The objective of this dissertation is to provide a framework to discover biofilm-specific antifungal drug targets of the opportunistic human fungal pathogen Candida albicans by answering two main questions: (1) which genes are functionally important for biofilm formation? (2) what are the primary roles of these genes during biofilm formation? In chapter one, I provide fundamental background information about C. albicans. In chapter two, I summarize the current understanding of the transcriptional regulatory circuits that govern three major developmental processes in C. albicans: biofilm formation, the white-opaque cell type switch, and the commensal to pathogen switch. In chapter three, I describe the findings of my main thesis project and highlight a set of genes that are part of the C. albicans biofilm regulatory network that are required for biofilm formation and play important roles in the different stages of biofilm development. Lastly, I conclude by summarizing the main points from each of the previous chapters and discussing their overall significance.

The primary project of my dissertation focused on studying the regulation of chlamydospores, a morphology formed by the common human fungal pathogen Candida albicans. C. albicans produces chlamydospores under stressful conditions, however, the biological functions of chlamydospores are still unknown. Since this important human fungal pathogen produces these enigmatic structures, I believe that chlamydospores must provide a selective advantage to C. albicans. I hypothesized that there must be underlying developmental and regulatory pathways dedicated to chlamydospore formation, and that identifying these pathways will be useful in understanding the biological functions of chlamydospores. Using forward genetics and genome-wide approaches including RNA-seq and ChIP-seq, I discovered that the C. albicans chlamydospore transcriptional regulatory network is highly interwoven comprised of nine core transcriptional regulators (i.e., transcription factors) controlling over 3,200 downstream target genes. Of these nine core regulators, I have found that six core transcription factor deletion mutant strains fail to form chlamydospores, while three core transcription factor deletion mutant strains form higher numbers of chlamydospores relative to the wildtype strain. Analysis of the chlamydospore regulatory network suggests roles for SNARE vesicular transport and fatty acid degradation pathways along with roles for enzymes involved in cell wall biosynthesis pathways. Preliminary network conservation analyses based on orthologous relationships of proteins within the chlamydospore network revealed that the network is comprised largely of “old” proteins (65%) interspersed with some “young” proteins (35%), indicative of the network being fairly well conserved. Further analysis of this regulatory network will be useful in identifying the biological functions of chlamydospores and will also give us insight into the regulation of C. albicans morphological transitions more generally. Another project of my dissertation focused on studying non-drug therapeutic strategies to target biofilm formation in C. albicans and Candida auris. C. albicans and C. auris form robust and drug resistant biofilms and treatment of biofilm infections caused by these species is challenging. I focused on exploring red, green and blue visible lights in combination with exogenous photosensitizing compounds as a non-drug therapeutic strategy against C. albicans and C. auris biofilm in vitro. I demonstrated that red, green and blue visible lights in combination with exogenous photosensitizing compounds are an effective non-drug therapeutic strategy against both Candida species biofilms. Blue light with and without photosensitizing compounds was the most effective treatment at inhibiting biofilm formation and also disrupting mature biofilms of both species, closely followed by red light in combination with photosensitizing compounds.

Biofilms are structured and densely packed communities of microbial cells attached to surfaces. They are considered the natural growth state for a vast majority of microorganisms. The ability to form biofilms is an important virulence factor for most pathogens, including the opportunistic human fungal pathogen Candida albicans. C. albicans is one of the most prevalent fungal species of the human microbiota. However, C. albicans can also cause severe and life-threatening infections when host conditions permit. Conventional C. albicans biofilms are often resistant to antifungal agents and the host immune response and can act as reservoirs of infectious cells, maintaining persistent infections and seeding new infections in a host. The majority of C. albicans clinical isolates are heterozygous (a/α) at the mating type-like (MTL) locus, which defines Candida mating types, and can form robust biofilms when cultured in vitro. These “conventional” biofilms, formed by MTL-heterozygous (a/α) cells, have been the primary focus of C. albicans biofilm research to date. Recent work in the field has uncovered novel mechanisms through which biofilms are generated by C. albicans cells that are homozygous or hemizygous (a/a, a/Δ, α/α, or α/Δ) at the MTL-locus. In these studies, the addition of mating pheromone of a particular mating type can induce cells of the other mating type to form specialized “sexual” biofilms. Although sexual biofilms are generally less robust than conventional biofilms, they could serve as a protective niche to promote the parasexual life cycle of mating-competent cells, and thus could be an adaptation which may increase population diversity in dynamic environments. In this work, we investigate the molecular and genetic mechanisms that enable sexual biofilm formation in C. albicans to gain insight into this unique biological process and establish a basis for comparison between sexual and conventional biofilms. Chapter 1 of this dissertation provides a detailed review of sexual biofilm formation in C. albicans. Chapter 2 of this dissertation provides detailed analyses and transcriptomics of several clinically isolated strains growing under sexual biofilm inducing conditions as well as co-cultures of white and opaque cells of various mating types. This led to the identification of several downstream genes that are involved in sexual biofilm formation. Chapter 3 of this dissertation identifies several TFs that are involved in regulating sexual biofilms. We find that many regulators of conventional biofilm formation are similarly involved in sexual biofilm formation. Interestingly, some transcription factors appear to switch from positive regulators of one system to negative regulators of the other. In addition, differential gene expression and gene ontology analyses reveal the unique role of these transcription factors in regulating sexual biofilm formation. Finally, Chapter 4 provides a detailed description of the conclusions and future directions for where other researchers can take this project, particularly to build clinical relevance for the sexual biofilm system and to uncover the transcription network of sexual biofilms and elucidate the evolutionary history of sexual and conventional biofilms.

The primary question of my dissertation is, “Does venom possess a microbiome specific to it as an ecosystem, and why?” Given the limited amount of literature on venom microbiomes, I selected the California Cone Snail, Californiconus californicus as a proposed, wild model system for studying venom-microbe interactions in the process of investigating hypothesized microbial interactions with host venom.

First, I present a data-driven approach for how sampling sites of venomous animals of interest can be selected in conjunction with the current trend to rely on anecdotal information. This work integrates curated museum collections, crowd-sourced data through digital mediums, knowledge through scientific literature, and personal research. This segment delves deeper into our understanding of C. californicus across space and time, dating back to the Pleistocene. We identify relationships between shell morphology and temperature, contributing foundational knowledge for this species and prospective context of venom microbiome coevolution.

Second, I characterize the seawater and sediment coastal microbial ecology of the initial known sampling site (Puerto Nuevo, Mexico) in which this species is commonly found. We sampled several sites along a gradient of exposure to urbanization (0.45 km) and characterized the core microbial communities for archaea, bacteria, and microbial eukaryotes using 16S and 18S amplicon sequencing. While only representing one time point and location, our experimental design allows us to demonstrate consistency in the literature in that we identify functionally relevant microbial taxa specific to different environmental types and distance. This work contributes as a preliminary example for the determination of how and where microbes in the venom may be sourced from the wild.

Third, I outline the main findings of the venom microbiome for C. californicus. Model organisms used today are common, simplified points of reference for downstream application. The California Cone Snail is a commonly found neogastropod along the California-Baja coast by the 100s. It can be cultured and maintained in a laboratory, acting well for experiments in the wild and in vivo or in vitro. We sampled C. californicus for three major sites for geographical variation: Puerto Nuevo, San Diego, and Monterey. We sampled summer, winter, and summer again, as well as three consecutive days in Puerto Nuevo for temporal variation. We sampled adult and eggs to compare microbial communities across life stage. We then compared venom microbial communities in a lab setting by testing for different hydrostatic pressures, axenic conditions, and exposure to prey. In summary, we find a specific microbial community (16S and 18S) found in and along the venom gland when compared to other environments, tissues, and conditions.

Finally, I tie extensions of science outreach together with scientific practice through communication, education, policy, and the first venom-microbe consortium. These initiatives act as proof-of-concept for strengths in democratically practiced open-source, interdisciplinary research through inclusion across demographics and educational and professional stages.