Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation

Abstract

Epigenetic factors play a role in the expression of virulence traits in Apicomplexa. Apicomplexan genomes encode putative DNA cytosine methylation enzymes. To assess the presence of cytosine methylation of Toxoplasma gondii and Cryptosporidium parvum DNA, we used mass spectrometry analysis and confirmed that these organisms lack detectable methylcytosine in their DNA.

FIG. 1.

Distribution of cytosine methylation as shown by HELP data from a representative experiment using T. gondii, Rattus norvegicus liver, and Mus musculus round spermatid DNA samples. The log intensity of the data from the HpaII (methylation-sensitive) genomic representation of these three samples is plotted versus HpaII tiny fragments size (15) in the three upper panels. The same data are shown for the MspI (methylation-insensitive) representation in the middle row of panels and for the normalized HpaII/MspI ratios in the three lower panels. “Failed” signals are indicated by gray dots and are identified as those signals that fall within the level of background noise (median of random probes plus 2.5 median absolute deviations). The distribution of fragments indicates that T. gondii DNA is unmethylated (positive normalized HpaII/MspI ratio), whereas rat liver shows some methylation and mouse sperm DNA is enriched for methylation.

FIG. 2.

LC-MS analysis of genomic DNA of T. gondii mixed with human genomic DNA, T. gondii, C. parvum, and P. falciparum. (A) The two MS peaks correspond to cytosine (Cyt; m/z = 112) and its stable isotope (m/z = 115) for a representative experiment detecting cytosine in T. gondii DNA. (B, C, D, and E) Two peaks correspond to 5mC (m/z = 126) and its stable isotope (m/z = 130). A 1:1 mixture of T. gondii and human genomic DNAs has a 5mC peak (B). No 5mC is detected in T. gondii (C), C. parvum (D), or P. falciparum (E) DNA.