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Legionellapneumophila induces methylomic changes in ten-eleven translocation to ensure bacterial reproduction in human lung epithelial cells.
Journal of Medical Microbiology 2023 March
Introduction. Legionella pneumophila is a Gram-negative flagellated bacteria that can infect human lungs and cause a severe form of pneumonia named Legionnaires' disease. Hypothesis. We hypothesize that L. pneumophila infection induces methylomic changes in methylcytosine dioxygenases, ten-eleven translocation (TET) genes, and controls DNA methylation following infection. Aim. In the current research, we sought to further investigate DNA methylation changes in human lung epithelial cells upon L. pneumophila infection and determine how methylation inhibitor agents disturb L. pneumophila reproduction. Methodology. A549 cell line was used in L. pneumophila infection and inhibitors' treatment, including 5 - azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG). Results. Interestingly, DNA methylation analysis of infected A549 using sodium bisulfite PCR and the methylation-sensitive HpaII enzyme showed potential methylation activity within the promoter regions of ten-eleven translocation (TET) genes located on CpG/397-8 and CpG/385-6 of TET1 and TET3, respectively. Such methylation changes in TET effectors decreased their expression profile following infection, indicated by quantitative real-time PCR (RT-qPCR), immunoblotting and flow cytometry. Furthermore, pre-treatment of A549 cells with 5-AZA or EGCG significantly decreased the bacterial reproduction characterized by the expression of L. pneumophila 16S ribosomal RNA and the c.f.u. ml-1 of bacterial particles. Moreover, both methylation inhibitors showed potent inhibition of methionine synthase (MS) expression, which was further confirmed by the docking analysis of inhibitor ligands and crystal structure of MS protein. Conclusion. These data provide evidence for the methylomic changes in the promoter region of TET1 and TET3 by L. pneumophila infection in the A549 cell line and suggest the anti-bacterial properties of 5-AZA and EGCG, as methylation inhibitors, are due to targeting the epigenetic effector methionine synthase.
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