Spivey, V.L.; (2011) Functional analysis of the serine-threonine protein kinase PknF and its substrate, the ABC transporter Rv1747, in Mycobacterium tuberculosis. Doctoral thesis , UCL (University College London).

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Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis exists in a number of different environmental states. It must therefore have gene regulatory systems which are specific for virulence. One major signalling method is through reversible phosphorylation of proteins, mediated by protein kinases and phosphatases. This study focuses on the function of one serine-threonine protein kinase, PknF, and its substrate, the ABC transporter Rv1747 which is necessary for growth in a virulent infection. This kinase is known to interact with both of the fork-head associated (FHA) domains of Rv1747 in a phosphorylation dependent manner. The aims of this study were to analyse the function of Rv1747 particularly in relation to its requirement for a virulent infection and to investigate how PknF is controlling Rv1747 function. pknF was shown to be co-transcribed with Rv1747 and the stimulus sensed by the kinase was investigated. Phenotypic analysis linked the function of Rv1747 to properties of the cell wall. Transcriptional microarray analysis of pknF and Rv1747 mutants showed altered expression levels of genes involved in cell wall functions. Moreover, thin layer chromatography revealed changes in lipid profiles between wild type and mutant, but these differences could not be confirmed to be due to the mutation since they were not restored by complementation. Cell wall structure, however, appeared normal by transmission electron microscopy. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues; mutation analysis indicated that these are likely to be the only residues phosphorylated. The involvement of the FHA domains in this regulation was demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. Furthermore, FHA-1 domain mutation resulted in attenuation in macrophages highlighting its critical role in Rv1747 function. Infection experiments in macrophages and in mice have been performed using the threonine mutants to determine the in vivo consequences of phosphorylation and hence construct a model of how this regulation takes place. This study has revealed that PknF positively regulates the function of Rv1747 which is required for growth in both the lungs and spleens of mice.

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