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Candida auris has become a serious threat to public health. The mechanisms of how this fungal pathogen adapts to the mammalian host are poorly understood. Here we report the rapid evolution of an adaptive C. auris multicellular aggregative morphology in the murine host during systemic infection. C. auris aggregative cells accumulate in the brain and exhibit obvious advantages over the single-celled yeast-form cells during systemic infection. Genetic mutations, specifically de novo point mutations in genes associated with cell division or budding processes, underlie the rapid evolution of this aggregative phenotype. Most mutated C. auris genes are associated with the regulation of cell wall integrity, cytokinesis, cytoskeletal properties, and cellular polarization. Moreover, the multicellular aggregates are notably more recalcitrant to the host antimicrobial peptides LL-37 and PACAP relative to the single-celled yeast-form cells. Overall, to survive in the host, C. auris can rapidly evolve a multicellular aggregative morphology via genetic mutations. Bing et al. report that Candida auris undergoes rapid evolution via de novo genetic mutations and forms multicellular aggregates that exhibit a survival advantage over the single-celled yeast-form phenotype during host infection. [ABSTRACT FROM AUTHOR]

Genetic regulatory networks (GRNs) regulate the flow of genetic information from the genome to expressed messenger RNAs (mRNAs) and thus are critical to controlling the phenotypic characteristics of cells. Numerous methods exist for profiling mRNA transcript levels and identifying protein-DNA binding interactions at the genome-wide scale. These enable researchers to determine the structure and output of transcriptional regulatory networks, but uncovering the complete structure and regulatory logic of GRNs remains a challenge. The field of GRN inference aims to meet this challenge using computational modeling to derive the structure and logic of GRNs from experimental data and to encode this knowledge in Boolean networks, Bayesian networks, ordinary differential equation (ODE) models, or other modeling frameworks. However, most existing models do not incorporate dynamic transcriptional data since it has historically been less widely available in comparison to "static" transcriptional data. We report the development of an evolutionary algorithm-based ODE modeling approach (named EA) that integrates kinetic transcription data and the theory of attractor matching to infer GRN architecture and regulatory logic. Our method outperformed six leading GRN inference methods, none of which incorporate kinetic transcriptional data, in predicting regulatory connections among TFs when applied to a small-scale engineered synthetic GRN in Saccharomyces cerevisiae. Moreover, we demonstrate the potential of our method to predict unknown transcriptional profiles that would be produced upon genetic perturbation of the GRN governing a two-state cellular phenotypic switch in Candida albicans. We established an iterative refinement strategy to facilitate candidate selection for experimentation; the experimental results in turn provide validation or improvement for the model. In this way, our GRN inference approach can expedite the development of a sophisticated mathematical model that can accurately describe the structure and dynamics of the in vivo GRN. Author summary: The establishment of distinct transcriptional programs, where specific sets of genes are activated or repressed, is fundamental to all forms of life. Sequence-specific DNA-binding proteins, often referred to as regulatory transcription factors, form interconnected gene regulatory networks (GRNs) which underlie the establishment and maintenance of specific transcriptional programs. Since their discovery, many modeling approaches have sought to understand the structure and regulatory behaviors of these GRNs. The field of GRN inference uses experimental measurements of transcript abundance to predict how regulatory transcription factors interact with their downstream target genes to establish specific transcriptional programs. However, most prior approaches have been limited by the exclusive use of "static" or steady-state measurements. We have developed a unique approach which incorporates dynamic transcriptional data into a sophisticated ordinary differential equation model to infer GRN structures that give rise to distinct transcriptional programs. Our model not only outperforms six other leading models, it also is capable of accurately predicting how changes in GRN structure will impact the resulting transcriptional programs. These notable features of our model, in conjunction with experimental validation of our predictions in real-world scenarios, contribute to an advancement in the field of gene regulatory network inference. [ABSTRACT FROM AUTHOR]

Sexual reproduction is prevalent in eukaryotic organisms and plays a critical role in the evolution of new traits and in the generation of genetic diversity. Environmental factors often have a direct impact on the occurrence and frequency of sexual reproduction in fungi. The regulatory effects of atmospheric relative humidity (RH) on sexual reproduction and pathogenesis in plant fungal pathogens and in soil fungi have been extensively investigated. However, the knowledge of how RH regulates the lifecycles of human fungal pathogens is limited. In this study, we report that low atmospheric RH promotes the development of mating projections and same-sex (homothallic) mating in the human fungal pathogen Candida albicans. Low RH causes water loss in C. albicans cells, which results in osmotic stress and the generation of intracellular reactive oxygen species (ROS) and trehalose. The water transporting aquaporin Aqy1, and the G-protein coupled receptor Gpr1 function as cell surface sensors of changes in atmospheric humidity. Perturbation of the trehalose metabolic pathway by inactivating trehalose synthase or trehalase promotes same-sex mating in C. albicans by increasing osmotic or ROS stresses, respectively. Intracellular trehalose and ROS signal the Hog1-osmotic and Hsf1-Hsp90 signaling pathways to regulate the mating response. We, therefore, propose that the cell surface sensors Aqy1 and Gpr1, intracellular trehalose and ROS, and the Hog1-osmotic and Hsf1-Hsp90 signaling pathways function coordinately to regulate sexual mating in response to low atmospheric RH conditions in C. albicans. [ABSTRACT FROM AUTHOR]

Candida vulturna belongs to the Candida haemulonii species complex and is phylogenetically related to C. auris. We report a C. vulturna outbreak among persons in Shanxi Province, China, during 2019-2022. Isolates were resistant to multiple antifungal drugs and exhibited enhanced adhesion and biofilm formation properties. [ABSTRACT FROM AUTHOR]

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 als1Δ/Δ, als3Δ/Δ, als1Δ/Δ/als3Δ/Δ, and hwp1Δ/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 als1Δ/Δ or hwp1Δ/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. [ABSTRACT FROM AUTHOR]

Candida auris is an emerging multidrug-resistant fungal pathogen and a new global threat to human health. A unique morphological feature of this fungus is its multicellular aggregating phenotype, which has been thought to be associated with defects in cell division. In this study, we report a new aggregating form of two clinical C. auris isolates with increased biofilm forming capacity due to enhanced adherence of adjacent cells and surfaces. Unlike the previously reported aggregating morphology, this new aggregating multicellular form of C. auris can become unicellular after treatment with proteinase K or trypsin. Genomic analysis demonstrated that amplification of the subtelomeric adhesin gene ALS4 is the reason behind the strain's enhanced adherence and biofilm forming capacities. Many clinical isolates of C. auris have variable copy numbers of ALS4, suggesting that this subtelomeric region exhibits instability. Global transcriptional profiling and quantitative real-time PCR assays indicated that genomic amplification of ALS4 results in a dramatic increase in overall levels of transcription. Compared to the previously characterized nonaggregative/yeast-form and aggregative-form strains of C. auris, this new Als4-mediated aggregative-form strain of C. auris displays several unique characteristics in terms of its biofilm formation, surface colonization, and virulence. Author summary: The emerging fungal pathogen Candida auris has rapidly spread worldwide in the past several years. This fungus is considered to be a "superbug" pathogen due to its multidrug-resistant properties and high rates of transmission in clinical settings. Unlike other pathogenic species in the Candida genus, many clinical isolates of C. auris can form a unique morphology, namely a multicellular aggregating phenotype, that is thought to be caused by defects in cell division. In this study, we report a new aggregating form observed from two clinical isolates of C. auris with increased adhesion and biofilm forming capacity. Genomic analysis demonstrated that amplification of the subtelomeric adhesin gene ALS4 is the reason behind the strain's enhanced adherence and biofilm forming capacities. Compared to the previously characterized nonaggregative/yeast-form and aggregative-form strains, this new Als4-mediated aggregative-form strain of C. auris displays many unique characteristics in terms of its biofilm formation, surface colonization, and virulence. [ABSTRACT FROM AUTHOR]

Candida species, the most common fungal pathogens affecting humans, cause not only superficial infections but also life-threatening invasive infections, particularly in immunocompromised individuals. Although Candida albicans remains the most frequent cause of candidiasis, infections caused by nonalbicans Candida species have been increasingly reported in clinical settings over the past two decades. Recently, species of the Metschnikowiaceae clade including the "superbug" Candida auris and other members of the Candida haemulonii species complex have attracted substantial attention for their multidrug resistance and high rates of transmission in clinical settings. In this review, we summarize the epidemiology, biology, virulence, and drug resistance of the C. haemulonii species complex and discuss potential reasons for the recent increase in the prevalence of infections caused by non-albicans species in clinical settings. [ABSTRACT FROM AUTHOR]

The recently emerged fungal pathogen Candida auris often displays resistance to one or more antifungal drugs. Its infections have been identified in at least 40 countries on six continents to date. Here we report a case of C. auris candidemia in a patient in Xiamen, a city in south China. We also review currently reported cases of C. auris infection in China and compare the genetic and biological features of C. auris strains isolated from this country. Our phylogenetic analysis indicates that there are at least two C. auris genetic clades present in China (the South African clade and the south Asian clade) that display opposite mating type loci (one is MTLa and the other is MTLα). We also found that there are several distinct features among the clinical isolates studied, including the expression of virulence factors, antifungal susceptibilities, and cellular morphologies, and that these features could be associated with the mating-type of the isolate. For example, C. auris MTLa isolates generally secreted higher levels of secreted aspartyl proteases (Saps) at ambient environmental temperatures. Taken together, this study demonstrates that C. auris clinical isolates from China exhibit diversity in both biological and genetic features. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Author summary: Candida albicans is a pathogenic organism that commonly causes infection in humans, particularly in immunocompromised individuals, and patients in hospitals. A key mechanism mediating its infectiousness is a morphological change from single yeast cells into branching cell collectives called hyphae. C. albicans cells undergo this transition in response to multiple environmental signals, including pH, temperature, serum levels, or other molecules. Understanding how the cells process these environmental signals is critical to understanding how C. albicans adapts to thrive in human hosts. Here, we built and analyzed a mathematical model of the C. albicans yeast to hyphal transition, integrating multiple environmental factors and regulatory mechanisms. 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. We probed this model to identify interventional control strategies that can block the transition. We experimentally validate two of these control predictions: deletion of the transcription factor UME6, and deletion of the histone deacetylase HDA1. This model can be used to identify alternative hypotheses, enabling progress toward a systems biology understanding of C. albicans morphological changes. [ABSTRACT FROM AUTHOR]

Candida albicans is one of the most common fungal pathogens of humans. Prior work introduced the planarian Schmidtea mediterranea as a new model system to study the host response to fungal infection at the organismal level. In the current study, we analyzed host–pathogen changes that occurred in situ during early infection with C. albicans. We found that the transcription factor Bcr1 and its downstream adhesin Als3 are required for C. albicans to adhere to and colonize the planarian epithelial surface, and that adherence of C. albicans triggers a multi-system host response that is mediated by the Dectin signaling pathway. This infection response is characterized by two peaks of stem cell divisions and transcriptional changes in differentiated tissues including the nervous and the excretory systems. This response bears some resemblance to a wound-like response to physical injury; however, it takes place without visible tissue damage and it engages a distinct set of progenitor cells. Overall, we identified two C. albicans proteins that mediate epithelial infection of planarians and a comprehensive host response facilitated by diverse tissues to effectively clear the infection. [ABSTRACT FROM AUTHOR]

Candida albicans, a diploid polymorphic fungus, has evolved a unique heritable epigenetic program that enables reversible phenotypic switching between two cell types, referred to as “white” and “opaque”. These cell types are established and maintained by distinct transcriptional programs that lead to differences in metabolic preferences, mating competencies, cellular morphologies, responses to environmental signals, interactions with the host innate immune system, and expression of approximately 20% of genes in the genome. Transcription factors (defined as sequence specific DNA-binding proteins) that regulate the establishment and heritable maintenance of the white and opaque cell types have been a primary focus of investigation in the field; however, other factors that impact chromatin accessibility, such as histone modifying enzymes, chromatin remodelers, and histone chaperone complexes, also modulate the dynamics of the white-opaque switch and have been much less studied to date. Overall, the white-opaque switch represents an attractive and relatively “simple” model system for understanding the logic and regulatory mechanisms by which heritable cell fate decisions are determined in higher eukaryotes. Here we review recent discoveries on the roles of chromatin accessibility in regulating the C. albicans white-opaque phenotypic switch. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Candida albicans is a commensal member of the human microbiota that colonizes multiple niches in the body including the skin, oral cavity, and gastrointestinal and genitourinary tracts of healthy individuals. It is also the most common human fungal pathogen isolated from patients in clinical settings. C. albicans can cause a number of superficial and invasive infections, especially in immunocompromised individuals. The ability of C. albicans to succeed as both a commensal and a pathogen, and to thrive in a wide range of environmental niches within the host, requires sophisticated transcriptional regulatory programs that can integrate and respond to host specific environmental signals. Identifying and characterizing the transcriptional regulatory networks that control important developmental processes in C. albicans will shed new light on the strategies used by C. albicans to colonize and infect its host. Here, we discuss the transcriptional regulatory circuits controlling three major developmental processes in C. albicans : biofilm formation, the white-opaque phenotypic switch, and the commensal-pathogen transition. Each of these three circuits are tightly knit and, through our analyses, we show that they are integrated together by extensive regulatory crosstalk between the core regulators that comprise each circuit. [ABSTRACT FROM AUTHOR]

The human fungal pathogen Candida albicans maintains pathogenic and commensal states primarily through cell wall functions. The echinocandin antifungal drug caspofungin inhibits cell wall synthesis and is widely used in treating disseminated candidiasis. Signaling pathways are critical in coordinating the adaptive response to cell wall damage (CWD). C. albicans executes a robust transcriptional program following caspofungin-induced CWD. A comprehensive analysis of signaling pathways at the transcriptional level facilitates the identification of prospective genes for functional characterization and propels the development of novel antifungal interventions. This review article focuses on the molecular functions and signaling crosstalk of the C. albicans transcription factors Sko1, Rlm1, and Cas5 in caspofungin-induced CWD signaling. [ABSTRACT FROM AUTHOR]

First described in 2009 in Japan, the emerging multidrug-resistant fungal pathogen Candida auris is becoming a worldwide public health threat that has been attracting considerable attention due to its rapid and widespread emergence over the past decade. The reasons behind the recent emergence of this fungus remain a mystery to date. Genetic analyses indicate that this fungal pathogen emerged simultaneously in several different continents, where 5 genetically distinct clades of C. auris were isolated from distinct geographical locations. Although C. auris belongs to the CTG clade (its constituent species translate the CTG codon as serine instead of leucine, as in the standard code), C. auris is a haploid fungal species that is more closely related to the haploid and often multidrug-resistant species Candida haemulonii and Candida lusitaniae and is distantly related to the diploid and clinically common fungal pathogens Candida albicans and Candida tropicalis. Infections and outbreaks caused by C. auris in hospitals settings have been rising over the past several years. Difficulty in its identification, multidrug resistance properties, evolution of virulence factors, associated high mortality rates in patients, and long-term survival on surfaces in the environment make C. auris particularly problematic in clinical settings. Here, we review progress made over the past decade on the biological and clinical aspects of C. auris. Future efforts should be directed toward understanding the mechanistic details of its biology, epidemiology, antifungal resistance, and pathogenesis with a goal of developing novel tools and methods for the prevention, diagnosis, and treatment of C. auris infections. [ABSTRACT FROM AUTHOR]

Mucus is an important host innate defense factor that lines most epithelial cell layers of the body and provides crucial physical and biological protection against pathogenic microorganisms. Mucins are the main glycoproteins of mucus that are responsible for interacting with microorganisms and are critical for the antimicrobial properties of mucus. The mechanisms by which microorganisms interact with mucins are poorly understood, especially in terms of fungi, and these interactions are continually evolving. Work in bacterial pathogens has shown that mucins inhibit bacterial virulence traits, including quorum sensing, toxin secretion and biofilm formation. Among the fungal clade, the common opportunistic human fungal pathogen and commensal Candida albicans engages in constant battle with the host innate immune system. This battle creates strong selective pressures for C. albicans to evolve in response to the host. Recent work in C. albicans found that mucins inhibit specific virulence traits, such as surface adherence, filamentation, biofilm formation and the production of secreted proteases. Here we review the current knowledge of microbial interactions with mucins, with a special emphasis on the interactions between C. albicans and mucins. [ABSTRACT FROM AUTHOR]

he 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. [ABSTRACT FROM AUTHOR]

The human fungal pathogen Candida albicans is constantly exposed to environmental challenges impacting the cell wall. Signaling pathways coordinate stress adaptation and are essential for commensalism and virulence. The transcription factors Sko1, Cas5, and Rlm1 control the response to cell wall stress caused by the antifungal drug caspofungin. Here, we expand the Sko1 and Rlm1 transcriptional circuit and demonstrate that Rlm1 activates Sko1 cell wall stress signaling. Caspofungin-induced transcription of SKO1 and several Sko1-dependent cell wall integrity genes are attenuated in an rlm1Δ/Δ mutant strain when compared to the treated wild-type strain but not in a cas5Δ/Δ mutant strain. Genome-wide chromatin immunoprecipitation (ChIP-seq) results revealed numerous Sko1 and Rlm1 directly bound target genes in the presence of caspofungin that were undetected in previous gene expression studies. Notable targets include genes involved in cell wall integrity, osmolarity, and cellular aggregation, as well as several uncharacterized genes. Interestingly, we found that Rlm1 does not bind to the upstream intergenic region of SKO1 in the presence of caspofungin, indicating that Rlm1 indirectly controls caspofungin-induced SKO1 transcription. In addition, we discovered that caspofungin-induced SKO1 transcription occurs through self-activation. Based on our ChIP-seq data, we also discovered an Rlm1 consensus motif unique to C. albicans. For Sko1, we found a consensus motif similar to the known Sko1 motif for Saccharomyces cerevisiae. Growth assays showed that SKO1 overexpression suppressed caspofungin hypersensitivity in an rlm1Δ/Δ mutant strain. In addition, overexpression of the glycerol phosphatase, RHR2, suppressed caspofungin hypersensitivity specifically in a sko1Δ/Δ mutant strain. Our findings link the Sko1 and Rlm1 signaling pathways, identify new biological roles for Sko1 and Rlm1, and highlight the complex dynamics underlying cell wall signaling. Author summary: Candida albicans is the most common human fungal pathogen isolated in clinical settings. The echinocandin drug caspofungin is used to treat invasive candidiasis; however, the emergence of increasing echinocandin resistance underscores the need for new antifungal strategies. Elucidating the signaling mechanisms that govern caspofungin-induced tolerance has the potential to identify candidate proteins that could serve as novel therapeutic targets. Here, we expand the Rlm1 and Sko1 cell wall transcriptional network and find that Rlm1 indirectly regulates Sko1 signaling. Furthermore, we identify Sko1- and Rlm1-specific biological roles in caspofungin adaptation, such as osmoregulation and secretion. Lastly, we discover a protective role for glycerol in caspofungin tolerance. Overall, these findings provide mechanistic insight into the genetic and cellular bases underlying cell wall signaling in C. albicans. [ABSTRACT FROM AUTHOR]

Biofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections. [ABSTRACT FROM AUTHOR]

Biofilms, structured and densely packed communities of microbial cells attached to surfaces, are considered to be 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 that asymptomatically colonizes healthy individuals. However, C. albicans can also cause severe and life-threatening infections when host conditions permit (e.g., through alterations in the host immune system, pH, and resident microbiota). Like many other pathogens, this ability to cause infections depends, in part, on the ability to form biofilms. Once formed, C. albicans biofilms are often resistant to antifungal agents and the host immune response, and can act as reservoirs to maintain persistent infections as well as to seed 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 are capable of forming 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, however, has uncovered novel mechanisms through which biofilms are generated by C. albicans cells that are homozygous or hemizygous (a/a, a/D, α/α, or α/D) at the MTL locus. In these studies, the addition of pheromones of the opposite mating type can induce the formation of specialized "sexual" biofilms, either through the addition of synthetic peptide pheromones to the culture, or in response to co-culturing of cells of the opposite mating types. Although sexual biofilms are generally less robust than conventional biofilms, they could serve as a protective niche to support genetic exchange between mating-competent cells, and thus may represent an adaptive mechanism to increase population diversity in dynamic environments. Although conventional and sexual biofilms appear functionally distinct, both types of biofilms are structurally similar, containing yeast, pseudohyphal, and hyphal cells surrounded by an extracellular matrix. Despite their structural similarities, conventional and sexual biofilms appear to be governed by distinct transcriptional networks and signaling pathways, suggesting that they may be adapted for, and responsive to, distinct environmental conditions. Here we review sexual biofilms and compare and contrast them to conventional biofilms of C. albicans. [ABSTRACT FROM AUTHOR]

Microbial communities control numerous biogeochemical processes critical for ecosystem function and health. Most analyses of coastal microbial communities focus on the characterization of bacteria present in either sediment or seawater, with fewer studies characterizing both sediment and seawater together at a given site, and even fewer studies including information about non-bacterial microbial communities. As a result, knowledge about the ecological patterns of microbial biodiversity across domains and habitats in coastal communities is limited–despite the fact that archaea, bacteria, and microbial eukaryotes are present and known to interact in coastal habitats. To better understand microbial biodiversity patterns in coastal ecosystems, we characterized sediment and seawater microbial communities for three sites along the coastline of Puerto Nuevo, Baja California, Mexico using both 16S and 18S rRNA gene amplicon sequencing. We found that sediment hosted approximately 500-fold more operational taxonomic units (OTUs) for bacteria, archaea, and microbial eukaryotes than seawater (p < 0.001). Distinct phyla were found in sediment versus seawater samples. Of the top ten most abundant classes, Cytophagia (bacterial) and Chromadorea (eukaryal) were specific to the sediment environment, whereas Cyanobacteria and Bacteroidia (bacterial) and Chlorophyceae (eukaryal) were specific to the seawater environment. A total of 47 unique genera were observed to comprise the core taxa community across environment types and sites. No archaeal taxa were observed as part of either the abundant or core taxa. No significant differences were observed for sediment community composition across domains or between sites. For seawater, the bacterial and archaeal community composition was statistically different for the Major Outlet site (p < 0.05), the site closest to a residential area, and the eukaryal community composition was statistically different between all sites (p < 0.05). Our findings highlight the distinct patterns and spatial heterogeneity in microbial communities of a coastal region in Baja California, Mexico. [ABSTRACT FROM AUTHOR]

Fungal pathogens such as Candida albicans exhibit several survival mechanisms to evade attack by antifungals and colonise host tissues. Rta3, a member of the Rta1-like family of lipid-translocating exporters has a 7-transmembrane domain topology, similar to the G-protein-coupled receptors and is unique to the fungal kingdom. Our findings point towards a role for the plasma membrane localised Rta3 in providing tolerance to miltefosine, an analogue 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. [ABSTRACT FROM AUTHOR]

Candida albicansandStreptococcus oralisare ubiquitous oral commensal organisms. Under host-permissive conditions these organisms can form hypervirulent mucosal biofilms.C. albicansbiofilm formation is controlled by 6 master transcriptional regulators: Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1. The objective of this work was to test whether any of these regulators play a role in cross-kingdom interactions betweenC. albicansandS. oralisin oral mucosal biofilms, and identify downstream target gene(s) that promote these interactions. Organotypic mucosal constructs and a mouse model of oropharyngeal infection were used to analyze mucosal biofilm growth and fungal gene expression. By screening 6C. albicanstranscription regulator reporter strains we discovered thatEFG1was strongly activated by interaction withS. oralisin late biofilm growth stages.EFG1gene expression was increased in polymicrobial biofilms on abiotic surfaces, mucosal constructs and tongue tissues of mice infected with both organisms.EFG1was required for robustCandida-streptococcal biofilm growth in organotypic constructs and mouse oral tissues.S. oralisstimulatedC. albicans ALS1gene expression in anEFG1-dependent manner, and Als1 was identified as a downstream effector of the Efg1 pathway which promotedC. albicans-S. oraliscoaggregation interactions in mixed biofilms. We conclude thatS. oralisinduces an increase inEFG1expression inC. albicansin late biofilm stages. This in turn increases expression ofALS1, which promotes coaggregation interactions and mucosal biofilm growth. Our work provides novel insights onC. albicansgenes which play a role in cross-kingdom interactions withS. oralisin mucosal biofilms. [ABSTRACT FROM AUTHOR]

Morphological transitions and metabolic regulation are critical for the human fungal pathogen Candida albicans to adapt to the changing host environment. In this study, we generated a library of central metabolic pathway mutants in the tricarboxylic acid (TCA) cycle, and investigated the functional consequences of these gene deletions on C. albicans biology. Inactivation of the TCA cycle impairs the ability of C. albicans to utilize non-fermentable carbon sources and dramatically attenuates cell growth rates under several culture conditions. By integrating the Ras1-cAMP signaling pathway and the heat shock factor-type transcription regulator Sfl2, we found that the TCA cycle plays fundamental roles in the regulation of CO2 sensing and hyphal development. The TCA cycle and cAMP signaling pathways coordinately regulate hyphal growth through the molecular linkers ATP and CO2. Inactivation of the TCA cycle leads to lowered intracellular ATP and cAMP levels and thus affects the activation of the Ras1-regulated cAMP signaling pathway. In turn, the Ras1-cAMP signaling pathway controls the TCA cycle through both Efg1- and Sfl2-mediated transcriptional regulation in response to elevated CO2 levels. The protein kinase A (PKA) catalytic subunit Tpk1, but not Tpk2, may play a major role in this regulation. Sfl2 specifically binds to several TCA cycle and hypha-associated genes under high CO2 conditions. Global transcriptional profiling experiments indicate that Sfl2 is indeed required for the gene expression changes occurring in response to these elevated CO2 levels. Our study reveals the regulatory role of the TCA cycle in CO2 sensing and hyphal development and establishes a novel link between the TCA cycle and Ras1-cAMP signaling pathways. [ABSTRACT FROM AUTHOR]

Candida albicans is the most common cause of life-threatening fungal infections in humans, especially in immunocompromised individuals. Crucial to its success as an opportunistic pathogen is the considerable dynamismof its genome,which readily undergoes genetic changes generating new phenotypes and shaping the evolution of new strains. Candida africana is an intriguing C. albicans biovariant strain that exhibits remarkable genetic and phenotypic differences when compared with standard C. albicans isolates. Candida africana is well-known for its low degree of virulence comparedwith C. albicans and for its inability to produce chlamydospores that C. albicans, characteristically, produces under certain environmental conditions. Chlamydospores are large, spherical structures,whose biological function is still unknown. For this reason, we have sequenced, assembled, and annotated the whole transcriptomes obtained from an efficient C. albicans chlamydospore-producing clinical strain (GE1), compared with the natural chlamydospore-negative C. africana clinical strain (CBS 11016). The transcriptomes of both C. albicans (GE1) and C. africana (CBS 11016) clinical strains, grown under chlamydospore-inducing conditions, were sequenced and assembled into 7,442 (GE1 strain) and 8,370 (CBS 11016 strain) high quality transcripts, respectively. The release of the first assembly of the C. africana transcriptome will allow future comparative studies to better understand the biology and evolution of this important human fungal pathogen. [ABSTRACT FROM AUTHOR]

Microorganisms rarely exist as single species in natural environments. The opportunistic fungal pathogen Candida albicans and lactic acid bacteria (LAB) are common members of the microbiota of several human niches such as the mouth, gut and vagina. Lactic acid bacteria are known to suppress filamentation, a key virulence feature of C. albicans, through the production of lactic acid and other metabolites. Here we report that C. albicans cells switch between two heritable cell types, white and opaque, to undergo filamentation to adapt to diversified environments. We show that acidic pH conditions caused by LAB and low temperatures support opaque cell filamentation, while neutral pH conditions and high temperatures promote white cell filamentation. The cAMP signalling pathway and the Rfg1 transcription factor play major roles in regulating the responses to these conditions. This cell type-specific response of C. albicans to different environmental conditions reflects its elaborate regulatory control of phenotypic plasticity. [ABSTRACT FROM AUTHOR]

We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. © 2016 IUBMB Life, 68(8):621-628, 2016 [ABSTRACT FROM AUTHOR]

Background: In this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovariant of Candida albicans that is naturally unable to assimilate the GlcNAc. Results: DNA sequence analysis of these genes revealed a number of characteristic nucleotide substitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic pathway. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24-48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media. Conclusions: Our results show that C. africana is a natural HXK1 mutant, displaying a number of phenotypic characteristics distinct from typical C. albicans isolates. [ABSTRACT FROM AUTHOR]

In humans, microbial cells (including bacteria, archaea, and fungi) greatly outnumber host cells. Candida albicans is the most prevalent fungal species of the human microbiota; this species asymptomatically colonizes many areas of the body, particularly the gastrointestinal and genitourinary tracts of healthy individuals. Alterations in host immunity, stress, resident microbiota, and other factors can lead to C. albicans overgrowth, causing a wide range of infections, from superficial mucosal to hematogenously disseminated candidiasis. To date, most studies of C. albicans have been carried out in suspension cultures; however, the medical impact of C. albicans (like that of many other microorganisms) depends on its ability to thrive as a biofilm, a closely packed community of cells. Biofilms are notorious for forming on implanted medical devices, including catheters, pacemakers, dentures, and prosthetic joints, which provide a surface and sanctuary for biofilm growth. C. albicans biofilms are intrinsically resistant to conventional antifungal therapeutics, the host immune system, and other environmental perturbations, making biofilm-based infections a significant clinical challenge. Here, we review our current knowledge of biofilms formed by C. albicans and closely related fungal species. [ABSTRACT FROM AUTHOR]

C andida albicans biofilms are composed of highly adherent and densely arranged cells with properties distinct from those of free-floating (planktonic) cells. These biofilms are a significant medical problem because they commonly form on implanted medical devices, are drug resistant and are difficult to remove. C . albicans biofilms are not static structures; rather they are dynamic and develop over time. Here we characterize gene expression in biofilms during their development, and by comparing them to multiple planktonic reference states, we identify patterns of gene expression relevant to biofilm formation. In particular, we document time-dependent changes in genes involved in adhesion and metabolism, both of which are at the core of biofilm development. Additionally, we identify three new regulators of biofilm formation, Flo8, Gal4, and Rfx2, which play distinct roles during biofilm development over time. Flo8 is required for biofilm formation at all time points, and Gal4 and Rfx2 are needed for proper biofilm formation at intermediate time points. [ABSTRACT FROM AUTHOR]

Members of the tristetraprolin ( TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in S accharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in S chizosaccharomyces pombe, Zfs1, regulates cell-cell interactions. In the human pathogen C andida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3′ UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS 3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often 'disappeared' with increasing evolutionary distance from the parental species. C . albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species. [ABSTRACT FROM AUTHOR]

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans. [ABSTRACT FROM AUTHOR]

: This study describes a novel “white-gray-opaque” tristable phenotypic switching system in the human fungal pathogen Candida albicans, revealing additional complexity in this organism's ability to adapt to changing environments. [ABSTRACT FROM AUTHOR]

The human fungal pathogen C andida albicans can switch between two phenotypic cell types, termed 'white' and 'opaque'. Both cell types are heritable for many generations, and the switch between the two types occurs epigenetically, that is, without a change in the primary DNA sequence of the genome. Previous work identified six key transcriptional regulators important for white-opaque switching: Wor1, Wor2, Wor3, Czf1, Efg1, and Ahr1. In this work, we describe the structure of the transcriptional network that specifies the white and opaque cell types and governs the ability to switch between them. In particular, we use a combination of genome-wide chromatin immunoprecipitation, gene expression profiling, and microfluidics-based DNA binding experiments to determine the direct and indirect regulatory interactions that form the switch network. The six regulators are arranged together in a complex, interlocking network with many seemingly redundant and overlapping connections. We propose that the structure (or topology) of this network is responsible for the epigenetic maintenance of the white and opaque states, the switching between them, and the specialized properties of each state. [ABSTRACT FROM AUTHOR]