Mycobacterium avium subsp. paratuberculosis induces differential cytosine methylation at miR-21 transcription start site region

Mycobacterium avium subspecies paratuberculosis (MAP), as an obligate intracellular bacterium, causes paratuberculosis (Johne’s disease) in ruminants. Plus, MAP has consistently been isolated from Crohn’s disease (CD) lesions in humans; a notion implying possible direct causative effect for MAP in CD development. Infections caused by MAP are refractory to treatment and in many cases the treatment does not easily resolve the infection. Studying the molecular mechanisms of host-pathogen interaction is helpful in identifying possible drug targets. In this line, it has already been shown that in macrophages infected with various bacteria, including mycobacteria, micro RNA 21 (miR-21) is upregulated, a change that results in diminished macrophages clearance ability and favours pathogens survival within the cells. However, the molecular mechanism(s) by which the intracellular bacteria induce miR-21 expression is not known. In order to verify possible effects from epigenetic changes induced by intracellular bacteria, we studied the cytosine methylation changes at the transcription start regions of miR-21 in THP-1 macrophages infected with MAP. For this purpose, genomic DNA was extracted from infected cells and the methylation status at the region of interest was evaluated by bisulfite conversion method. Our work showed that MAP directs de-methylation of the cystosines at CpG di-nucleotides in this region, while non-CpG cytosines of this region did not show significant changes. Interestingly, the CpG cytosines that were differentially methylated in the infected macrophages occur at the binding sites of the transcription factors already known to regulate miR-21 expression.