Background and Aim

CpG motifs in DNA sequences are recognized by TLR9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine (5mC)) and modified adenine bases (6-methyladenine (6mA)). It is well-known that 5mC inhibits immune activation by CpG DNA; however, it is still unclear whether 6mA inhibits immune activation by CpG DNA. In this study, we tried to examine whether the existence of 6mA affected the immunostimulatory activity of gDNA.

Materials and Methods

We focused on adenine methyltransferases (MTases) expressed by Mycobacterium bovis BCG (BCG). Each adenine MTases has specific target sites. Therefore, we used oligodeoxynucleotides (ODNs) with 6mA on the target sequences of MTases from BCG. We stimulated murine bone-marrow derived macrophages (BMDMs) with ODNs harboring 6mA or ODNs without 6mA. We then compared the immunostimulatory activity of ODNs with 6mA to ODNs without 6mA using. We also developed adenine MTase-deficient BCG. We analyzed whether 6mA on gDNA from BCG is involved in the immunostimulatory activity of BCG gDNA.


Our results showed that 6mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced IL-12p40 production from BMDMs stimulated with CpG DNA.

We also observed that gDNA from mamA-deficient BCG induced less IL-12p40 from BMDMs than gDNA from mamA-intact BCG.


We concluded from these results that adenine methylation on ODNs may enhance immune activity induced by bacterial gDNA.