Generation of phenotypic diversity in the fungal pathogen Candida Albicans

Abstract

Microbial organisms have a diverse array of mechanisms to obtain phenotypic variation. Phenotypic variation not only enhances population fitness and competitiveness for a specific niche but it is also critical for the survival of a population to unexpected environmental changes. Further, in pathogenic organisms, phenotypic variation is directly associated with virulence. Therefore, besides of the contribution to our understanding of microbial evolution, dissecting the mechanisms that lead to phenotypic variation in pathogenic organisms is very clinically relevant. Candida albicans is the most successful opportunistic fungi that infect humans. C. albicans is an obligate diploid yeast with an almost exclusive clonal form of reproduction. In the absence of meiosis to introduce variation in the population, C. albicans needs alternative mechanisms to achieve variability, such as the colony morphology phenotypic switching (CMPS). CMPS is the formation of colonies that have an altered, heritable, and low frequency reversible morphology. CMPS is associated with pathogenesis in C. albicans: variant colony morphologies have been isolated during infections in humans and show an altered expression of diverse virulence factors, including the secretion of hydrolytic enzymes and resistance to antifungal drugs. Despite the potential role of CMPS in the pathogenesis of C. albicans, little is known about the mechanisms that regulate this phenomenon. In our lab, we serendipitously identified a negative regulator of CMPS in C. albicans: the Kelch protein Mds3. Mds3 had been previously associated with other morphogenetic processes in fungi, but the biological role of Mds3 in the cell was unknown. Therefore, my goals in this dissertation were to understand the function of Mds3 in the cell and to use this knowledge to gain insights into C. albican's CMPS mechanisms and regulation. Through a combination of bioinformatic, biochemical, and genetic analyses we found that Mds3 appears to be a large Kelch/BTB cytoplasmic scaffold protein that functions as a regulator of two major signaling cascades, the TOR and Ras pathways (Chapters 2, 3, and 5). With this information, I was able to identify more CMPS regulators that belong to these pathways and environmental signals that regulate CMPS and which are all strongly associated with signaling through these pathways (Chapters 4 and 5). Analyses of morphologically switched mds3delta/delta strains indicated that the phenotypic switch is accompanied by an increase in the sensitivity to the TOR inhibitor rapamycin, which suggests an increased dependence on TOR function in the switched strains. Further, the phenotypic switch was also accompanied by increased sensitivity to genotoxic agents and sometimes also by karyotypic rearrangements and aneuploidies (Chapter 4). Increased DNA damage and genomic instability are mechanisms associated with phenotypic variation in several highly diverse organisms, and could also be mechanisms leading to the phenotypic switch in C. albicans (Chapter 4). Taken together, I propose a model for CMPS in C. albicans in which defects in the signaling through the TOR and Ras signal transduction pathways as cells become nutrient limited and stressed lead to the accumulation of genetic and epigenetic alterations that eventually cause the phenotypic switch.