De Maria, Francesca (2015) Mycobacterium tuberculosis adaptive response to DNA methylation stress. [Tesi di dottorato]

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Tuberculosis (TB) is an infectious disease caused by the micro-organism Mycobacterium tuberculosis (MTB) that currently represents a global health emergency with around 9 million people affected and nearly 2 million deaths each year (1). The present therapeutic treatment for TB consists in the use of multiple anti-mycobacterial drugs such as rifampicin and isoniazid. This treatment is carried out for a long time leading to the onset of drug- and multidrug-resistant strains of MTB. Therefore new therapeutic models are needed to overcome this problem (2). During MTB infection, the host antimicrobial response generates several metabolically activated DNA alkylating agents leading to severe DNA-damaging injuries on MTB cells (3, 4). Protection of the DNA molecule from chemical damages then strictly depends on the MTB repair mechanisms. Recent studies identified an adaptive response mechanism in MTB homologous to that already described in E.coli (5). Being the adaptive response a fundamental biological process for MTB viability whereas it is missing in human, this process may represent a putative therapeutic target to explore in search for new TB treatments. Previous studies in MTB identified four genes which encode the proteins constituent of the adaptive response mechanism homologous to the same process already defined in E. coli. Exposure of E. coli to sublethal concentrations of alkylating agents induces the expression of four genes (ada, alkA, alkB and aidB). The activation of these genes increases the resistance to the cytotoxic and mutagenic effects of alkylating molecules (6). Unlike E. coli, the DNA repair/protection systems in MBT have not been investigated and it is still largely unknown. Aims This thesis project focuses on the investigation of the adaptive response to DNA methylation stress in MTB, in order to identify possible innovative therapeutic targets against tubercular infection. A non-pathogenic, reference strain of Mycobacteria, M. smegmatis, was used to perform all the experiments. The effect of methylating molecules on M. smegmatis was evaluated using MMS, a common laboratory methylating molecule. Moreover, Busulfan was chosen as methylating reference drug. First the effect of alkylating agents was investigated on M. smegmatis cells by evaluating the effect of MMS on cell survival, DNA alkylation and on the global proteomic response. Second, structural and functional characterization of the recombinant Ada-Alka and Ogt proteins was carried out because of their involvement in the cellular response to alkylating agents. Finally, functional proteomic experiments on M.smegmatis Ogt protein was performed in order to shine a light on its biological function through the identification of its protein partners in vivo.

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