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Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. C. albicans is the most prevalent Candida pathogen of humans, living in and on a majority of the world’s population. Interrogation of key virulence-related processes in C. albicans requires production of continually improved tools. We describe two methods, the Galleria mellonella waxworm infection model and an automated, high-throughput platform for assessing the individual components of biofilm formation, that significantly improved our analyses. We highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. The second method is used to demonstrate that media profoundly altered all biofilm-related phenotypes among a diverse collection of isolates, whereas temperature minimally impacted these traits. Importantly, this work showed that the extent of biofilm formation correlated significantly with the additive score for its component phenotypes under some conditions. In addition, the response of the genome reference strain, SC5314, was an extreme outlier compared to all other strains, suggesting it may not be representative of the species.These tools, along with a number of other techniques, were applied to the study of gene family expansions in C. albicans. Gene families often expand in function or regulation of processes which are critical to an organism’s lifestyle. As such, expanded gene families have been the target of intense study, but the individual contributions of single paralogs and the extent of sequence diversity is often unexplored across multiple strain backgrounds. Critical to C. albicans success is its ability to survive and thrive under a wide range of nutrient and stress conditions across the human body. Coincidentally, the telomere-associated (TLO) gene family has undergone an extensive expansion from a single gene in most Candida species to 14 members in C. albicans. To determine TLO genetic diversity, we sequenced TLO genes across 23 clinical isolates and obtained 189 TLO gene sequences that can be placed into three previously described architectural groups. Interestingly, this analysis identified that most TLOs from the alpha and beta groups contain predicted prion-like domain, indicating they may be capable of forming liquid-like droplets which can engage in transcription factor interactions. Furthermore, each TLO group (alpha, beta, and gamma) appears to be evolving under different evolutionary constraints to produce a range of distinct genes within the same gene family in the same species. To identify biological functions associated with each C. albicans TLO found in the genome reference strain SC5314, expression of individual family members was regulated using a Tet-ON system and the strains were assessed across a wide range of simple and complex phenotypes. All TLOs affected multiple phenotypes and a single phenotype was often affected by multiple TLOs. No phenotype was regulated by all TLOs. Therefore, expansion of the TLO gene family has conferred unique regulatory properties to each paralog such that it influences a range of phenotypes. In an ongoing collaboration with Drs. Derek Sullivan and Gary Moran, we have conducted preliminary characterization of the TLO-null strain background which presents strong drug tolerance, slower growth rates, and increased aggregation relative to both its parental strain and any individually induced TLO. |
