. Assay of the phenotypes of single and double deletion mutants of Acr1 and Acr2 in different in vitro conditions failed to show any evidence that the two chaperones are functionally redundant. Indeed, experiments on intracellular infection of macrophages showed no phenotypic consequences resulted from loss of Acr1 but deletion of Acr2 resulted in an altered cytotoxic effect on the host cell.