Candida albicans is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of C. albicans depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections. Here, we present several in vitro biofilm protocols, including protocols that are optimized for high-throughput screening of mutant libraries and antifungal compounds. We also present protocols to examine specific stages of biofilm development and protocols to evaluate interspecies biofilms that C. albicans forms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.

Fungal pathogens like Candida albicans exhibit several survival mechanisms to evade attack by antifungals and colonize host tissues. Rta3, a member of the Rta1-like family of lipid-translocating exporters has a 7-transmembrane domain (7TMD) topology, similar to the G-protein-coupled receptors (GPCR) and is unique to the fungal kingdom. Our findings point towards a role for the plasma membrane localized Rta3 in providing tolerance to miltefosine, an analog of alkylphosphocholine, by maintaining mitochondrial energetics. Concurrent with miltefosine susceptibility, the rta3Δ/Δ strain displays increased inward translocation (flip) of fluorophore-labelled phosphatidylcholine (PC) across the plasma membrane attributed to enhanced PC-specific flippase activity. We also assign a novel role to Rta3 in the Bcr1-regulated pathway for in vivo biofilm development. Transcriptome analysis reveals that Rta3 regulates expression of Bcr1 target genes involved in cell surface properties, adhesion, and hyphal growth. We show that rta3Δ/Δ mutant is biofilm-defective in a rat venous catheter model of infection and that BCR1 overexpression rescues this defect, indicating that Bcr1 functions downstream of Rta3 to mediate biofilm formation in C. albicans. The identification of this novel Rta3-dependent regulatory network that governs biofilm formation and PC asymmetry across the plasma membrane will provide important insights into C. albicans pathogenesis.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER‐resident protein, inositol‐requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1‐mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence‐related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin‐induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1‐mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

Fungal infections are increasing in prevalence worldwide, especially in immunocompromised individuals. Given the emergence of drug-resistant fungi and the fact that there are only three major classes of antifungal drugs available to treat invasive fungal infections, there is a need to develop alternative therapeutic strategies effective against fungal infections. Candida albicans is a commensal of the human microbiota that is also one of the most common fungal pathogens isolated from clinical settings. C. albicans possesses several virulence traits that contribute to its pathogenicity, including the ability to form drug-resistant biofilms, which can make C. albicans infections particularly challenging to treat. Here, we explored red, green, and blue visible lights alone and in combination with common photosensitizing compounds for their efficacies at inhibiting and disrupting C. albicans biofilms. We found that blue light inhibited biofilm formation and disrupted mature biofilms on its own and that the addition of photosensitizing compounds improved its antibiofilm potential. Red and green lights, however, inhibited biofilm formation only in combination with photosensitizing compounds but had no effects on disrupting mature biofilms. Taken together, these results suggest that photodynamic therapy may be an effective non-drug treatment for fungal biofilm infections that is worthy of further exploration.

Fungal infections are increasing in prevalence worldwide. The paucity of available antifungal drug classes, combined with the increased occurrence of multidrug resistance in fungi, has led to new clinical challenges in the treatment of fungal infections. Candida auris is a recently emerged multidrug resistant human fungal pathogen that has become a worldwide public health threat. C. auris clinical isolates are often resistant to one or more antifungal drug classes, and thus, there is a high unmet medical need for the development of new therapeutic strategies effective against C. auris. Additionally, C. auris possesses several virulence traits, including the ability to form biofilms, further contributing to its drug resistance, and complicating the treatment of C. auris infections. Here we assessed red, green, and blue visible lights alone and in combination with photosensitizing compounds for their efficacies against C. auris biofilms. We found that (1) blue light inhibited and disrupted C. auris biofilms on its own and that the addition of photosensitizing compounds improved its antibiofilm potential; (2) red light inhibited and disrupted C. auris biofilms, but only in combination with photosensitizing compounds; and (3) green light inhibited C. auris biofilms in combination with photosensitizing compounds, but had no effects on disrupting C. auris biofilms. Taken together, our findings suggest that photodynamic therapy could be an effective non-drug therapeutic strategy against multidrug resistant C. auris biofilm infections.

CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an “AddTag” sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.

RNA‐sequencing (RNA‐seq) is a gold‐standard method to profile genome‐wide changes in gene expression. RNA‐seq uses high‐throughput sequencing technology to quantify the amount of RNA in a biological sample. With the increasing popularity of RNA‐seq, many variations on the protocol have been proposed to extract unique and relevant information from biological samples. 3′ Tag‐Seq (also called TagSeq, 3′ Tag‐RNA‐Seq, and Quant‐Seq 3′ mRNA‐Seq) is one RNA‐seq variation where the 3′ end of the transcript is selected and amplified to yield one copy of cDNA from each transcript in the biological sample. We present a simple, easy‐to‐use, and publicly available computational workflow to analyze 3′ Tag‐Seq data. The workflow begins by trimming sequence adapters from raw FASTQ files. The trimmed sequence reads are checked for quality using FastQC and aligned to the reference genome, and then read counts are obtained using STAR. Differential gene expression analysis is performed using DESeq2, based on differential analysis of gene count data. The outputs of this workflow are MA plots, tables of differentially expressed genes, and UpSet plots. This protocol is intended for users specifically interested in analyzing 3′ Tag‐Seq data, and thus normalizations based on transcript length are not performed within the workflow. Future updates to this workflow could include custom analyses based on the gene counts table as well as data visualization enhancements. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Running the 3′ Tag‐Seq workflow Support Protocol: Generating genome indices

A normal resident of healthy humans and warm-blooded animals, C. albicans is a commensal fungus that is also among the most common opportunistic pathogens of humans. C. albicans forms unique morphological structures called chlamydospores, which are large, spherical, thick-walled structures formed at the ends of hyphae that have unknown biological function. My goal is to discover the regulatory network controlling chlamydospore formation in C. albicans. By determining this network, we can gain insight into the biological roles of chlamydospores in the C. albicans lifestyle, better understand C. albicans morphological transitions, and determine the selective advantage (if any) provided by chlamydospores to this pathogenic fungus. To determine this regulatory network, I have screened a library of 211 C. albicans transcription factor (TF) homozygous deletion mutants to assay for their abilities to form chlamydospores under standard chlamydospore-inducing growth conditions. I have identified seven TF mutants that fail to produce any chlamydospores andthree TF mutants that produce high levels of chlamydospores relative to WT. To characterize the transcriptional changes occurring during chlamydospore formation, I have performed RNA sequencing (RNA-seq) on these identified regulator mutants to uncover the differentially regulated target genes of each chlamydospore regulator. I will use genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) on epitope-tagged versions of these regulators to determine which genes are directly under the control of each TF. RNA-seq coupled with ChIP-seq will allow me to determine the regulatory network controlling chlamydospore formation in C. albicans.