Molecular genetics of mycobacteria

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Worldwide more people die from tuberculosis (TB) than any other infectious disease. The causative agent, Mycobacterium tuberculosis, can persist in apparently healthy infected people and re-initiate active disease decades later should the immune system become compromised. Failure to comply with the six months treatment required can result in the emergence of multi-drug resistant strains. The agent responsible, Mycobacterium tuberculosis, survives within the very cells of the immune system designed to eliminate invading organisms. One of the questions we are addressing is: How does M. tuberculosis adapt to and withstand the conditions it encounters there? A clearer understanding of this is important for the development of new drugs to treat TB.||Adaptation depends on the regulation of expression of particular genes in response to specific conditions. In bacteria, most such regulation is controlled by proteins which affect the synthesis of RNA. We are studying some of the regulatory proteins that act in this way. This includes altering the amount of the regulatory protein in the cell and examining the effect this has on the expression of other genes, to identify regulatory networks, as well as on the ability of the bacteria to cause disease in infection models. We are also isolating the components of the regulatory system to study their interactions in the test tube.||M. tuberculosis can withstand conditions which kill many other bacteria. One of the bacterial components that these conditions damage is DNA. As the repair of damaged DNA is crucial to survival, we are investigating the importance of various DNA repair enzymes to the virulence of M. tuberculosis. We have constructed a panel of strains in which individual DNA repair genes have been inactivated and are comparing the survival of these strains in infection models.

Technical Summary

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an intra-cellular pathogen which survives and replicates within the hostile environment of the macrophage, part of the hosts defence system. In addition, it can persist asymptomatically in the infected host, termed latent disease, in a state from which it can emerge to cause active disease at a later date following weakening of the immune system. Understanding how M. tuberculosis adapts to and withstands these conditions is important to the development of new and improved drugs to treat TB.||One of the bacterial components that are damaged following activation of the macrophage is DNA. Whilst damaged lipids and proteins can be replaced, it is essential that damaged DNA is repaired to avoid mutation and to ensure that replication can proceed. To investigate the role of DNA repair genes in M. tuberculosis pathogenesis, we have constructed a panel of strains in which individual DNA repair genes have been inactivated by allelic exchange. The phenotypes of these mutant strains are being compared with that of the wild-type following DNA damaging stresses in vitro and in models of infection. By targeting members of different DNA repair pathways, we hope to gain an insight into their relative importance. The identification of DNA repair genes affecting virulence, coupled with the definition of their in vitro susceptibility to particular kinds of DNA damaging agents, will shed light on the conditions to which M. tuberculosis is exposed during infection.||A key aspect of bacterial survival in hostile conditions is adaptation to the new environment by regulation of gene expression. We have discovered that a novel mechanism (or mechanisms) of gene regulation in response to DNA damage exists in M. tuberculosis and we wish to identify the components involved. We are focusing on transcriptional regulators which themselves are DNA-damage inducible. We are determining which genes are under the control of individual regulatory proteins using a combination of in vivo and in vitro approaches. We have constructed strains of M. tuberculosis in which the expression level of individual regulatory proteins has been modified by over-expression or mutation, and are examining the effects of these changes on the expression of other genes by DNA microarray analysis. The phenotypes of these strains following in vitro stresses and in infection models are also under investigation. In a complementary approach aimed at identifying those genes that are directly controlled, the regulatory proteins of interest are being expressed in heterologous systems, so that purified protein can be used to assess protein-DNA and/or protein-protein interactions.

Publications

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