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Global regulation in Mycobacterium: Role of Lsr2

Final Report Summary - MYCO-REG (Global regulation in Mycobacterium: Role of Lsr2)

Project context and objectives

The aim of MYCOREG project was to investigate the global regulation of Lsr2 in mycobacteria. We had earlier reported that Lsr2 negatively regulates Glycopeptidolipid (GPL). GPLs are a small lipid molecule present on the outer surface of different mycobacterial species. Our first approach was transcriptome profiling of Lsr2 mutant along with wild type and complemented strains to understand its role. We performed the microarray experiments in collaboration with the 'Microarray platform' of the Pasteur Institute, Paris, headed by Dr. J.-Y. Coppee. During the course of action, we faced some serious background trouble of PFGRC microarrays chip, as the results obtained were not similar in each set of experiments. We tried all the possible options (including consultation with PFGRC team) to overcome the problem. On the contrary, the results were not satisfying and this forces us to shift slightly from the proposed project.
We then started to investigate the direct relation of Lsr2 with GPL regulation as we had reported Lsr2 as a negative regulator of GPL locus. We designed the further experiment to identify the active promoter region of GPL locus. Using different beta-gal reporter fusions and beta-galactosidase quantitative assay, we identified the active promoter region, which is required for activation of GPL locus. To further explore if Lsr2 binds in this active promoter region, we conducted DNA-cell extract binding assay and very surprisingly we did not observe any protein correspondent to 12 KDa proteins which are the size of Lsr2. We observed a 40 KDa protein specifically binding to the active promoter region. This might be showing that Lsr2 protein binding is indirect and also possibly that some other factor is assisting the function. To identify this unknown 40 KDa protein, we collaborated with Dr. Sebastien Gallien (Laboratoire de Spectrometrie de Masse Bio-Organique, Strasbourg, France). Using the Ortho-proteomic approach, we identified MOXR (a putative transcriptional regulator). To understand the mechanisms of GPL regulation and possible role of MOXR, we mutated the MOXR gene and as a result found that it is an essential gene in M. Smegmatis. Using quantitative RT-PCR, phenotypical (congo red assay, sliding motility assay etc) and biochemical assays, we established that MOXR positively regulates the GPL expression in M. Smegmatis.

Project results

To better understand the pathogenesis of NTM, it is necessary to elucidate the molecular structure, biochemical features and regulation of GPLs. GPLs play a crucial role in the physiology of bacilli by controlling the envelope architecture through their effects on permeability, exerting an anti-phagocytic effect and promoting TNF-alfa synthesis (3-5). Our presented study sought to bring novel information on the molecular mechanisms implicated in the regulation of GPLs in M. Smegmatis. It needs to be developed further to realise the regulatory relations between Lsr2 and MOXR proteins. However, it will be very useful towards better understanding the lipid regulation in mycobacteria and could be useful as a further drug target. In addition, as the Lsr2 and MOXR are conserved in all mycobacterial species sequences to date (including M. leprae), it is hence likely that further study will be transposable to the pathogenic species relevant in human health.

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