Impact of RNAi and unisexual reproduction on Cryptococcus evolution, drug resistance, and pathogenesis

摘要

Abstract Cryptococcus neoformans and Cryptococcus gattii are globally distributed fungal pathogens that cause >220,000 life-threatening infections each year in immunocompromised and immunocompetent patients, leading to >180,000 deaths, >15% of all HIV/AIDS-related deaths, and >70% mortality in low-income countries. In studies supported by this award, the scope and functions of a- and - sexual reproduction on the evolution and pathogenesis of Cryptococcus were defined. Sexual reproduction governs population structure, enabling genetic exchange and promoting clonality, and results in the production of infectious spores. During this sustained funding period, significant advances were achieved characterizing the mechanisms and impact of sexual reproduction on these pathogens and their virulence: 1) discovery of roles of RNAi in controlling transposon movement and transgene silencing during mitosis and meiosis and characterization of the consequences of RNAi loss on genome structure and stability; 2) studies on generation and affect of aneuploidy on drug resistance, pathogenicity, and development, and engineering of a ploidy sensor to analyze genetic factors contributing to endoreplication during unisexual reproduction; 3) delineation of isolates causing the C. gattii outbreak in the Pacific Northwest and closely aligned hypermutator lineages, and 4) demonstration that unisexual reproduction generates genetic diversity de novo and enhances competitiveness for nutrients and mating partners. In the current proposal, we hypothesize 1) RNAi has been retained and lost in unique isolates and species, influencing genomic stability and driving evolution of drug resistance and pathogenesis, and 2) unisexual reproduction is a major force in evolution, global distribution, and impact of Cryptococcus pathogens. Our studies reveal unique facets of RNAi and sexual reproduction enabling us to propose new aims to test these hypotheses. Aim 1 focuses on RNAi in genome stability, elucidating components and mechanisms by which RNAi controls transposons and silences repetitive DNA sequences, and studying the short-term and long- term consequences of RNAi loss on drug resistance and pathogenesis in C. neoformans hypermutator clinical isolates and the RNAi-deficient C. deuterogattii outbreak species. Aim 2 will elucidate unisexual reproduction pathways and mechanisms involving 1) unisexual reproduction occurring in mixed mating-type populations and in strains with rearranged genomes as a model of speciation, 2) genetic and environmental factors that promote unisexual reproduction of global serotype A lineages and analysis of infectious spores in animal models, and 3) overcoming the Hill-Robertson effect to enable linkage of beneficial mutations and separation of detrimental and beneficial mutations to enhance fitness and pathogenesis. These studies will advance the understanding of how genome stability and unisexual reproduction drive pathogen evolution and population structure, development of drug resistance, and infectious spore production and virulence, with direct implications for infectious disease evolution, treatment, and prevention.