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Candida albicans and Streptococcus mutans are known to synergistically interact with each other in the oral cavity. For example, glucosyltransferase B (GtfB), secreted by S. mutans , can bind to the C. albicans cell surface, promoting dual-species biofilm formation. However, the fungal factors mediating interactions with S. mutans are unknown. The C. albicans adhesins Als1, Als3, and Hwp1 are key players in C. albicans single-species biofilm formation, but their roles, if any, in interacting with S. mutans have not been assessed. Here, we investigated the roles of the C. albicans cell wall adhesins Als1, Als3, and Hwp1 on forming dual-species biofilms with S. mutans . We assessed the abilities of the C. albicans wild-type als1 Δ/Δ, als3 Δ/Δ, als1 Δ/Δ/ als3 Δ/Δ, and hwp1 Δ/Δ strains to form dual-species biofilms with S. mutans by measuring optical density, metabolic activity, cell enumeration, biomass, thickness, and architecture of the biofilms. We observed that the C. albicans wild-type strain formed enhanced dual-species biofilms in the presence of S. mutans in these different biofilm assays, confirming that C. albicans and S. mutans synergistically interact in the context of biofilms. Our results reveal that C. albicans Als1 and Hwp1 are major players in interacting with S. mutans , since dual-species biofilm formation was not enhanced when the als1 Δ/Δ or hwp1 Δ/Δ strains were cultured with S. mutans in dual-species biofilms. Als3, however, does not seem to play a clear role in interacting with S. mutans in dual-species biofilm formation. Overall, our data suggest that the C. albicans adhesins Als1 and Hwp1 function to modulate interactions with S. mutans and could be potential targets for future therapeutics.

Catheter-associated urinary tract infections (CAUTIs) account for 40% of hospital-acquired infections (HAIs). As 20 to 50% of hospitalized patients receive catheters, CAUTIs are one of the most common HAIs, resulting in increased morbidity, mortality, and health care costs. Candida albicans is the second most common CAUTI uropathogen, yet relative to its bacterial counterparts, little is known about how fungal CAUTIs are established. Here, we show that the catheterized bladder environment induces Efg1- and fibrinogen (Fg)-dependent biofilm formation that results in CAUTI. In addition, we identify the adhesin Als1 as the critical fungal factor for C. albicans Fg-urine biofilm formation. Furthermore, we show that in the catheterized bladder, a dynamic and open system, both filamentation and attachment are required, but each by themselves are not sufficient for infection. Our study unveils the mechanisms required for fungal CAUTI establishment, which may aid in the development of future therapies to prevent these infections.

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., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Infections caused by the Candida species of human fungal pathogens are a significant medical problem because they can disseminate to nearly every organ of the body. In addition, there are only a few classes of antifungal drugs available to treat patients with invasive fungal infections. Candida infections that are associated with biofilms can withstand much higher concentrations of antifungal drugs compared with infections caused by planktonic cells, thus making biofilm infections particularly challenging to treat. Candida albicans is among the most prevalent fungal species of the human microbiota, asymptomatically colonizing several niches of the body, including the gastrointestinal tract, genitourinary tract, mouth, and skin. Immunocompromised health conditions, dysbiosis of the microbiota, or environmental changes, however, can lead to C. albicans overgrowth, causing infections that range from superficial mucosal infections to severe hematogenously disseminated infections. Here, we review the current knowledge of antifungal drug-resistance mechanisms occurring in Candida biofilms., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This protocol is focused on using the recently established planarian infection model system to study host-pathogen interactions during fungal infection. Here, we describe in detail the infection of the planarian Schmidtea mediterranea with the human fungal pathogen Candida albicans. This simple and reproducible model system allows for rapid visualization of tissue damage throughout different infection timepoints. We note that this model system has been optimized for use with C. albicans, but should also be applicable for use with other pathogens of interest., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide. It can cause life-threatening disseminated infections in humans, with mortality rates upwards of 50%. The molecular mechanisms underlying its multidrug resistance and pathogenic properties are largely unknown. Few methods exist for genome editing in C. auris, all of which rely on selectable markers that limit the number of modifications that can be made. Here, we present a markerless CRISPR/Cas9-mediated genome editing system in C. auris. Using this system, we successfully deleted genes of interest and subsequently reconstituted them at their native loci in isolates across all five C. auris clades. This system also enabled us to introduce precision genome edits to create translational fusions and single point mutations. Using Cas5 as a test case for this system, we discovered a conserved role for Cas5 in the caspofungin response between Candida albicans and C. auris. Overall, the development of a system for precise and facile genome editing in C. auris that can allow edits to be made in a high-throughput manner is a major step forward in improving our understanding of this important human fungal pathogen. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris. Here, we present a markerless genome editing system for C. auris that relies on CRISPR/Cas9 technology and works to modify the genomes of all known C. auris clades. Using this system, we discovered a conserved role for Cas5 in the caspofungin response between C. albicans and C. auris. Overall, the development of a system for facile genome editing in C. auris is a major step forward in improving our understanding of this important human fungal pathogen.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

A striking feature of pathogenic Candida species is morphological plasticity that facilitates environmental adaptation and host infection. Candida auris is an emerging multidrug-resistant fungal pathogen first described in Japan in 2009. In this study, we demonstrate that clinical isolates of C. auris have multiple colony and cellular morphologies including the yeast, filamentous, aggregated, and elongated forms. This phenotypic diversity has been observed in eight clinical isolates of C. auris representing four major genetic clades, suggesting that it could be a general characteristic. We further demonstrate that different cell types of C. auris exhibit distinct antifungal resistance and virulence properties in a Galleria mellonella infection model. Our findings imply that morphological diversity is an important biological feature of C. auris and could be a contributor to its emergence and rapid prevalence worldwide., Lay Summary: Candida auris is an emerging multidrug-resistant fungal pathogen. Morphological analyses indicate that filamentation is a general feature of clinical isolates of C. auris. This ability is associated with antifungal resistance and virulence., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The human fungal pathogen Candida albicans can form biofilms on biotic and abiotic surfaces, which are inherently resistant to antifungal drugs. We screened the Chembridge Small Molecule Diversity library containing 30,000 "drug-like" small molecules and identified 45 compounds that inhibited biofilm formation. These 45 compounds were then tested for their abilities to disrupt mature biofilms and for combinatorial interactions with fluconazole, amphotericin B, and caspofungin, the three antifungal drugs most commonly prescribed to treat Candida infections. In the end, we identified one compound that moderately disrupted biofilm formation on its own and four compounds that moderately inhibited biofilm formation and/or moderately disrupted mature biofilms only in combination with either caspofungin or fluconazole. No combinatorial interactions were observed between the compounds and amphotericin B. As members of a diversity library, the identified compounds contain "drug-like" chemical backbones, thus even seemingly "weak hits" could represent promising chemical starting points for the development and the optimization of new classes of therapeutics designed to target Candida biofilms.

Candida albicans is an opportunistic fungal pathogen of humans known for its ability to cause a wide range of infections. One major virulence factor of C. albicans is its ability to form hyphae that can invade host tissues and cause disseminated infections. Here, we introduce a method based on atomic force microscopy to investigate C. albicans hyphae in situ on silicone elastomer substrates, focusing on the effects of temperature and antifungal drugs. Hyphal growth rates differ significantly for measurements performed at different physiologically relevant temperatures. Furthermore, it is found that fluconazole is more effective than caspofungin in suppressing hyphal growth. We also investigate the effects of antifungal drugs on the mechanical properties of hyphal cells. An increase in Young's modulus and a decrease in adhesion force are observed in hyphal cells subjected to caspofungin treatment. Young's moduli are not significantly affected following treatment with fluconazole; the adhesion force, however, increases. Overall, our results provide a direct means of observing the effects of environmental factors and antifungal drugs on C. albicans hyphal growth and mechanics with high spatial resolution. IMPORTANCE Candida albicans is one of the most common pathogens of humans. One important virulence factor of C. albicans is its ability to form elongated hyphae that can invade host tissues and cause disseminated infections. Here, we show the effect of different physiologically relevant temperatures and common antifungal drugs on the growth and mechanical properties of C. albicans hyphae using atomic force microscopy. We demonstrate that minor temperature fluctuations within the normal range can have profound effects on hyphal cell growth and that different antifungal drugs impact hyphal cell stiffness and adhesion in different ways., (Copyright © 2020 Çolak et al.)

The sensing and efficient utilization of environmental nutrients are critical for the survival of microorganisms in environments where nutrients are limited, such as within mammalian hosts. Candida albicans is a common member of the human microbiota as well as an opportunistic fungal pathogen. The amide derivative sugar N-acetlyglucosamine (GlcNAc) is an important signaling molecule for C. albicans that could be a major nutrient source for this fungus in host settings. In this article, we review progress made over the past two decades on GlcNAc utilization, sensing, and functions in C. albicans and its related fungal species. GlcNAc sensing and catabolic pathways have been intensively studied in C. albicans . The C. albicans protein Ngt1 represents the first identified GlcNAc-specific transporter in eukaryotic organisms. In C. albicans , GlcNAc not only induces morphological transitions including the yeast to hyphal transition and the white to opaque phenotypic switch, but it also promotes fungal cell death. The Ras-cAMP/PKA signaling pathway plays critical roles in regulating these processes. Given the importance of GlcNAc sensing and utilization in C. albicans , targeting GlcNAc associated pathways and key pathway components could be promising in the development of new antifungal strategies.

Candida albicans is a major human fungal pathogen, capable of switching among a range of morphological types, such as the yeast form, including white and opaque cell types and the GUT (gastrointestinally induced transition) cell type, the filamentous form, including hyphal and pseudohyphal cell types, and chlamydospores. This ability is associated with its commensal and pathogenic life styles. In response to pheromone, C. albicans cells are able to form long mating projections resembling filaments. This filamentous morphology is required for efficient sexual mating. In the current study, we report the genetic regulatory mechanisms controlling the development of mating projections in C. albicans . Ectopic expression of MTL α1 in " a " cells induces the secretion of α-pheromone and promotes the development of mating projections. Using this inducible system, we reveal that members of the pheromone-sensing pathway (including the pheromone receptor), the Ste11-Hst7-Cek1/2 mediated MAPK signalling cascade, and the RAM pathway are essential for the development of mating projections. However, the cAMP/PKA signalling pathway and a number of key regulators of filamentous growth such as Hgc1, Efg1, Flo8, Tec1, Ume6, and Rfg1 are not required for mating projection formation. Therefore, despite the phenotypic similarities between filaments and mating projections in C. albicans , distinct mechanisms are involved in the regulation of these two morphologies.

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., (© 2018 John Wiley & Sons, Inc.)

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these experiments.