The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

IMPORTANCE: Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans . The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1 946S allele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1 946S allele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1 946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1 946P allele establishes a commensal state while the ROB1 946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. Importance: Candida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Rewilding of laboratory mice enhances granulopoiesis and immunity through intestinal fungal colonization.

The paucity of blood granulocyte populations such as neutrophils in laboratory mice is a notable difference between this model organism and humans, but the cause of this species-specific difference is unclear. We previously demonstrated that laboratory mice released into a seminatural environment, referred to as rewilding, display an increase in blood granulocytes that is associated with expansion of fungi in the gut microbiota. Here, we find that tonic signals from fungal colonization induce sustained granulopoiesis through a mechanism distinct from emergency granulopoiesis, leading to a prolonged expansion of circulating neutrophils that promotes immunity. Fungal colonization after either rewilding or oral inoculation of laboratory mice with Candida albicans induced persistent expansion of myeloid progenitors in the bone marrow. This increase in granulopoiesis conferred greater long-term protection from bloodstream infection by gram-positive bacteria than by the trained immune response evoked by transient exposure to the fungal cell wall component ß-glucan. Consequently, introducing fungi into laboratory mice may restore aspects of leukocyte development and provide a better model for humans and free-living mammals that are constantly exposed to environmental fungi.

Candida albicans Isolates 529L and CHN1 Exhibit Stable Colonization of the Murine Gastrointestinal Tract.

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences among mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of single nucleotide polymorphisms (SNPs) and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice modestly increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche. IMPORTANCE Understanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intraspecies variation as well as host antimicrobial defense mechanisms in defining the outcome of commensal interactions.

Evaluation of Biofilm Formation in Candida tropicalis Using a Silicone-Based Platform with Synthetic Urine Medium.

Molecular mechanisms of biofilm formation in Candida tropicalis and current methods for biofilm analyses in this fungal pathogen are limited. (2) Methods: Biofilm biomass and crystal violet staining of the wild-type and each gene mutant strain of C. tropicalis were evaluated on silicone under synthetic urine culture conditions. (3) Results: Seven media were tested to compare the effects on biofilm growth with or without silicone. Results showed that biofilm cells of C. tropicalis were unable to form firm biofilms on the bottom of 12-well polystyrene plates. However, on a silicone-based platform, Roswell Park Memorial Institute 1640 (RPMI 1640), yeast nitrogen base (YNB) + 1% glucose, and synthetic urine media were able to induce strong biofilm growth. In particular, replacement of Spider medium with synthetic urine in the adherence step and the developmental stage is necessary to gain remarkably increased biofilms. Interestingly, unlike Candida albicans, the C. tropicalis ROB1 deletion strain but not the other five biofilm-associated mutants did not cause a significant reduction in biofilm formation, suggesting that the biofilm regulatory circuits of the two species are divergent. (4) Conclusions: This system for C. tropicalis biofilm analyses will become a useful tool to unveil the biofilm regulatory network in C. tropicalis.

Hemizygosity Enables a Mutational Transition Governing Fungal Virulence and Commensalism.

Candida albicans is a commensal fungus of human gastrointestinal and reproductive tracts, but also causes life-threatening systemic infections. The balance between colonization and pathogenesis is associated with phenotypic plasticity, with alternative cell states producing different outcomes in a mammalian host. Here, we reveal that gene dosage of a master transcription factor regulates cell differentiation in diploid C. albicans cells, as EFG1 hemizygous cells undergo a phenotypic transition inaccessible to "wild-type" cells with two functional EFG1 alleles. Notably, clinical isolates are often EFG1 hemizygous and thus licensed to undergo this transition. Phenotypic change corresponds to high-frequency loss of the functional EFG1 allele via de novo mutation or gene conversion events. This phenomenon also occurs during passaging in the gastrointestinal tract with the resulting cell type being hypercompetitive for commensal and systemic infections. A "two-hit" genetic model therefore underlies a key phenotypic transition in C. albicans that enables adaptation to host niches.

The Impact of Gene Dosage and Heterozygosity on The Diploid Pathobiont Candida albicans.

Candida albicans is a fungal species that can colonize multiple niches in the human host where it can grow either as a commensal or as an opportunistic pathogen. The genome of C. albicans has long been of considerable interest, given that it is highly plastic and can undergo a wide variety of alterations. These changes play a fundamental role in determining C. albicans traits and have been shown to enable adaptation both to the host and to antifungal drugs. C. albicans isolates contain a heterozygous diploid genome that displays variation from the level of single nucleotides to largescale rearrangements and aneuploidy. The heterozygous nature of the genome is now increasingly recognized as being central to C. albicans biology, as the relative fitness of isolates has been shown to correlate with higher levels of overall heterozygosity. Moreover, loss of heterozygosity (LOH) events can arise frequently, either at single polymorphisms or at a chromosomal level, and both can alter the behavior of C. albicans cells during infection or can modulate drug resistance. In this review, we examine genome plasticity in this pathobiont focusing on how gene dosage variation and loss of heterozygosity events can arise and how these modulate C. albicans behavior.

The antimicrobial photodynamic inactivation resistance of Candida albicans is modulated by the Hog1 pathway and the Cap1 transcription factor.

Candida albicans is the most important fungal pathogen afflicting humans, particularly immunocompromised patients. However, currently available antifungal drugs are limited and ineffective against drug-resistant strains. The development of new drugs or alternative therapeutic approaches to control fungal infections is urgent and necessary. Photodynamic inactivation (PDI) is a new promising therapy for eradicating microorganism infections through combining visible light, photosensitizers, and oxygen to generate reactive oxygen species (ROS). Although cytoprotective responses induced by photodynamic therapy (PDT) have been well studied in cancer cells, the mechanisms by which C. albicans responds to PDI are largely unknown. In this study, we first demonstrated that PDI induces C. albicans Hog1p activation. Deletion of any of the SSK2, PBS2, and HOG1 genes significantly decreased the survival rate after photochemical reactions, indicating that the Hog1 SAPK pathway is required for tolerance to PDI. Furthermore, the basic leucine zipper transcription factor Cap1 that regulates several downstream antioxidant genes was highly expressed during the response to PDI, and loss of CAP1 also resulted in decreased C. albicans survival rates. This study demonstrates the importance of the Hog1 SAPK and the Cap1 transcription factor, which regulates in resistance to PDI-mediated oxidative stress in C. albicans. Understanding the mechanisms by which C. albicans responds to PDI and consequently scavenges ROS will be very useful for the further development of therapeutics to control fungal infectious diseases, particularly those of the skin and mucosal infections.

The conserved dual phosphorylation sites of the Candida albicans Hog1 protein are crucial for white-opaque switching, mating, and pheromone-stimulated cell adhesion.

Candida albicans is an opportunistic human pathogen capable of causing life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cells differ in virulence, mating capability, biofilm formation, and host-cell interaction. Previous studies revealed that deletion of the SSK2, PBS2, or HOG1 gene resulted in 100% opaque cell formation and suppressed the mating response. Thr-174 and Tyr-176 of the Hog1 protein are important phosphoacceptors and can be activated in response to stimuli. In this study, we first demonstrated the importance of two conserved phosphorylation sites in white-opaque switching, mating, and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including nonphosphorylated strains (Hog1(T174A), Hog1(Y176F), and Hog1(T174A,Y176F)) and negatively charged phosphorylated strains (Hog1(T174D), Hog1(Y176D), and Hog1(T174D,Y176D)). Point mutation on Thr-174, Tyr-176 or in combination with the Hog1 protein in C. albicans MTL homozygous strains stimulated opaque cell formation at a frequency of 100%. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies and pheromone-stimulated cell adhesive numbers were lower than those of the wild-type. By investigating the effects of Hog1 phosphorylation in ssk1Δ and sln1Δ, we also demonstrate that the phosphorylation intensity of Hog1p is directly involved in the white-opaque switching. Taken together, the results of our study demonstrate that dual phosphorylation sites of C. albicans are crucial for white-opaque transition, sexual mating, and pheromone-induced cell adhesion.

A novel function for Hog1 stress-activated protein kinase in controlling white-opaque switching and mating in Candida albicans.

Candida albicans is a commensal in heathy people but has the potential to become an opportunistic pathogen and is responsible for half of all clinical infections in immunocompromised patients. Central to understanding C. albicans behavior is the white-opaque phenotypic switch, in which cells can undergo an epigenetic transition between the white state and the opaque state. The phenotypic switch regulates multiple properties, including biofilm formation, virulence, mating, and fungus-host interactions. Switching between the white and opaque states is associated with many external stimuli, such as oxidative stress, pH, and N-acetylglucosamine, and is directly regulated by the Wor1 transcriptional circuit. The Hog1 stress-activated protein kinase (SAPK) pathway is recognized as the main pathway for adapting to environmental stress in C. albicans. In this work, we first show that loss of the HOG1 gene in A: / A: and α/α cells, but not A: /α cells, results in 100% white-to-opaque switching when cells are grown on synthetic medium, indicating that switching is repressed by the A1: /α2 heterodimer that represses WOR1 gene expression. Indeed, switching in the hog1Δ strain was dependent on the presence of WOR1, as a hog1Δ wor1Δ strain did not show switching to the opaque state. Deletion of PBS2 and SSK2 also resulted in C. albicans cells switching from white to opaque with 100% efficiency, indicating that the entire Hog1 SAPK pathway is involved in regulating this unique phenotypic transition. Interestingly, all Hog1 pathway mutants also caused defects in shmoo formation and mating efficiencies. Overall, this work reveals a novel role for the Hog1 SAPK pathway in regulating white-opaque switching and sexual behavior in C. albicans.