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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. albicanscells, 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. albicansby 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.

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. albicansand 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.

Candida albicans is among the most prevalent fungal species of the human microbiota and asymptomatically colonizes healthy individuals. However, it is also an opportunistic pathogen that can cause severe, and often fatal, bloodstream infections. The medical impact of C. albicans typically depends on its ability to form biofilms, which are closely packed communities of cells that attach to surfaces, such as tissues and implanted medical devices. In this Review, we provide an overview of the processes involved in the formation of C. albicans biofilms and discuss the core transcriptional network that regulates biofilm development. We also consider some of the advantages that biofilms provide to C. albicans in comparison with planktonic growth and explore polymicrobial biofilms that are formed by C. albicans and certain bacterial species.

ABSTRACTCandida albicansand Streptococcus 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 between C. albicansand S. 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 6 C. albicanstranscription regulator reporter strains we discovered that EFG1was strongly activated by interaction with S. 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 robust Candida-streptococcal biofilm growth in organotypic constructs and mouse oral tissues. S. oralisstimulated C. albicans ALS1gene expression in an EFG1-dependent manner, and Als1 was identified as a downstream effector of the Efg1 pathway which promoted C. albicans-S. oraliscoaggregation interactions in mixed biofilms. We conclude that S. oralisinduces an increase in EFG1expression in C. albicansin late biofilm stages. This in turn increases expression of ALS1, which promotes coaggregation interactions and mucosal biofilm growth. Our work provides novel insights on C. albicansgenes which play a role in cross-kingdom interactions with S. oralisin mucosal biofilms.

Candida albicansis a commensal microorganism of the human microbiome; it is also the most prevalent fungal pathogen of humans. Many infections caused by C. albicansare a direct consequence of its proclivity to form biofilms—resilient, surface-associated communities of cells where individual cells acquire specialized properties that are distinct from those observed in suspension cultures. We recently identified the transcriptional network that orchestrates the formation of biofilms in C. albicans. These results set the stage for understanding how biofilms are formed and, once formed, how the specialized properties of biofilms are elaborated. This information will provide new insight for understanding biofilms in more detail and may lead to improvements in preventing and treating biofilm-based infections in the future.

Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1 mutant is defective in retention of nonadherent bcr1/bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1/bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.

ABSTRACTHwp1 is a well-characterized Candida albicanscell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1mutant is defective in retention of nonadherent bcr1/bcr1mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1in a bcr1/bcr1mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicansbiofilm formation in vivo and is thus an excellent therapeutic target.

Biofilm formation plays a key role in the life cycles and subsistence of many microorganisms. For the human fungal pathogen Candida albicans, biofilm development is arguably a virulence trait, because medical implants that serve as biofilm substrates are significant risk factors for infection. The development of C. albicans biofilms in vitro proceeds through an early phase, in which yeast cells populate a substrate, an intermediate phase, in which pseudohyphal and hyphal cell types are produced, and a maturation phase, in which continued cell growth is accompanied by accumulation of an extracellular matrix. Here we report the results of a screen for C. albicans biofilm-defective mutants, in which homozygous insertions in NUP85, MDS3, KEM1, and SUV3 were found to block biofilm development. Confocal microscopic examination suggests that nup85, suv3, and mds3 mutations cause early-phase arrest, whereas the kem1 mutation causes intermediate-phase arrest. All of the mutants are defective in hypha production in several media. Analysis of mixed-biofilm development indicates that all of the mutants are defective in the production of hyphae in the context of a biofilm. Because all of the mutants are defective in the retention of cells in the biofilm, we infer that hyphae provide an adherent scaffold that stabilizes the biofilm structure.

ABSTRACTBiofilm formation plays a key role in the life cycles and subsistence of many microorganisms. For the human fungal pathogen Candida albicans, biofilm development is arguably a virulence trait, because medical implants that serve as biofilm substrates are significant risk factors for infection. The development of C. albicansbiofilms in vitro proceeds through an early phase, in which yeast cells populate a substrate, an intermediate phase, in which pseudohyphal and hyphal cell types are produced, and a maturation phase, in which continued cell growth is accompanied by accumulation of an extracellular matrix. Here we report the results of a screen for C. albicansbiofilm-defective mutants, in which homozygous insertions in NUP85, MDS3, KEM1, and SUV3were found to block biofilm development. Confocal microscopic examination suggests that nup85, suv3, and mds3mutations cause early-phase arrest, whereas the kem1mutation causes intermediate-phase arrest. All of the mutants are defective in hypha production in several media. Analysis of mixed-biofilm development indicates that all of the mutants are defective in the production of hyphae in the context of a biofilm. Because all of the mutants are defective in the retention of cells in the biofilm, we infer that hyphae provide an adherent scaffold that stabilizes the biofilm structure.

Abstract The scorpion, Centruroides vittatus, lives in groups, which may allow females to discriminate among mates. We examine possible female mate discrimination based on the duration of the “kiss” phase of courtship, during which sperm is transferred, and on the duration of the “promenade a deux” phase of courtship that precedes sperm transfer. Larger males spent less time during the promenade and more time during the kiss phases of courtship. We discuss the implications of this to sexual selection in scorpions.

Antimicrobial peptides and proteins play critical roles in the host defense against invading pathogens. We recently discovered that recombinantly expressed human and mouse serum amyloid A1 (rhSAA1 and rmSAA1, respectively) proteins have potent antifungal activities against the major human fungal pathogen Candida albicans. At high concentrations, rhSAA1 disrupts C. albicansmembrane integrity and induces rapid fungal cell death. In the present study, we find that rhSAA1 promotes cell aggregation and targets the C. albicanscell wall adhesin Als3.

Mammalian serum amyloid A (SAA) is a major acute phase protein that shows a massive increase in plasma concentration during inflammation. In the present study, we demonstrate that the expression of mouse SAA1 in serum was increased when infected with Candida albicans, a major human fungal pathogen, in a systemic infection model. We then set out to investigate the antifungal activity of SAA proteins against C. albicans.

Candida albicansis 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. albicansdepends, 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 vitrobiofilm 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. albicansforms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.

ABSTRACTCandida albicansbiofilms have a significant medical impact due to their rapid growth on implanted medical devices, their resistance to antifungal drugs, and their ability to seed disseminated infections. Biofilm assays performed in vitroallow for rapid, high-throughput screening of gene deletion libraries or antifungal compounds and typically serve as precursors to in vivostudies. Here, we compile and discuss the protocols for several recently published C. albicansin vitrobiofilm assays. We also describe improved versions of these protocols as well as novel in vitroassays. Finally, we consider some of the advantages and disadvantages of these different types of assays.

ABSTRACTCandida albicansis a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicanscells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicansinfections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicansbiofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicansbiofilms; we also show that these activities are upregulated in other Candidaclade pathogens. Deletion of the SAP5and SAP6genes in C. albicanscompromised biofilm development in vitroin standard biofilm assays and in vivoin a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candidabiofilm formation.IMPORTANCEBiofilm formation by the opportunistic fungal pathogen C. albicansis a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicansbiofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicansbiofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicansand also from additional pathogenic Candidaspecies. Furthermore, SAP5and SAP6deletions confirm that both proteases are required for proper biofilm development in vitroand in vivo. We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candidabiofilm-associated infections.

ABSTRACTThe human commensal and opportunistic pathogen Candida albicanscan switch between two distinct, heritable cell types, named “white” and “opaque,” which differ in morphology, mating abilities, and metabolic preferences and in their interactions with the host immune system. Previous studies revealed a highly interconnected group of transcriptional regulators that control switching between the two cell types. Here, we identify Ssn6, the C. albicansfunctional homolog of the Saccharomyces cerevisiaetranscriptional corepressor Cyc8, as a new regulator of white-opaque switching. In aor α mating type strains, deletion of SSN6results in mass switching from the white to the opaque cell type. Transcriptional profiling of ssn6deletion mutant strains reveals that Ssn6 represses part of the opaque cell transcriptional program in white cells and the majority of the white cell transcriptional program in opaque cells. Genome-wide chromatin immunoprecipitation experiments demonstrate that Ssn6 is tightly integrated into the opaque cell regulatory circuit and that the positions to which it is bound across the genome strongly overlap those bound by Wor1 and Wor2, previously identified regulators of white-opaque switching. This work reveals the next layer in the white-opaque transcriptional circuitry by integrating a transcriptional regulator that does not bind DNA directly but instead associates with specific combinations of DNA-bound transcriptional regulators.IMPORTANCEThe most common fungal pathogen of humans, C. albicans, undergoes several distinct morphological transitions during interactions with its host. One such transition, between cell types named “white” and “opaque,” is regulated in an epigenetic manner, in the sense that changes in gene expression are heritably maintained without any modification of the primary genomic DNA sequence. Prior studies revealed a highly interconnected network of sequence-specific DNA-binding proteins that control this switch. We report the identification of Ssn6, which defines an additional layer of transcriptional regulation that is critical for this heritable switch. Ssn6 is necessary to maintain the white cell type and to properly express the opaque cell transcriptional program. Ssn6 does not bind DNA directly but rather associates with specific combinations of DNA-bound transcriptional regulators to control the switch. This work is significant because it reveals a new level of regulation of an important epigenetic switch in the predominant fungal pathogen of humans.

ABSTRACTSingle-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicansis a common member of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicansgained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicansuses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicanscells normally enter the G0phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomal biogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans’ more efficient colonization of the gut.IMPORTANCEThe ability to rapidly and appropriately respond to nutrients in the environment is crucial to free-living microorganisms. To maximize the use of available nutrients, microorganisms often use a limiting nutritional component as a signal to coordinate multiple biological processes. The human fungal pathogen Candida albicansuses N-acetylglucosamine (GlcNAc) as a signal for the availability of external nutrient resources. GlcNAc induces rapid cell death in C. albicansdue to the constitutive activation of oxidative metabolism and accumulation of reactive oxygen species (ROS), and multiple pathways are involved in its regulation. This study sheds light on the mechanisms of niche specialization of pathogenic fungi and raises the possibility that this cell death pathway could be an unexplored therapeutic target.

ABSTRACTCandida albicansis the most prevalent fungal pathogen of humans, causing a variety of diseases ranging from superficial mucosal infections to deep-seated systemic invasions. Mucus, the gel that coats all wet epithelial surfaces, accommodates C. albicansas part of the normal microbiota, where C. albicansresides asymptomatically in healthy humans. Through a series of in vitroexperiments combined with gene expression analysis, we show that mucin biopolymers, the main gel-forming constituents of mucus, induce a new oval-shaped morphology in C. albicansin which a range of genes related to adhesion, filamentation, and biofilm formation are downregulated. We also show that corresponding traits are suppressed, rendering C. albicansimpaired in forming biofilms on a range of different synthetic surfaces and human epithelial cells. Our data suggest that mucins can manipulate C. albicansphysiology, and we hypothesize that they are key environmental signals for retaining C. albicansin the host-compatible, commensal state.IMPORTANCEThe yeast Candida albicanscauses both superficial infections of the mucosa and life-threatening infections upon entering the bloodstream. However, C. albicansis not always harmful and can exist as part of the normal microbiota without causing disease. Internal body surfaces that are susceptible to infection by C. albicansare coated with mucus, which we hypothesize plays an important role in preventing infections. Here, we show that the main components of mucus, mucin glycoproteins, suppress virulence attributes of C. albicansat the levels of gene expression and the corresponding morphological traits. Specifically, mucins suppress attachment to plastic surfaces and human cells, the transition to cell-penetrating hyphae, and the formation of biofilms (drug-resistant microbial communities). Additionally, exposure to mucins induces an elongated morphology that physically resembles the mating-competent opaque state but is phenotypically distinct. We suggest that mucins are potent antivirulence molecules that have therapeutic potential for suppressing C. albicansinfections.

ABSTRACTBiofilms are resilient, surface-associated communities of cells with specialized properties (e.g., resistance to drugs and mechanical forces) that are distinct from those of suspension (planktonic) cultures. Biofilm formation by the opportunistic human fungal pathogen Candida albicansis medically relevant because C. albicansinfections are highly correlated with implanted medical devices, which provide efficient substrates for biofilm formation; moreover, biofilms are inherently resistant to antifungal drugs. Biofilms are also important for C. albicansto colonize diverse niches of the human host. Here, we describe four core members of a conserved histone deacetylase complex in C. albicans(Set3, Hos2, Snt1, and Sif2) and explore the effects of their mutation on biofilm formation. We find that these histone deacetylase complex members are needed for proper biofilm formation, including dispersal of cells from biofilms and multifactorial drug resistance. Our results underscore the importance of the physical properties of biofilms in contributing to drug resistance and dispersal and lay a foundation for new strategies to target biofilm dispersal as a potential antifungal intervention.IMPORTANCEThrough the formation of biofilms—surface-associated communities of cells—microorganisms can establish infections, become drug resistant, and evade the host immune system. Here we investigate how four core members of a conserved histone deacetylase complex mediate biofilm formation by Candida albicans, the major fungal pathogen of humans. We show that this histone deacetylase complex is required for biofilm dispersal, a process through which cells leave the biofilm to establish new infections. We also show that the deacetylase complex mediates biofilm drug resistance. This work provides new insight into how the physical properties of biofilms affect dispersal and drug resistance and suggests new potential antifungal strategies that could be effective against biofilms.