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1. Host-Induced Genome Instability Rapidly Generates Phenotypic Variation across Candida albicans Strains and Ploidy States

   https://doi.org/10.1128/mSphere.00433-20  

   Smith, Amanda C.; Hickman, Meleah A. (June 2020, mSphere)

   Mitchell, Aaron P. (Ed.)

   ABSTRACT Candida albicans is an opportunistic fungal pathogen of humans that is typically diploid yet has a highly labile genome tolerant of large-scale perturbations including chromosomal aneuploidy and loss-of-heterozygosity events. The ability to rapidly generate genetic variation is crucial for C. albicans to adapt to changing or stressful environments, like those encountered in the host. Genetic variation occurs via stress-induced mutagenesis or can be generated through its parasexual cycle, in which tetraploids arise via diploid mating or stress-induced mitotic defects and undergo nonmeiotic ploidy reduction. However, it remains largely unknown how genetic background contributes to C. albicans genome instability in vitro or in the host environment. Here, we tested how genetic background, ploidy, and the host environment impacts C. albicans genome stability. We found that host association induced both loss-of-heterozygosity events and genome size changes, regardless of genetic background or ploidy. However, the magnitude and types of genome changes varied across C. albicans strain background and ploidy state. We then assessed if host-induced genomic changes resulted in fitness consequences on growth rate and nonlethal virulence phenotypes and found that many host-derived isolates significantly changed relative to their parental strain. Interestingly, diploid host-associated C. albicans predominantly decreased host reproductive fitness, whereas tetraploid host-associated C. albicans increased host reproductive fitness. Together, these results are important for understanding how host-induced genomic changes in C. albicans alter its relationship with the host. IMPORTANCE Candida albicans is an opportunistic fungal pathogen of humans. The ability to generate genetic variation is essential for adaptation and is a strategy that C. albicans and other fungal pathogens use to change their genome size. Stressful environments, including the host, induce C. albicans genome instability. Here, we investigated how C. albicans genetic background and ploidy state impact genome instability, both in vitro and in a host environment. We show that the host environment induces genome instability, but the magnitude depends on C. albicans genetic background. Furthermore, we show that tetraploid C. albicans is highly unstable in host environments and rapidly reduces in genome size. These reductions in genome size often resulted in reduced virulence. In contrast, diploid C. albicans displayed modest host-induced genome size changes, yet these frequently resulted in increased virulence. Such studies are essential for understanding how opportunistic pathogens respond and potentially adapt to the host environment. 

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2. Host defense mechanisms induce genome instability leading to rapid evolution in an opportunistic fungal pathogen

   https://doi.org/10.1128/IAI.00328-21  

   Smith, Amanda; Morran, Levi; Hickman, Meleah A. (January 2022, Infection and Immunity)

   The ability to generate genetic variation facilitates rapid adaptation in stressful environments. The opportunistic fungal pathogen Candida albicans frequently undergoes large-scale genomic changes, including aneuploidy and loss-of heterozygosity (LOH), following exposure to host environments. However, the specific host factors inducing C. albicans genome instability remain largely unknown. Here, we leveraged the genetic tractability of nematode hosts to investigate whether innate immune components, including antimicrobial peptides (AMPs) and reactive oxygen species (ROS), induced host-associated C. albicans genome instability. C. albicans associated with immunocompetent hosts carried multiple large-scale genomic changes including LOH, whole chromosome, and segmental aneuploidies. In contrast, C. albicans associated with immunocompromised hosts deficient in AMPs or ROS production had reduced LOH frequencies and fewer, if any, additional genomic changes. To evaluate if extensive host-induced genomic changes had long-term consequences for C. albicans adaptation, we experimentally evolved C. albicans in either immunocompetent or immunocompromised hosts and selected for increased virulence. C. albicans evolved in immunocompetent hosts rapidly increased virulence, but not in immunocompromised hosts. Taken together, this work suggests that host-produced ROS and AMPs induces genotypic plasticity in C. albicans which facilitates rapid evolution. 

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3. Mathematical modeling of the Candida albicans yeast to hyphal transition reveals novel control strategies

   https://doi.org/10.1371/journal.pcbi.1008690  

   Wooten, David J.; Zañudo, Jorge Gómez; Murrugarra, David; Perry, Austin M.; Dongari-Bagtzoglou, Anna; Laubenbacher, Reinhard; Nobile, Clarissa J.; Albert, Réka (March 2021, PLOS Computational Biology)

   Thieffry, Denis (Ed.)

   Candida albicans , an opportunistic fungal pathogen, is a significant cause of human infections, particularly in immunocompromised individuals. Phenotypic plasticity between two morphological phenotypes, yeast and hyphae, is a key mechanism by which C . albicans can thrive in many microenvironments and cause disease in the host. Understanding the decision points and key driver genes controlling this important transition and how these genes respond to different environmental signals is critical to understanding how C . albicans causes infections in the host. Here we build and analyze a Boolean dynamical model of the C . albicans yeast to hyphal transition, integrating multiple environmental factors and regulatory mechanisms. We validate the model by a systematic comparison to prior experiments, which led to agreement in 17 out of 22 cases. The discrepancies motivate alternative hypotheses that are testable by follow-up experiments. Analysis of this model revealed two time-constrained windows of opportunity that must be met for the complete transition from the yeast to hyphal phenotype, as well as control strategies that can robustly prevent this transition. We experimentally validate two of these control predictions in C . albicans strains lacking the transcription factor UME6 and the histone deacetylase HDA1 , respectively. This model will serve as a strong base from which to develop a systems biology understanding of C . albicans morphogenesis. 

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4. An expanded toolkit of drug resistance cassettes for Candida glabrata , Candida auris , and Candida albicans leads to new insights into the ergosterol pathway

   https://doi.org/10.1128/msphere.00311-23  

   Gregor, Justin B.; Gutierrez-Schultz, Victor A.; Hoda, Smriti; Baker, Kortany M.; Saha, Debasmita; Burghaze, Madeline G.; Vazquez, Cynthia; Burgei, Kendra E.; Briggs, Scott D. (December 2023, mSphere)

   Mitchell, Aaron P. (Ed.)

   ABSTRACT The World Health Organization recently published the first list of priority fungal pathogens highlighting multipleCandidaspecies, includingCandida glabrata,Candida albicans, andCandida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes,NatMXandHphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit forC. glabrata,C. auris, andC. albicansto includeKanMXandBleMXwhen coupled with anin vitroassembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes forCandida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion ofERG3inC. glabrata,C. auris,andC. albicansprototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that anERG5deletion inC. glabratais azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when bothERG3andERG5are deleted inC. glabrata,which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates. IMPORTANCEThe increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies inCandida glabrata,Candida auris, andCandida albicanshave been mainly limited to the use ofNatMX/SAT1andHphMX/CaHygfor genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated thatNatMX/SAT1, HphMX, KanMX,and/orBleMXdrug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway ofC. glabrata,C. auris, andC. albicans. Moreover, the utility of these tools has provided new insights intoERGgenes and their relationship to azole resistance inCandida. Overall, we have expanded the toolkit forCandidapathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis. 

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5. Evolution and strain diversity advance exploration of Candida albicans biology

   https://doi.org/10.1128/msphere.00641-23  

   Anderson, Matthew Z; Dietz, Siobhan M (August 2024, mSphere)

   Mitchell, Aaron P (Ed.)

   ABSTRACT Fungi were some of the earliest organismal systems used to explore mutational processes and its phenotypic consequences on members of a species. Yeasts that cause significant human disease were quickly incorporated into these investigations to define the genetic and phenotypic drivers of virulence. AmongCandidaspecies,Candida albicanshas emerged as a model for studying genomic processes of evolution because of its clinical relevance, relatively small genome, and ability to tolerate complex chromosomal changes. Here, we describe major recent findings that used evolution of strains from defined genetic backgrounds to delineate mutational and adaptative processes and include how nascent exploration into naturally occurring variation is contributing to these conceptual frameworks. Ultimately, efforts to discern adaptive mechanisms used byC. albicanswill continue to divulge new biology and can better inform treatment regimens for the increasing prevalence of fungal disease. 

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