m6A methylation of IRGM transcripts in epithelial-Cryptosporidium interactions - Summary Cryptosporidium, an apicomplexan parasite that infects the gastrointestinal epithelium in humans, remains an important opportunistic pathogen in AIDS patients who do not have access to HAART treatment. This parasite is also a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. The N6- methyladenosine (m6 a reversible RNA post-transcriptional modification. Emerging A) RNA methylation is evidence from our lab and others has defined m6A RNA methylation as a crucial regulator in host antimicrobial response. The overall objective of this R21 proposal is to advance our understanding of the molecular mechanisms by which intestinal epithelial cells control the dynamic of m6A RNA modifications of mRNA transcripts to orchestrate IFN-γ-mediated epithelial anti-Cryptosporidium defense. We have recently demonstrated significant alterations in the topology of host m6A mRNA methylome in murine intestinal epithelial cells following Cryptosporidium infection. Although the RNA levels of several IFN-induced immunity- related GTPase family M (IRGM) genes are upregulated, a decreased in their m6A RNA methylation level and translation was identified in infected cells. In contrast, IFN-γ stimulation upregulates the RNA levels of IRGM genes with an increased m6A RNA methylation and importantly, m6A methylation of IRGM RNAs is associated with an increased translation. Given the fact that the IRGM proteins are critical effectors in IFN-γ-mediated cell- autonomous immunity in both mice and humans, we hypothesize that m6A methylation of IRGM mRNA transcripts promotes IFN-γ-mediated epithelial cell antimicrobial defense and to counteract IFN-γ-mediated host defense, Cryptosporidium has developed strategies to suppress m6A methylation of IRGM mRNA transcripts. We will use in vitro, ex vivo, and in vivo infection models and complementary biochemical, molecular, and morphologic approaches to elucidate the molecular mechanisms by which m6A methylation of IRGM mRNA transcripts promotes IFN-γ-mediated intestinal epithelial cell anti-Cryptosporidium defense (Aim 1) and determine how Cryptosporidium suppresses m6A methylation of IRGM mRNA transcripts, resulting in attenuation of IFN-γ-mediated epithelial anti-parasitic defence (Aim 2). Detailed mechanistic studies of the role m6A RNA methylation of IRGM transcripts in host-Cryptosporidium interactions could not only reveal new insights into the molecular immunology and immunopathology of intestinal Cryptosporidium infection but also establish a foundation on which to build long-term, mechanistic studies to define its pathogenesis and therapeutic interventaion.
