N6-methyladenosine in Candida white-opaque switching and oral infection

项目摘要/摘要

Project Summary / Abstract Candida albicans white-opaque switching is an epigenetic process that regulates its mating ability, virulence, and colonization of different niches in the human host. One of these niches is the oral cavity, where Candida species can cause painful infections called oropharyngeal candidiasis (OPC). While there are known environmental cues and transcriptional regulators that control the white-opaque switch, a mechanistic understanding of how specific environments trigger switching to one state or another and how this contributes to colonization and infection is lacking. This proposal will investigate how the RNA modification N6- methyladenosine (m6A) acts to mediate white-opaque switching and mating in C. albicans. Preliminary studies from my collaborator and I have found that cells lacking IME4, the m6A methyltransferase, are impaired in temperature-induced opaque to white switching and have an increased mating efficiency. As someone with a background in Candida white-opaque switching and m6A biology, I am uniquely qualified to investigate the hypothesis that m6A regulation mediates environmental signals that induce phenotypic changes that mediate infection by C. albicans. Aim 1 is to identify targets of m6A modification in white-opaque switching by using eCLIP to map single nucleotide sites of m6A modification and binding sites of MRB1, the m6A reader, as well as gene expression analysis in wild-type and ime4 mutant white and opaque cells. Aim 2 will determine how the environment regulates m6A in switching and filamentation by generating gene expression and metabolomic profiles of cells under environmental conditions such as high temperature or CO2 that induce white-opaque switching or filamentation. Aim 3 will investigate the role of m6A in oral colonization of C. albicans by using cell culture and mouse models of OPC. White-opaque switching regulates oral colonization, and we hypothesize that IME4 plays a role in this process. In this aim, I will also study how radiation affects Candida cells lacking IME4 and how it affects colonization of the murine oral niche. In aim 4 I will investigate the function of m6A in interactions with the oral microbiome. Many of the m6A modified genes I have identified are involved in interspecies interactions, suggesting that IME4 and m6A regulate these interactions within the host. This research plan will be the first to combine the fields of epitranscriptomics and Candida biology to study how m6A contributes to the lifestyle and pathogenicity of a clinically relevant fungal species, with the potential to pave the way for new anti-fungal drug treatments for OPC. I have proposed a comprehensive training program for the K99 phase of this proposal including acquisition of new skills in bioinformatics, metabolomics, animal models of oral infection, microbiome analyses, guidance from an advisory committee of prominent mentors and collaborators, and development of key professional and leadership skills. Collectively, this plan will facilitate my transition to independence during the R00 phase and prepare me for a career as an independent researcher.