Changes and differences of DNA methylation in human macrophages infected with virulent and avirulent Mycobacterium tuberculosis

Abstract

Objective: To profile DNA methylation and compare differentially methylated region (DMR) of macrophages infected with virulent and avirulent strains of Mycobacterium tuberculosis (MTB). Methods: PMA(50 ng/ml) treated THP-1 macrophages were left uninfected or infected with the virulent H37Rv(THP-1/Rv) or avirulent H37Ra(THP-1/Ra). The genomic DNA was then extracted and DNA methylation was profiled via Reduced Representation Bisulfite Sequencing (RRBS). The DNA methylation pattern and DMRs between the tested samples were indentified. DMRs-associated genes were annotated, clustered by Gene Ontology (GO), and the pathways analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG). mRNA expression of several DMR-associated genes were verified by Q-PCR, and Student's t-test was used to analyze the differential expression between either 2 samples. Results: The mostly DNA methylated C in 3 samples was in CG, accounting for 98% of all types of methylated C(CG, CGH, CHH). Besides, the average DNA methylation level in THP-1/Rv, THP-1/Ra and THP-1/NC were 7.72%, 7.38% and 7.58% in the promoter, and 9.90%, 9.57% and 9.80% in the CpG island (CGI), respectively. We identified 58 DMRs between H37Rv and H37Ra infected THP-1 macrophages with an average length of 152 bp. Six DMRs-associated genes were enriched. Among them, AP-1 and DDIT4 were differentially expressed in cells infected with H37Rv and H37Ra. Conclusions: Infection with MTB is not correlated with a large-scale gain or loss in DNA methylation in host cells. AP-1 and DDIT4 might be novel virulence-related genes regulated by DNA methylation of their corresponding promoter regions.