Functional analysis of the serine-threonine protein kinase PknF and its substrate, the ABC transporter Rv1747, in Mycobacterium tuberculosis

摘要

Mycobacterium tuberculosis, the causative agent of tuberculosis exists in a number ofuddifferent environmental states. It must therefore have gene regulatory systems which areudspecific for virulence. One major signalling method is through reversibleudphosphorylation of proteins, mediated by protein kinases and phosphatases. This studyudfocuses on the function of one serine-threonine protein kinase, PknF, and its substrate,udthe ABC transporter Rv1747 which is necessary for growth in a virulent infection. Thisudkinase is known to interact with both of the fork-head associated (FHA) domains ofudRv1747 in a phosphorylation dependent manner. The aims of this study were to analyseudthe function of Rv1747 particularly in relation to its requirement for a virulent infectionudand to investigate how PknF is controlling Rv1747 function.udpknF was shown to be co-transcribed with Rv1747 and the stimulus sensed by the kinaseudwas investigated. Phenotypic analysis linked the function of Rv1747 to properties of theudcell wall. Transcriptional microarray analysis of pknF and Rv1747 mutants showedudaltered expression levels of genes involved in cell wall functions. Moreover, thin layerudchromatography revealed changes in lipid profiles between wild type and mutant, butudthese differences could not be confirmed to be due to the mutation since they were notudrestored by complementation. Cell wall structure, however, appeared normal byudtransmission electron microscopy.udExperiments to determine how PknF regulates the function of Rv1747 demonstrated thatudphosphorylation occurs on two specific threonine residues; mutation analysis indicatedudthat these are likely to be the only residues phosphorylated. The involvement of the FHAuddomains in this regulation was demonstrated by isothermal titration calorimetry, usingudpeptides containing both phosphothreonine residues. Furthermore, FHA-1 domainudmutation resulted in attenuation in macrophages highlighting its critical role in Rv1747udfunction. Infection experiments in macrophages and in mice have been performed usingudthe threonine mutants to determine the in vivo consequences of phosphorylation andudhence construct a model of how this regulation takes place. This study has revealed thatudPknF positively regulates the function of Rv1747 which is required for growth in bothudthe lungs and spleens of mice.