Abstract
Tuberculosis kills an estimated 2 million people each year. The causative organism, Mycobacterium tuberculosis, has evolved the ability to survive within human macrophages and avoid the destructive action of the host immune response. Understanding the molecular mechanisms of intracellular survival will uncover new targets for effective drug and vaccine design. High-throughput screening of M. tuberculosis transposon mutants selected during macrophage infection was used to identify mutants which were unable to inhibit phagosome-lysosome fusion. One of the mutants carried an insertion in a gene, Rv2745c, encoding a putative transcriptional regulator. Targeted gene knock-out of Rv2745c and two other putative regulators (Rv3058c and Rv2548) was successfully achieved in an M. tuberculosis H37Rv background. These mutant strains were complemented by reintroduction of the wild-type gene and each strain was characterised for in vitro growth and growth in J774 macrophages. All the mutants had reduced survival following heat shock. Only ΔRv2745c showed any evidence of attenuation for intracellular growth. Microarray analysis was performed to generate transcriptional profiles of each mutant strain grown in culture compared with the wild-type H37Rv strain. Gene expression differences in the ΔRv3058c strain appeared to be associated with differences in growth phase of the mutant and wild-type strain. No direct regulon for Rv3058c could be identified from the transcriptional profile of the mutant. Thirty eight gene transcripts were significantly more abundant and none less abundant in the ΔRv2548 strain. This is consistent with Rv2548 functioning as the Ribonuclease component of a toxin-antitoxin system. Transcriptional profiling of the ΔRv2745c mutant demonstrated that 31 genes were up-regulated compared to the wild-type and 9 genes were down-regulated. Bioinformatic analysis revealed Rv2745c to be similar to the transcriptional activator ClgR from the related bacterial species Streptomyces and Corynebacterium. The putative binding site of ClgR is CGC-N5-GCG shown to be involved in cell cycle and growth regulation in Streptomyces. A bioinformatic analysis of those genes down-regulated in the ΔRv2745c mutant revealed the following genes to have ClgR-like binding motifs: acr2, Rv1043c, ptrB, clpP and clpC. Expression changes of these genes in the mutant were confirmed using quantitative real-time PCR (QRTPCR). Recombinant GST-Rv2745c (ClgR) was expressed and purified in Escherichia coli. Electrophoretic mobility shift assays demonstrated recombinant GST-Rv2745c to bind ClgR-like promoter regions of ptrB and acr2 genes. Together, this project demonstrates that M. tuberculosis controls expression of stress induced chaperones Acr2, ClpC and proteases ClpP, Rv1043c and PtrB using a transcriptional activator, Rv2745c (ClgR). These chaperones and proteases are likely to be important in regulation of the protein content of the cell and this study provides preliminary evidence that correct coordination of this function is required for the survival within the macrophage.