Fungal burden, dimorphic transition and candidalysin: Role in Candida albicans-induced vaginal cell damage and mitochondrial activation in vitro.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans.

The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

Regulation of copper uptake by the SWI/SNF chromatin remodeling complex in Candida albicans affects susceptibility to antifungal and oxidative stresses under hypoxia.

Candida albicans is a human colonizer and also an opportunistic yeast occupying different niches that are mostly hypoxic. While hypoxia is the prevalent condition within the host, the machinery that integrates oxygen status to tune the fitness of fungal pathogens remains poorly characterized. Here, we uncovered that Snf5, a subunit of the chromatin remodeling complex SWI/SNF, is required to tolerate antifungal stress particularly under hypoxia. RNA-seq profiling of snf5 mutant exposed to amphotericin B and fluconazole under hypoxic conditions uncovered a signature that is reminiscent of copper (Cu) starvation. We found that under hypoxic and Cu-starved environments, Snf5 is critical for preserving Cu homeostasis and the transcriptional modulation of the Cu regulon. Furthermore, snf5 exhibits elevated levels of reactive oxygen species and an increased sensitivity to oxidative stress principally under hypoxia. Supplementing growth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated reactive oxygen species levels in response to antifungal challenge. Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in separate pathways. Together, our data underlined a unique role of SWI/SNF complex as a potent regulator of Cu metabolism and antifungal stress under hypoxia.

Candida albicans accelerates atherosclerosis by activating intestinal hypoxia-inducible factor2α signaling.

The gut microbiota is closely linked to atherosclerosis. However, the role of intestinal fungi, essential members of the complex microbial community, in atherosclerosis is poorly understood. Herein, we show that gut fungi dysbiosis is implicated in patients with dyslipidemia, characterized by higher levels of Candida albicans (C. albicans), which are positively correlated with plasma total cholesterol and low-density lipoprotein-cholesterol (LDL-C) levels. Furthermore, C. albicans colonization aggravates atherosclerosis progression in a mouse model of the disease. Through gain- and loss-of-function studies, we show that an intestinal hypoxia-inducible factor 2α (HIF-2α)-ceramide pathway mediates the effect of C. albicans. Mechanistically, formyl-methionine, a metabolite of C. albicans, activates intestinal HIF-2α signaling, which drives increased ceramide synthesis to accelerate atherosclerosis. Administration of the HIF-2α selective antagonist PT2385 alleviates atherosclerosis in mice by reducing ceramide levels. Our findings identify a role for intestinal fungi in atherosclerosis progression and highlight the intestinal HIF-2α-ceramide pathway as a target for atherosclerosis treatment.

Differential alteration in Lactiplantibacillus plantarum subsp. plantarum quorum-sensing systems and reduced Candida albicans yeast survival and virulence gene expression in dual-species interaction.

Candida albicans (C. albicans) and Lactiplantibacillus plantarum subsp. plantarum (L. plantarum) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans, including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS. Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans, indicating the potential interruption of pathogenic activities by L. plantarum. Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans. The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.IMPORTANCEThe anti-Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans.

Phenylacetic acid, an anti-vaginitis metabolite produced by the vaginal symbiotic bacterium Chryseobacterium gleum.

The human microbiome contains genetic information that regulates metabolic processes in response to host health and disease. While acidic vaginal pH is maintained in normal conditions, the pH level increases in infectious vaginitis. We propose that this change in the vaginal environment triggers the biosynthesis of anti-vaginitis metabolites. Gene expression levels of Chryseobacterium gleum, a vaginal symbiotic bacterium, were found to be affected by pH changes. The distinctive difference in the metabolic profiles between two C. gleum cultures incubated under acidic and neutral pH conditions was suggested to be an anti-vaginitis molecule, which was identified as phenylacetic acid (PAA) by spectroscopic data analysis. The antimicrobial activity of PAA was evaluated in vitro, showing greater toxicity toward Gardnerella vaginalis and Candida albicans, two major vaginal pathogens, relative to commensal Lactobacillus spp. The activation of myeloperoxidase, prostaglandin E2, and nuclear factor-κB, and the expression of cyclooxygenase-2 were reduced by an intravaginal administration of PAA in the vaginitis mouse model. In addition, PAA displayed the downregulation of mast cell activation. Therefore, PAA was suggested to be a messenger molecule that mediates interactions between the human microbiome and vaginal health.

Biofilm-associated metabolism via ERG251 in Candida albicans.

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.

Sur7 mediates a novel pathway for PI4,5P2 regulation in C. albicans that promotes stress resistance and cell wall morphogenesis.

The human fungal pathogen Candida albicans can cause lethal systemic infections due to its ability to resist stress from the host and to undergo invasive hyphal growth. Previous studies showed that plasma membrane MCC/eisosome domains were important for virulence by promoting the ability of Sur7 to mediate normal cell wall morphogenesis and stress resistance. The sur7Δ mutant displayed abnormal clusters of PI4,5P2, suggesting that misregulation of this lipid underlies the sur7Δ phenotype. To test this, we increased PI4,5P2 levels by deleting combinations of the three PI4,5P2 5' phosphatase genes (INP51, INP52, and INP54) and found that some combinations, such as inp51Δ inp52Δ, gave phenotypes similar the sur7Δ mutant. In contrast, deleting one copy of MSS4, the gene that encodes the 5' kinase needed to create PI4,5P2, reduced the abnormal PI4,5P2 clusters and also decreased the abnormal cell wall and stress sensitive phenotypes of the sur7Δ mutant. Additional studies support a model that the abnormal PI4,5P2 patches recruit septin proteins, which in turn promote aberrant cell wall growth. These results identify Sur7 as a novel regulator of PI4,5P2 and highlight the critical role of PI4,5P2 in the regulation of C. albicans virulence properties.

Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

Lethal metabolism of Candida albicans respiratory mutants.

The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models.

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity. IMPORTANCE: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans, outcompete beneficial species, such as Streptococcus gordonii, in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries.

Drug Repurposing of ACT001 to Discover Novel Promising Sulfide Prodrugs with Improved Safety and Potent Activity for Neutrophil-Mediated Antifungal Immunotherapy.

Neutrophil-mediated immunotherapy is a promising strategy for treating Candida albicans infection due to its potential in dealing with drug-resistant events. Our previous study found that ACT001 exhibited good antifungal immunotherapeutic activity by inhibiting PD-L1 expression in neutrophils, but its strong cytotoxicity and high BBB permeability hindered its antifungal application. To address these deficiencies, a series of novel sulfide derivatives were designed and synthesized based on a slow-release prodrug strategy. Among these derivatives, compound 16 exhibited stronger inhibition of PD-L1 expression, less cytotoxicity to neutrophils, and lower BBB permeability than ACT001. Compound 16 also significantly enhanced neutrophil-mediated antifungal immunity in C. albicans infected mice, with acceptable pharmacokinetic properties and good oral safety. Moreover, pharmacological mechanism studies demonstrated that ACT001 and compound 16 reduced PD-L1 expression in neutrophils by directly targeting STAT3. Briefly, this study provided a novel prototype compound 16 which exhibited great potential in neutrophil-mediated antifungal immunotherapy.

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.

Divergent mitochondrial responses and metabolic signal pathways secure the azole resistance in Crabtree-positive and negative Candida species.

Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat to human health. Several reports have shown that mitochondria are associated with the virulence of pathogenic fungi. However, there are few studies on the mechanisms of mitochondria-mediated azoles resistance. Here, we first performed mitochondrial proteomic analysis on multiple Candida species (Candida albicans, Nakaseomyces glabrata, Pichia kudriavzevii, and Candida auris) and analyzed the differentially expressed mitochondrial proteins (DEMPs) between azole-sensitive and azole-resistant Candida species. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology analysis, and protein-protein interaction network analysis of DEMPs. Our results showed that a total of 417, 165, and 25 DEMPs were identified in resistant C. albicans, N. glabrata, and C. auris, respectively. These DEMPs were enriched in ribosomal biogenesis at cytosol and mitochondria, tricarboxylic acid cycle, glycolysis, transporters, ergosterol, and cell wall mannan biosynthesis. The high activations of these cellular activities, found in C. albicans and C. auris (at low scale), were mostly opposite to those observed in two fermenter species-N. glabrata and P. kudriavzevii. Several transcription factors including Rtg3 were highly produced in resistant C. albicans that experienced a complex I activation of mitochondrial electron transport chain (ETC). The reduction of mitochondrial-related activities and complex IV/V of ETC in N. glabrata and P. kudriavzevii was companying with the reduced proteins of Tor1, Hog1, and Snf1/Snf4.IMPORTANCECandida spp. are common organisms that cause a variety of invasive diseases. However, Candida spp. are resistant to azoles, which hinders antifungal therapy. Exploring the drug-resistance mechanism of pathogenic Candida spp. will help improve the prevention and control strategy and discover new targets. Mitochondria, as an important organelle in eukaryotic cells, are closely related to a variety of cellular activities. However, the role of mitochondrial proteins in mediating azole resistance in Candida spp. has not been elucidated. Here, we analyzed the mitochondrial proteins and signaling pathways that mediate azole resistance in Candida spp. to provide ideas and references for solving the problem of azole resistance. Our work may offer new insights into the connection between mitochondria and azoles resistance in pathogenic fungi and highlight the potential clinical value of mitochondrial proteins in the treatment of invasive fungal infections.

Alpha1-antitrypsin impacts innate host-pathogen interactions with Candida albicans by stimulating fungal filamentation.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic ß-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences.

The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.

Nanobody-mediated neutralization of candidalysin prevents epithelial damage and inflammatory responses that drive vulvovaginal candidiasis pathogenesis.

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.IMPORTANCEWorldwide, vaginal infections caused by Candida albicans (VVC) annually affect millions of women, with symptoms significantly impacting quality of life. Current treatments are based on anti-fungals and probiotics that target the fungus. However, in some cases, infections are recurrent, called recurrent VVC, which often fails to respond to treatment. Vaginal mucosal tissue damage caused by the C. albicans peptide toxin candidalysin is a key driver in the induction of hyperinflammatory responses that fail to clear the infection and contribute to immunopathology and disease severity. In this pre-clinical evaluation, we show that nanobody-mediated candidalysin neutralization reduces tissue damage and thereby limits inflammation. Implementation of candidalysin-neutralizing nanobodies may prove an attractive strategy to alleviate symptoms in complicated VVC cases.