Identification of an active RNAi pathway in Candida albicans.

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Ferrous sulphate triggers ferroptosis in Candida albicans and cures vulvovaginal candidiasis in a mouse model.

Candida albicans is the most leading cause of life-threatening fungal invasive infections, especially for vulvovaginal candidiasis (VVC). Resistance and tolerance to common fungicide has risen great demands on alternative strategies for treating C. albicans infections. In the present study, ferroptosis has been proven to occur in C. albicans by directly exposed to FeSO4 via induing hallmarks of ferroptosis, including Fe2+ overload burden, ROS eruption and lipid peroxidation. Transcriptomic profile gave the great hints of the possible mechanism for fungal ferroptosis that FeSO4 disturb pathways associated to ribosome, tyrosine metabolism, triglyceride metabolism and thiamine metabolism, thus mobilizing death-related gene synthesis. Inspired by the results, a FeSO4-loaded hydrogel was prepared as an antifungal agent to treat C. albicans infection. This hydrogel exhibited excellent dressing properties and maintained superior antifungal activity by characterization tests. Besides, mice treated by this composite hydrogel displayed excellent therapeutic efficacy. These results highlighted the potential therapeutic use of FeSO4 as an innovative strategy in treating C. albicans infections by targeting ferroptosis.

Strain background of Candida albicans interacts with SIR2 to alter phenotypic switching.

The genetic background between strains of a single species and within a single strain lineage can significantly impact the expression of biological traits. This genetic variation may also reshape epigenetic mechanisms of cell identity and environmental responses that are controlled by interconnected transcriptional networks and chromatin-modifying enzymes. Histone deacetylases, including sirtuins, are critical regulators of chromatin state and have been directly implicated in governing the phenotypic transition between the 'sterile' white state and the mating-competent opaque state in Candida albicans, a common fungal commensal and pathogen of humans. Here, we found that a previously ambiguous role for the sirtuin SIR2 in C. albicans phenotypic switching is likely linked to the genetic background of mutant strains produced in the RM lineage of SC5314. SIR2 mutants in a specific lineage of BWP17 displayed increased frequencies of switching to the opaque state compared to the wild-type. Loss of SIR2 in other SC5314-derived backgrounds, including newly constructed BWP17 sir2Δ/Δ mutants, failed to recapitulate the increased white-opaque switching frequencies observed in the original BWP17 sir2Δ/Δ mutant background. Whole-genome sequencing revealed the presence of multiple imbalanced chromosomes and large loss of heterozygosity tracts that likely interact with SIR2 to increase phenotypic switching in this BWP17 sir2Δ/Δ mutant lineage. These genomic changes are not found in other SC5314-derived sir2Δ/Δ mutants that do not display increased opaque cell formation. Thus, complex karyotypes can emerge during strain construction that modify mutant phenotypes and highlight the importance of validating strain background when interpreting phenotypes.

Genetic modification of Candida maltosa, a non-pathogenic CTG species, reveals EFG1 function.

Candida maltosa is closely related to important pathogenic Candida species, especially C. tropicalis and C. albicans, but it has been rarely isolated from humans. For this reason, through comparative studies, it could be a powerful model to understand the genetic underpinnings of the pathogenicity of Candida species. Here, we generated a cohesive assembly of the C. maltosa genome and developed genetic engineering tools that will facilitate studying this species at a molecular level. We used a combination of short and long-read sequencing to build a polished genomic draft composed of 14 Mbp, 45 contigs and close to 5700 genes. This assembly represents a substantial improvement from the currently available sequences that are composed of thousands of contigs. Genomic comparison with C. albicans and C. tropicalis revealed a substantial reduction in the total number of genes in C. maltosa. However, gene loss seems not to be associated to the avirulence of this species given that most genes that have been previously associated with pathogenicity were also present in C. maltosa. To be able to edit the genome of C. maltosa we generated a set of triple auxotrophic strains so that gene deletions can be performed similarly to what has been routinely done in pathogenic Candida species. As a proof of concept, we generated gene knockouts of EFG1, a gene that encodes a transcription factor that is essential for filamentation and biofilm formation in C. albicans and C. tropicalis. Characterization of these mutants showed that Efg1 also plays a role in biofilm formation and filamentous growth in C. maltosa, but it seems to be a repressor of filamentation in this species. The genome assembly and auxotrophic mutants developed here are a key step forward to start using C. maltosa for comparative and evolutionary studies at a molecular level.

Transcriptional analysis of oxidative and nitrosative stress on oral opportunistic Candida albicans.

The yeast Candida albicans is one of the most aggressive opportunistic pathogens in immunocompromised patients. The ability of the yeast to withstand stresses and radicals is of great concern. In the present study, four isolates of C. albicans were taken from patients with oral candidiasis and grown on RPMI for 24 hours at 37°C. Then, they were exposed to various concentrations of oxidative (H2O2) and nitrosative (HNO3) stress for two hours, and gene expression rates were measured through RT-PCR. After initial biofilm formation steps and growth validation, RNA extracted from the yeast and gene expression status were evaluated. Upon treatment with H2O2, the gene expression profile for ALS1, MLH1, and EXO1 showed approximately a fold increase in expression. While within HNO3 the yeast gene expression exhibited a dramatic increase in ALS1 up to 217 folds, while others such as MLH1, HWP1, and ERG11 showed a one-fold increase in the expression rate. The findings of this research indicate a considerable expression activity within the biofilm of Candida albicans, increased rate of DNA mismatch repair and break fixation may indicate the ability of the yeast to tolerate high concentrations of free radicals. It paves the way toward understanding the pathogenicity of the yeast and its survival capability inside macrophages. The study also revealed that the biofilm strategy of the yeast is more active within these stresses.

Highly Reactive Group I Introns Ubiquitous in Pathogenic Fungi.

Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris, demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target.

Secretory IgA reduced the ergosterol contents of Candida albicans to repress its hyphal growth and virulence.

Candida albicans, one of the most prevalent conditional pathogenic fungi, can cause local superficial infections and lethal systemic infections, especially in the immunocompromised population. Secretory immunoglobulin A (sIgA) is an important immune protein regulating the pathogenicity of C. albicans. However, the actions and mechanisms that sIgA exerts directly against C. albicans are still unclear. Here, we investigated that sIgA directs against C. albicans hyphal growth and virulence to oral epithelial cells. Our results indicated that sIgA significantly inhibited C. albicans hyphal growth, adhesion, and damage to oral epithelial cells compared with IgG. According to the transcriptome and RT-PCR analysis, sIgA significantly affected the ergosterol biosynthesis pathway. Furthermore, sIgA significantly reduced the ergosterol levels, while the addition of exogenous ergosterol restored C. albicans hyphal growth and adhesion to oral epithelial cells, indicating that sIgA suppressed the growth of hyphae and the pathogenicity of C. albicans by reducing its ergosterol levels. By employing the key genes mutants (erg11Δ/Δ, erg3Δ/Δ, and erg3Δ/Δ erg11Δ/Δ) from the ergosterol pathway, sIgA lost the hyphal inhibition on these mutants, while sIgA also reduced the inhibitory effects of erg11Δ/Δ and erg3Δ/Δ and lost the inhibition of erg3Δ/Δ erg11Δ/Δ on the adhesion to oral epithelial cells, further proving the hyphal repression of sIgA through the ergosterol pathway. We demonstrated for the first time that sIgA inhibited C. albicans hyphal development and virulence by affecting ergosterol biosynthesis and suggest that ergosterol is a crucial regulator of C. albicans-host cell interactions. KEY POINTS: • sIgA repressed C. albicans hyphal growth • sIgA inhibited C. albicans virulence to host cells • sIgA affected C. albicans hyphae and virulence by reducing its ergosterol levels.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

Functional annotation of Candida albicans hypothetical proteins: a bioinformatics approach.

Candida albicans is a member of the ascomycetes class of fungi and it is an opportunistic pathogen species responsible for a wide range of fungal infections in humans. Bioinformatics and sequencing analysis of Candida proteomics has disclosed that around 69% proteome is still uncharacterized which needs to be annotated with functions. The NCBI-Genome has termed them as hypothetical proteins (HPs) in the whole proteome of Candida. Interpretation of this substantial portion of the proteome can reveal novel pharmacological targets for markers, drug development, and other therapeutics and so on. In this article, we have assigned functional annotation to these hypothetical proteins using bioinformatics methodologies. The advanced and robust computational models have been used to assign the preliminary functions to these putative HPs with high level of confidence. The findings of this study unveil some novel pharmacological targets for drug therapy and vaccines and it would help to identify novel molecular mechanisms underlying the fungal pathogenesis.

Mechanisms of antifungal resistance and developments in alternative strategies to combat Candida albicans infection.

Candida albicans is a commensal fungus that infects the humans and becomes an opportunistic pathogen particularly in immuno-compromised patients. Among the Candida genus, yeast C. albicans is the most frequently incriminated species and is responsible for nearly 50-90% of human candidiasis, with vulvovaginal candidiasis alone, affecting about 75% of the women worldwide. One of the significant virulence traits in C. albicans is its tendency to alternate between the yeast and hyphae morphotypes, accounting for the development of multi-drug resistance in them. Thus, a thorough comprehension of the decision points and genes controlling this transition is necessary, to understand the pathogenicity of this, naturally occurring, pernicious fungus. Additionally, the formation of C. albicans biofilm is yet another pathogenesis trait and a paramount cause of invasive candidiasis. Since 1980 and in 90 s, wide spread use of immune-suppressing therapies and over prescription of fluconazole, a drug used to treat chronic fungal infections, triggered the emergence of novel anti-fungal drug development. Thus, this review thoroughly elucidates the diseases associated with C. albicans infection as well as the anti-fungal resistance mechanism associated with them and identifies the emerging therapeutic agents, along with a rigorous discussion regarding the future strategies that can possibly be adopted for the cure of this deleterious pathogen.

Metagenetic analysis of the bacterial diversity of Kazakh koumiss and assessment of its anti-Candida albicans activity.

Koumiss, a five-thousand-year-old fermented mare's milk beverage, is widely recognized for its beneficial nutrient and medicinal properties. The microbiota of Chinese and Mongolian koumiss have been largely characterized in recent years, but little is known concerning Kazakh koumiss despite this drink historically originates from the modern Kazakhstan territory. In addition, while koumiss is regarded as a drink with therapeutic potential, there are also no data on koumiss anti-Candida activity. In this context, the aims of the present study were to investigate the bacterial diversity and anti-Candida albicans activity of homemade Kazakh koumiss samples as well as fermented whey and cow's milk, derived from koumiss and propagated for several months. Koumiss bacterial communities were largely dominated by lactic acid bacteria including Lactobacillus sensu lato spp. (69% of total reads), Streptococcus (8.0%) and Lactococcus (6.1%), while other subdominant genera included Acetobacter (2.6%), Enterobacter (2.4%), and Klebsiella (1.5%). Several but not all koumiss samples as well as fermented whey and cow's milk showed antagonistic activities towards C. albicans. Linear discriminant effect size (LEfSe) analysis showed that their bacterial communities were characterized by a significantly higher abundance of amplicon sequence variants (ASV) belonging to the genus Acetobacter. In conclusion, this study allowed to identify the key microorganisms of Kazakh koumiss and provided new information on the possible underestimated contribution of acetic acid bacteria to its probiotic properties.

Inhibition of Biofilm Formation in Cutibacterium acnes, Staphylococcus aureus, and Candida albicans by the Phytopigment Shikonin.

Skin microbiota, such as acne-related Cutibacterium acnes, Staphylococcus aureus, and fungal Candida albicans, can form polymicrobial biofilms with greater antimicrobial tolerance to traditional antimicrobial agents and host immune systems. In this study, the phytopigment shikonin was investigated against single-species and multispecies biofilms under aerobic and anaerobic conditions. Minimum inhibitory concentrations of shikonin were 10 µg/mL against C. acnes, S. aureus, and C. albicans, and at 1-5 µg/mL, shikonin efficiently inhibited single biofilm formation and multispecies biofilm development by these three microbes. Shikonin increased porphyrin production in C. acnes, inhibited cell aggregation and hyphal formation by C. albicans, decreased lipase production, and increased hydrophilicity in S. aureus. In addition, shikonin at 5 or 10 µg/mL repressed the transcription of various biofilm-related genes and virulence-related genes in C. acnes and downregulated the gene expression levels of the quorum-sensing agrA and RNAIII, α-hemolysin hla, and nuclease nuc1 in S. aureus, supporting biofilm inhibition. In addition, shikonin prevented multispecies biofilm development on porcine skin, and the antimicrobial efficacy of shikonin was recapitulated in a mouse infection model, in which it promoted skin regeneration. The study shows that shikonin inhibits multispecies biofilm development by acne-related skin microbes and might be useful for controlling bacterial infections.

Use of a Candida albicans SC5314 PacBio HiFi reads dataset to close gaps in the reference genome assembly, reveal a subtelomeric gene family, and produce accurate phased allelic sequences.

Candida albicans SC5314 is the most-often used strain for molecular manipulation of the species. The SC5314 reference genome sequence is the result of considerable effort from many scientists and has advanced research into fungal biology and pathogenesis. Although the resource is highly developed and presented in a phased diploid format, the sequence includes gaps and does not extend to the telomeres on its eight chromosome pairs. Accurate SC5314 genome assembly is complicated by the presence of extensive repeated sequences and considerable allelic length variation at some loci. Advances in genome sequencing technology provide the tools to obtain highly accurate long-read data that span even the most-difficult-to-assemble genome regions. Here, we describe derivation of a PacBio HiFi data set and creation of a collapsed haploid telomere-to-telomere assembly of the SC5314 genome (ASM3268872v1) that revealed previously unknown features of the strain. ASM3268872v1 subtelomeric distances were up to 19 kb larger than in the reference genome and revealed a family of highly conserved DNA helicase-encoding genes at 10 of the 16 chromosome ends. We also describe alignments of individual HiFi reads to deduce accurate diploid sequences for the most notoriously difficult-to-assemble C. albicans genes: the agglutinin-like sequence (ALS) gene family. We provide a tutorial that demonstrates how the HiFi reads can be visualized to explore any region of interest. Availability of the HiFi reads data set and the ASM3268872v1 comparative guide assembly will streamline research efforts because accurate diploid sequences can be derived using simple in silico methods rather than time-consuming laboratory-bench approaches.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium Moorena producens JHB Discovered via Coculture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was cocultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that coculture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

Inhibiting pathogenicity of vaginal Candida albicans by lactic acid bacteria and MS analysis of their extracellular compounds.

Maintaining healthy vaginal microflora post-puberty is critical. In this study we explore the potential of vaginal lactic acid bacteria (LAB) and their extracellular metabolites against the pathogenicity of Candida albicans. The probiotic culture free supernatant (PCFS) from Lactobacillus crispatus, L. gasseri, and L. vaginalis exhibit an inhibitory effect on budding, hyphae, and biofilm formation of C. albicans. LGPCFS manifested the best potential among the LAB PCFS, inhibiting budding for 24 h and restricting hyphae formation post-stimulation. LGPCFS also pre-eminently inhibited biofilm formation. Furthermore, L. gasseri itself grew under RPMI 1640 stimulation suppressing the biofilm formation of C. albicans. The PCFS from the LAB downregulated the hyphal genes of C. albicans, inhibiting the yeast transformation to fungi. Hyphal cell wall proteins HWP1, ALS3, ECE1, and HYR1 and transcription factors BCR1 and CPH1 were downregulated by the metabolites from LAB. Finally, the extracellular metabolome of the LAB was studied by LC-MS/MS analysis. L.gasseri produced the highest antifungal compounds and antibiotics, supporting its best activity against C. albicans. Vaginal LAB and their extracellular metabolites perpetuate C. albicans at an avirulent state. The metabolites produced by these LAB in vitro have been identified, and can be further exploited as a preventive measure against vaginal candidiasis.

Proteome dataset of Candida albicans (ATCC10231) opaque cell.

OBJECTIVES: Candida albicans, a polymorphic yeast, is one of the most common, opportunistic fungal pathogens of humans. Among the different morphological forms, opaque form is one of the least-studied ones. This opaque phenotype is essential for mating and is also reported to be involved in colonizing the gastrointestinal tract. Considering the significance of the clinical and sexual reproduction of C. albicans, we have investigated the morphophysiological modulations in opaque form using a proteomic approach. DATA DESCRIPTION: In the current investigation, we have used Micro-Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis to create a protein profile for opaque-specific proteins. Whole-cell proteins from C. albicans (ATCC10231) cells that had been cultured for seven days on synthetic complete dextrose (SCD) medium in both as an opaque (test) and as a white (control) form cells were extracted, digested, and identified using LC-MS/MS. This information is meant to serve the scientific community and represents the proteome profile (SWATH Spectral Libraries) of C. albicans opaque form.

The transcription factor Ofi1 is critical for white-opaque switching in natural MTLa/α isolates of Candida albicans.

Candida albicans, an opportunistic fungal pathogen, is able to switch between two distinct cell types: white and opaque. While white-to-opaque switching is typically repressed by the a1/α2 heterodimer in MTLa/α cells, it was recently reported that switching can also occur in some natural MTLa/α strains under certain environmental conditions. However, the regulatory program governing white-opaque switching in MTLa/α cells is not fully understood. Here, we collected 90 clinical isolates of C. albicans, 16 of which possess the ability to form opaque colonies. Among the known regulators implicated in white-opaque switching, only OFI1 exhibited significantly higher expression in these 16 strains compared to the reference strain SC5314. Importantly, ectopic expression of OFI1 in both clinical isolates and laboratory strains promoted switching frequency even in the absence of N-acetylglucosamine and high CO2 , the optimal condition for white-to-opaque switching in MTLa/α strains. Deleting OFI1 resulted in a reduction in opaque-formation frequency and the stability of the opaque cell in MTLa/α cells. Ofi1 binds to the promoters of WOR1 and WOR3 to induce their expression, which facilitates white-to-opaque switching. Ofi1 is conserved across the CTG species. Altogether, our study reported the identification of a transcription factor Ofi1 as the critical regulator that promotes white-to-opaque switching in natural MTLa/α isolates of C. albicans.

Impaired neutrophil extracellular trap-forming capacity contributes to susceptibility to chronic vaginitis in a mouse model of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, is characterized by aberrant inflammation by polymorphonuclear neutrophils (PMNs) in the vaginal lumen. Data from the established murine model shows that despite potent antifungal properties, PMNs fail to clear C. albicans due to local heparan sulfate that inhibits the interaction between PMNs and C. albicans, resulting in chronic vaginal immunopathology. To understand the role of neutrophil extracellular traps (NETs) in defense against C. albicans at the vaginal mucosa, we investigated the NET-forming capacity of PMNs in chronic VVC-susceptible (CVVC-S/C3H) and -resistant (CVVC-R/CD-1) mouse strains. Immunofluorescence revealed the formation of NETs (release of DNA with PMN-derived antimicrobial proteins) in PMN-C. albicans cocultures using vaginal conditioned medium (VCM) generated from CVVC-R/CD-1 mice, similar to NET-inducing positive controls. Under these NETotic conditions, PMNs released high levels of double-stranded DNA bound with NET-associated proteins, concomitant with substantial C. albicans killing activity. In contrast, PMN-C. albicans cocultures in VCM from CVVC-S/C3H mice lacked NET formation together with reduced antifungal activity. Similar results were observed in vivo: active NET-C. albicans interaction followed by fungal clearance in inoculated CVVC-R/CD-1 mice, and sustained high vaginal fungal burden and no evidence of NETs in inoculated CVVC-S/C3H mice. Furthermore, the level of Ki67 expression, a putative NETotic PMN marker, was significantly reduced in vaginal lavage fluid from CVVC-S/C3H mice compared to CVVC-R/CD-1 mice. Finally, scanning electron microscopy revealed that PMNs in CVVC-R, but not CVVC-S, conditions exhibited NETs in direct contact with C. albicans hyphae in vitro and in vivo. These results suggest that VVC-associated immunopathology includes impaired NET-mediated antifungal activity.

A CUG codon-adapted anchor-away toolkit for functional analysis of genes in Candida albicans.

Promoter shutoff of essential genes in the diploid Candida albicans has often been insufficient to create tight, conditional null alleles due to leaky expression and has been a stumbling block in pathogenesis research. Moreover, homozygous deletion of non-essential genes has often been problematic due to the frequent aneuploidy in the mutant strains. Rapid, conditional depletion of essential genes by the anchor-away strategy has been successfully employed in Saccharomyces cerevisiae and other model organisms. Here, rapamycin mediates the dimerization of human FK506-binding protein (FKBP12) and FKBP12-rapamycin-binding (FRB) domain-containing target protein, resulting in relocalization to altered sub-cellular locations. In this work, we used the ribosomal protein Rpl13 as the anchor and took two nuclear proteins as targets to construct a set of mutants in a proof-of-principle approach. We first constructed a rapamycin-resistant C. albicans strain by introducing a dominant mutation in the CaTOR1 gene and a homozygous deletion of RBP1, the ortholog of FKBP12, a primary target of rapamycin. The FKBP12 and the FRB coding sequences were then CUG codon-adapted for C. albicans by site-directed mutagenesis. Anchor-away strains expressing the essential TBP1 gene or the non-essential SPT8 gene as FRB fusions were constructed. We found that rapamycin caused rapid cessation of growth of the TBP-AA strain within 15 minutes and the SPT8-AA strain phenocopied the constitutive filamentous phenotype of the spt8Δ/spt8Δ mutant. Thus, the anchor-away toolbox for C. albicans developed here can be employed for genome-wide analysis to identify gene function in a rapid and reliable manner, further accelerating anti-fungal drug development in C. albicans. IMPORTANCE: Molecular genetic studies thus far have identified ~27% open-reading frames as being essential for the vegetative growth of Candida albicans in rich medium out of a total 6,198 haploid set of open reading frames. However, a major limitation has been to construct rapid conditional alleles of essential C. albicans genes with near quantitative depletion of encoded proteins. Here, we have developed a toolbox for rapid and conditional depletion of genes that would aid studies of gene function of both essential and non-essential genes.

RFX transcription factor in the human-associated yeast Candida albicans regulates adhesion to oral epithelium.

Adhesion to mucosal surfaces is a critical step in many bacterial and fungal infections. Here, using a mouse model of oral infection by the human fungal pathobiont Candida albicans, we report the identification of a novel regulator of C. albicans adhesion to the oral mucosa. The regulator is a member of the regulatory factor X (RFX) family of transcription factors, which control cellular processes ranging from genome integrity in model yeasts to tissue differentiation in vertebrates. Mice infected with the C. albicans rfx1 deletion mutant displayed increased fungal burden in tongues compared to animals infected with the reference strain. High-resolution imaging revealed RFX1 transcripts being expressed by C. albicans cells during infection. Concomitant with the increase in fungal burden, the rfx1 mutant elicited an enhanced innate immune response. Transcriptome analyses uncovered HWP1, a gene encoding an adhesion protein that mediates covalent attachment to buccal cells, as a major RFX1-regulated locus. Consistent with this result, we establish that C. albicans adhesion to oral cells is modulated by RFX1 in an HWP1-dependent manner. Our findings expand the repertoire of biological processes controlled by the RFX family and illustrate a mechanism whereby C. albicans can adjust adhesion to the oral epithelium.