Differential gene expression in mouse bone-marrow derived macrophages following infection with Mycobacterium tuberculosis pathogenic and non-pathogenic strains.
Currently, more than one-third of the world’s population is infected with Mycobacterium tuberculosis (M. tb), which has made it a major threat to global health. Treatment has been hindered not only the bacterium rapidly acquiring resistance to first and second-line drugs, but also by the spreading of these multi-drug resistant (MDR) and extensively drug resistant (XDR) strains. Due to the fact that TB drug research and therapy is largely focussed on targeting the bacteria directly, new and innovative therapeutic strategies are required. Analysis of the host response to M.tb infection may provide new targets and avenues of treatment, as drug resistance is unlikely to develop against host components. The objective of this study is to investigate differential gene expression in mouse bone-marrow derived macrophages after 12 h of infection with various pathogenic and non-pathogenic M.tb strains. RNAseq will be employed to assess these genetic changes in the host macrophage after infection. Subsequently; qPCR will be used to determine which of the top transcripts exhibit sustained up-regulation. It is from this data that we plan to knock-down specific genes through the use of siRNAs, not only to determine the genes responsible for M.tb survival and growth, but also to determine their effect on host toxicity and therefore their value as possible therapeutic targets.
Central Nitrogen Metabolism
Central nitrogen metabolism, in slow growing mycobacteria, controls the homeostasis of glutamate through the enzymatic reactions of glutamate dehydrogenase (GDH) and glutamate synthase (GltS). GDH converts glutamate to α-ketoglutarate and ammonia whilst GltS converts glutamine and α-ketoglutarate to glutamate. L-glutamate acts as a nitrogen donor to all nitrogen based compounds in the cell, furthermore the homeostasis of glutamate and glutamine has been implicated in the virulence of M. tuberculosis. Our objective is to understand the role of glutamate homeostasis in the survival of slow growing mycobacteria by studying the deregulation of glutamate in M. bovis BCG, a close relative to M. tuberculosis. Given the central role glutamate plays within the metabolism of these organisms, the deregulation thereof may elucidate metabolic vulnerabilities within BCG which may subsequently be applicable to M. tuberculosis as well. Ultimately we aim to identify novel drug targets to combat M. tuberculosis infection.
Development of novel anti-TB drugs and Characterisation of their mechanisms of action
This project aims to identify novel or new compounds that have activity against M. tuberculosis. To achieve this, are trying to set up a luminescence-based high-throughput assay system for routine antimycobacterial screening of putative compounds against H37Rv, as well as mono-drug resistant and multi-drug resistant clinical isolates. Secondly, we want to investigate the mechanism of action of any compounds identified and thus we plan on using sequence-tagged transposon mutagenesis and whole-genome in-silico subtractive hybridisation (WISH) to identify any targets of these compounds.
The role of Ergothioneine in mycobacteria
Enzymes involved in the synthesis of mycobacterial components that are essential for the physiology and/or virulence of Mtb are potential targets for the development of new drugs. Enzymes of the mycothiol and ergothioneine biosynthetic pathways are targets of choice not only because they are unique to mycobacteria but also because it protects mycobacteria against reactive oxygen species. Recent data from our lab suggest that ergothioneine is over-expressed in mutants carrying a deletion of mshA encoding the first enzyme of the mycothiol pathway. This suggests that ergothioneine acts in synergy with mycothiol and can even compensate for a lack of mycothiol. This will allow us to decipher the precise role of these two components before conceiving new drugs against the different enzymes involved in their synthesis. Thus far, we have knocked out a gene coding for EgtD (an enzyme involved in ergothioneine biosynthesis) from the wild type (mc2155) and the mycothiol deficient mutant (∆mshA) strains. We discovered that EgtD is imperative for biosynthesis since the mutants were found to be deficient in ergothioneine. In stress assays we found that the ergothioneine deficient single mutant (∆egtD) and mycothiol deficient single mutant (∆mshA) were slightly sensitive to oxidative stress conditions generated by cumene hydroperoxide relatively to the wild type, while the double mutant (∆mshA/egtD) was significantly affected. This suggests a synergistic anti-oxidative role of ergothioneine and mycothiol in mycobacteria. In addition to that, as opposed to previous reports on mycothiol, we could detect extracellular ergothioneine and we have proof that extracellular ergothioneine results from an excretion/secretion system. The M.tb mutants have been generated and are currently being investigated.