Fungal pathogens are responsible for millions of infections worldwide every year. These infections are caused by several fungal species, yet the Candida species of fungal pathogens are the most frequently isolated from humans. They cause superficial to life-threatening disseminated infections, where individuals with compromised immune systems, individuals with changes in their resident microbiota, and those undergoing antibiotic treatments are at highest risk for developing severe Candida infections. A longstanding limitation in the field is the ability to make precise genome edits in the Candida species to identify genes and pathways involved in virulence and antifungal resistance. Chapter 1 of this dissertation describes the identification of Candida albicans transcriptional regulators controlling cell aggregation in response to the antifungal drug caspofungin. This led to the identification of a novel adhesin mediating aggregation and a cell wall protein critical for the caspofungin response. Chapter 2 describes the development and optimization of the first markerless CRISPR/Cas9 system for genome editing in the emerging fungal pathogen Candida auris. I show the versatility of this tool to perform genome edits in clinical isolates and identify a conserved role for a transcription factor involved in the caspofungin response. Chapter 3 describes my work on the efficacy of a novel oxidizing disinfectant agent capable of disrupting biofilms, surface attached microbial communities, of pathogenic fungal and bacterial species. Finally, Chapter 4 summarizes my conclusions from the first three chapters and discusses future directions for the work. Overall, this body of work provides new tools for the genetic manipulation of an emerging fungal pathogen and expands our understanding of how Candida species respond to antifungal drug treatment.

Candida albicans is a commensal member of the human microbiota that is also capable of causing superficial and disseminated infections and is known to be the predominant fungal pathogen of humans in clinical settings. Mucosal linings are critical for protecting the host against infections, and it is the biological and viscoelastic properties of mucus, predominantly attributed to glycoproteins called mucins, that provide this protection. Chapter 1 introduces how mucins are involved in maintaining a healthy microbial balance on mucosal linings when directly interacting with microorganisms. Oftentimes, invading microorganisms colonize mucosal surfaces by forming microbial communities called biofilms. Biofilms can have detrimental impacts on the host because, once formed, they are highly resistant to chemical and mechanical perturbations. Chapter 2 aims to discover novel regulators that mediate the ability of C. albicans to interact with mucin in the context of biofilms. We screened a library of 211 transcription factor deletion mutants using in vitro biofilm assays in the presence of mucin to identify the regulators involved in governing C. albicans interactions with mucin. A total of 12 “master” regulators were found to play roles in interacting with mucins. Using a combination of genetic and genome-wide approaches, we mapped the regulatory connections of each transcription factor to one another and to downstream target genes to determine the regulatory network controlling the ability of C. albicans to interact with mucin. Understanding how C. albicans modulates its interactions with mucin will shed new light on the regulatory control that evolved to mediate this important interaction with the host. In Chapter 3, we introduce a host-pathogen model system to study the interactions between C. albicans and the flatworm Schmidtea mediterranea. This model is a unique invertebrate model that allows for the study of an early C. albicans epithelial infection in the presence of secreted mucus produced by the flatworm. Using this model, we identified two C. albicans proteins that mediate epithelial infection in S. mediterranea as well as a comprehensive host response facilitated by diverse host tissues to clear the infection.

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.

Transcription is a complex process underlying many cellular functions. DNA structure was discovered by Rosalind Franklin over 50 years ago. Since then, we have been steadily dissecting our understanding of the biological logic governing all life on Earth. Ultimately, Francis Crick discovered the central dogma of molecular biology in 1970. Early studies on Escherichia coli began formulating the idea of gene regulation as the basis of information flow in biological systems. Relatively soon, due to advancements in computing power, computer aided analyses of DNA and RNA were introduced to understand regulatory sequences. Since then, many improvements in experimental and computation protocols have accelerated our understanding of the control of biological information flow.

My thesis work, which uses the fungal species Candida albicans as a model, is the latest advancement in our understanding of gene regulation. In chapter one, I present my work on identifying the gene regulatory networks controlling biofilm formation in C. albicans across the biofilm life cycle. I explain my novel workflow that utilizes sequence-based as well as DNA-shape based features to predict transcription factor binding sites (TFBSs) genome-wide in C. albicans. In chapter two, I present CUT&RUN sequencing data implemented to assess binding events for specific TFs in C. albicans. I also present my novel CUT&RUN computational pipeline to analyze CUT&RUN sequencing data. In chapter three, I present a computational workflow to analyze 3’ Tag-Seq data. In this 3’ Tag-Seq, the 3’ end of the transcript is selected and amplified to yield one copy of cDNA from each transcript in a biological sample.

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.