Morphogenetic transitions in the adaptation of Candida albicans to the mammalian gut.

Candida albicans is a pathobiont in humans that forms part of the mycobiota in healthy individuals and can cause different pathologies upon alterations of the host defenses. The mammalian gut is clinically relevant as this niche is the most common pool for bloodstream-derived infections. The ability of C. albicans to switch from yeast to hypha has been related to the commensal-to-pathogen transition and is, therefore, considered relevant in virulence. Recently, filaments have been implicated in the humoral response in the gut. C. albicans exhibits other morphologies that play different roles in pathogenicity and commensalism. This review focuses on the role of these morphological transitions in C. albicans proliferation and its establishment as a commensal in the mammalian gut, paying special attention to the transcription factors involved in their regulation.

The defective gut colonization of Candida albicans hog1 MAPK mutants is restored by overexpressing the transcriptional regulator of the white opaque transition WOR1.

The transcriptional master regulator of the white opaque transition of Candida albicans WOR1 is important for the adaptation to the commensal lifestyle in the mammalian gut, a major source of invasive candidiasis. We have generated cells that overproduce Wor1 in mutants defective in the Hog1 MAP kinase, defective in several stress responses and unable to colonize the mice gut. WOR1 overexpression allows hog1 to be established as a commensal in the murine gut in a commensalism model and even compete with wild-type C. albicans cells for establishment. This increased fitness correlates with an enhanced ability to adhere to biotic surfaces as well as increased proteinase and phospholipase production and a decrease in filamentation in vitro. We also show that hog1 WOR1OE are avirulent in a systemic candidiasis model in mice.

Overexpression of the White Opaque Switching Master Regulator Wor1 Alters Lipid Metabolism and Mitochondrial Function in Candida albicans.

Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans; increased colonization of this yeast in this niche has implicated the master regulator of the white-opaque transition, Wor1, by mechanisms not completely understood. We have addressed the role that this transcription factor has on commensalism by the characterization of strains overexpressing this gene. We show that WOR1 overexpression causes an alteration of the total lipid content of the fungal cell and significantly alters the composition of structural and reserve molecular species lipids as determined by lipidomic analysis. These cells are hypersensitive to membrane-disturbing agents such as SDS, have increased tolerance to azoles, an augmented number of peroxisomes, and increased phospholipase activity. WOR1 overexpression also decreases mitochondrial activity and results in altered susceptibility to certain oxidants. All together, these changes reflect drastic alterations in the cellular physiology that facilitate adaptation to the gastrointestinal tract environment.

Identification of Clinical Isolates of Candida albicans with Increased Fitness in Colonization of the Murine Gut.

The commensal and opportunistic pathogen Candida albicans is an important cause of fungal diseases in humans, with the gastrointestinal tract being an important reservoir for its infections. The study of the mechanisms promoting the C. albicans commensal state has attracted considerable attention over the last few years, and several studies have focused on the identification of the intestinal human mycobiota and the characterization of Candida genes involved in its establishment as a commensal. In this work, we have barcoded 114 clinical C. albicans isolates to identify strains with an enhanced fitness in a murine gastrointestinal commensalism model. The 114 barcoded clinical isolates were pooled in four groups of 28 to 30 strains that were inoculated by gavage in mice previously treated with antibacterial therapy. Eight strains that either exhibited higher colonization load and/or remained in the gut after antibiotic removal were selected. The phenotypic analysis of these strains compared to an RFP-tagged SC5314 wild type strain did not reveal any specific trait associated with its increased colonization; all strains were able to filament and six of the eight strains displayed invasive growth on Spider medium. Analysis of one of these strains, CaORAL3, revealed that although mice required previous bacterial microbiota reduction with antibiotics to be able to be colonized, removal of this procedure could take place the same day (or even before) Candida inoculation. This strain was able to colonize the intestine of mice already colonized with Candida without antibiotic treatment in co-housing experiments. CaORAL3 was also able to be established as a commensal in mice previously colonized by another (CaHG43) or the same (CaORAL3) C. albicans strain. Therefore, we have identified C. albicans isolates that display higher colonization load than the standard strain SC5314 which will surely facilitate the analysis of the factors that regulate fungal colonization.

Hog1 Controls Lipids Homeostasis Upon Osmotic Stress in Candida albicans.

As opportunistic pathogen, Candida albicans adapts to different environmental conditions and its corresponding stress. The Hog1 MAPK (Mitogen Activated Protein Kinase) was identified as the main MAPK involved in the response to osmotic stress. It was later shown that this MAPK is also involved in the response to a variety of stresses and therefore, its role in virulence, survival to phagocytes and establishment as commensal in the mouse gastrointestinal tract was reported. In this work, the role of Hog1 in osmotic stress is further analyzed, showing that this MAPK is involved in lipid homeostasis. The hog1 mutant accumulates lipid droplets when exposed to osmotic stress, leading to an increase in cell permeability and delaying the endocytic trafficking routes. Cek1, a MAPK also implicated in the response to osmotic challenge, did not play a role in lipid homeostasis indicating that Hog1 is the main MAP kinase in this response. The alteration on lipid metabolism observed in hog1 mutants is proposed to contribute to the sensitivity to osmotic stress.

The Fungicidal Action of Micafungin is Independent on Both Oxidative Stress Generation and HOG Pathway Signaling in Candida albicans.

In fungi, the Mitogen-Activated Protein kinase (MAPK) pathways sense a wide variety of environmental stimuli, leading to cell adaptation and survival. The HOG pathway plays an essential role in the pathobiology of Candida albicans, including the colonization of the gastrointestinal tract in a mouse model, virulence, and response to stress. Here, we examined the role of Hog1 in the C. albicans response to the clinically relevant antifungal Micafungin (MF), whose minimum inhibitory concentration (MIC) was identical in the parental strain (RM100) and in the isogenic homozygous mutant hog1 (0.016 mg/L). The cell viability was impaired without significant differences between the parental strain, the isogenic hog1 mutant, and the Hog1+ reintegrant. This phenotype was quite similar in a collection of hog1 mutants constructed in a different C. albicans background. MF-treated cells failed to induce a relevant increase of both reactive oxygen species (ROS) formation and activation of the mitochondrial membrane potential in parental and hog1 cells. MF was also unable to trigger any significant activation of the genes coding for the antioxidant activities catalase (CAT1) and superoxide dismutase (SOD2), as well as on the corresponding enzymatic activities, whereas a clear induction was observed in the presence of Amphotericin B (AMB), introduced as a positive control of Hog1 signaling. Furthermore, Hog1 was not phosphorylated by the addition of MF, but, notably, this echinocandin caused Mkc1 phosphorylation. Our results strongly suggest that the toxic effect of MF on C. albicans cells is not mediated by the Hog1 MAPK and is independent of the generation of an internal oxidative stress in C. albicans.

The MAPK Hog1 mediates the response to amphotericin B in Candida albicans.

The HOG MAP kinase pathway plays a crucial role in the response to different stresses in the opportunistic pathogen Candida albicans. The polyene amphotericin B (AMB) has been reported to trigger oxidative stress in several pathogenic fungi, including C. albicans. In the present work, we have analyzed the role of the MAPK Hog1 in sensing and survival to AMB treatment. Mutants lacking Hog1 are more susceptible to AMB than their parental strains and Hog1 became phosphorylated in the presence of this polyene. A set of mutated versions of Hog1 revealed that both the kinase activity and phosphorylation of Hog1 are required to cope with AMB treatment. Flow cytometry analysis showed that AMB induced intracellular ROS accumulation in both parental and hog1 null mutant strains. In addition, AMB triggered a Hog1-independent synthesis of trehalose. The addition of rotenone to AMB-treated cells improved cell viability, decreased intracellular ROS and prevented intracellular trehalose accumulation, suggesting that AMB-induced ROS is associated to a functional electron transport chain but the presence of rotenone did not impair Hog1 phosphorylation in AMB-treated cells. Our results indicate that Hog1 is necessary during AMB treatment to increase its survival.

Cooperative Role of MAPK Pathways in the Interaction of Candida albicans with the Host Epithelium.

Candida albicans is an important human fungal pathogen responsible for tens of millions of infections as well as hundreds of thousands of severe life-threatening infections each year. MAP kinase (MAPK) signal transduction pathways facilitate the sensing and adaptation to external stimuli and control the expression of key virulence factors such as the yeast-to-hypha transition, the biogenesis of the cell wall, and the interaction with the host. In the present study, we have combined molecular approaches and infection biology to analyse the role of C. albicans MAPK pathways during an epithelial invasion. Hog1 was found to be important for adhesion to abiotic surfaces but was dispensable for damage to epithelial cells. The Mkc1 cell wall integrity (CWI) and Cek1 pathways, on the other hand, were both required for oral epithelial damage. Analysis of the ability to penetrate nutrient-rich semi-solid media revealed a cooperative role for Cek1 and Mkc1 in this process. Finally, cek2Δ (as well as cek1Δ) but not mkc1Δ or hog1Δ mutants, exhibited elevated ß-glucan unmasking as revealed by immunofluorescence studies. Therefore, the four MAPK pathways play distinct roles in adhesion, epithelial damage, invasion and cell wall remodelling that may contribute to the pathogenicity of C. albicans.

Deletion of the SKO1 Gene in a hog1 Mutant Reverts Virulence in Candida albicans.

Candida albicans displays the ability to adapt to a wide variety of environmental conditions, triggering signaling pathways and transcriptional regulation. Sko1 is a transcription factor that was previously involved in early hypoxic response, cell wall remodeling, and stress response. In the present work, the role of sko1 mutant in in vivo and ex vivo studies was explored. The sko1 mutant behaved as its parental wild type strain regarding the ability to colonize murine intestinal tract, ex vivo adhesion to murine gut epithelium, or systemic virulence. These observations suggest that Sko1 is expendable during commensalism or pathogenesis. Nevertheless, the study of the hog1 sko1 double mutant showed unexpected phenotypes. Previous researches reported that the deletion of the HOG1 gene led to avirulent C. albicans mutant cell, which was, therefore, unable to establish as a commensal in a gastrointestinal murine model. Here, we show that the deletion of sko1 in a hog1 background reverted the virulence of the hog1 mutant in a systemic infection model in Galleria mellonella larvae and slightly improved the ability to colonize the murine gut in a commensalism animal model compared to the hog1 mutant. These results indicate that Sko1 acts as a repressor of virulence related genes, concluding that Sko1 plays a relevant role during commensalism and systemic infection.

New insights of CRISPR technology in human pathogenic fungi.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems have emerged as a powerful tool for genome manipulation. Class 2 type II CRISPR/CAS9 is so far the most studied system and has been implemented in many biological systems such as mammalian cells, plants, fungi and bacteria. Fungi are important causes of human diseases worldwide. Genetic manipulation of pathogenic fungi is critical to develop new therapeutic approaches and novel antifungals. We will review here the progress done with CRISPR/CAS9 systems in human pathogenic fungi, with emphasis in Candida albicans and the main modifications that have improved their usefulness in biological research. We finally discuss possible future outcomes and applications to the developed in a near future.

Implementation of a CRISPR-Based System for Gene Regulation in Candida albicans.

Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast Candida albicans By fusing an allele of Streptococcus pyogenes Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene CAT1, encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of CAT1 controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen.IMPORTANCE CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in Candida albicans using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Overexpression of the Transcriptional Regulator WOR1 Increases Susceptibility to Bile Salts and Adhesion to the Mouse Gut Mucosa in Candida albicans.

The transcriptional regulator Wor1 has been shown to induce the GUT transition, an environmentally triggered process that increases the fitness of Candida albicans in the mouse gastrointestinal tract. We have developed strains where the expression of this gene is driven from the strong and tightly regulated tetracycline promoter. These cells retain the main characteristics reported for GUT cells albeit they show defects in the initial stages of colonization. They also show a differential colonization along the gastrointestinal tract compared to isogenic strains, which is probably caused by their susceptibility to bile salts. We also show that WOR1 overexpressing cells have an altered metabolic activity, as revealed by a different susceptibility to inhibitors of respiration, and an enhanced adhesion to the mouse mucosa. We propose that this may contribute to their long-term favored ability to colonize the gastrointestinal tract.

Arsenic inorganic compounds cause oxidative stress mediated by the transcription factor PHO4 in Candida albicans.

Arsenic is a toxic metalloid widespread in nature. Recently, it has been demonstrated a main role of the transcription factor Pho4 in the acquisition of tolerance to arsenic-derived compounds, arsenite and arsenate in Candida albicans. Here, the effect of these compounds on this pathogenic yeast has been analyzed. In wild type cells, both arsenite and arsenate induced a marked increase in the endogenous production of Reactive Oxygen Species (ROS), together with the accumulation of intracellular trehalose and the activation of catalase, suggesting their role as generators of oxidative stress in this yeast. However, a pho4 null mutant showed a minor increase of intracellular ROS and a different kinetics of catalase activation upon exposure to arsenite and arsenate. Interestingly, the enzymatic activity of glutathione reductase and superoxide dismutase were exclusively triggered by arsenite but not by arsenate. pho4 mutant cells were also found to be sensitive to azide but significantly resistant to arsenate through a process dependent on an active electron transport chain and the alternative oxidase system. Therefore, arsenic-derived compounds induce a strong antioxidant response in C.albicans via different mechanisms.

Corrigendum: The Hog1 MAP Kinase Promotes the Recovery from Cell Cycle Arrest Induced by Hydrogen Peroxide in Candida albicans.

[This corrects the article on p. 2133 in vol. 7, PMID: 28111572.].

Role of catalase overproduction in drug resistance and virulence in Candida albicans.

AIM: To investigate the role of Cat1 overproduction in Candida albicans. MATERIALS & METHODS: Strains overproducing the CAT1 gene were constructed. RESULTS: Cells overproducing CAT1 were found to be more resistant to some oxidants and mammalian phagocytic cells. They also showed reduced intracellular reactive oxygen species generated by amphotericin B or ciclopirox olamine. CAT1 overproduction did not change the minimum inhibitory concentration of fungal cells to fungistatic or fungicidal azoles nor to amphotericin B although increased twofold the minimum inhibitory concentration to caspofungin. The role of Cat1 overproduction in virulence and colonization was also analyzed in mouse models. CONCLUSION: The overproduction of Cat1 protects against oxidants, phagocytes and certain antifungals at subinhibitory concentration but does not increase virulence in a systemic infection mouse model.

The Candida albicans Pho4 Transcription Factor Mediates Susceptibility to Stress and Influences Fitness in a Mouse Commensalism Model.

The Pho4 transcription factor is required for growth under low environmental phosphate concentrations in Saccharomyces cerevisiae. A characterization of Candida albicans pho4 mutants revealed that these cells are more susceptible to both osmotic and oxidative stress and that this effect is diminished in the presence of 5% CO2 or anaerobiosis, reflecting the relevance of oxygen metabolism in the Pho4-mediated response. A pho4 mutant was as virulent as wild type strain when assayed in the Galleria mellonella infection model and was even more resistant to murine macrophages in ex vivo killing assays. The lack of Pho4 neither impairs the ability to colonize the murine gut nor alters the localization in the gastrointestinal tract. However, we found that Pho4 influenced the colonization of C. albicans in the mouse gut in competition assays; pho4 mutants were unable to attain high colonization levels when inoculated simultaneously with an isogenic wild type strain. Moreover, pho4 mutants displayed a reduced adherence to the intestinal mucosa in a competitive ex vivo assays with wild type cells. In vitro competitive assays also revealed defects in fitness for this mutant compared to the wild type strain. Thus, Pho4, a transcription factor involved in phosphate metabolism, is required for adaptation to stress and fitness in C. albicans.

The Hog1 MAP Kinase Promotes the Recovery from Cell Cycle Arrest Induced by Hydrogen Peroxide in Candida albicans.

Eukaryotic cell cycle progression in response to environmental conditions is controlled via specific checkpoints. Signal transduction pathways mediated by MAPKs play a crucial role in sensing stress. For example, the canonical MAPKs Mkc1 (of the cell wall integrity pathway), and Hog1 (of the HOG pathway), are activated upon oxidative stress. In this work, we have analyzed the effect of oxidative stress induced by hydrogen peroxide on cell cycle progression in Candida albicans. Hydrogen peroxide was shown to induce a transient arrest at the G1 phase of the cell cycle. Specifically, a G1 arrest was observed, although phosphorylation of Mkc1 and Hog1 MAPKs can take place at all stages of the cell cycle. Interestingly, hog1 (but not mkc1) mutants required a longer time compared to wild type cells to resume growth after hydrogen peroxide challenge. Using GFP-labeled cells and mixed cultures of wild type and hog1 cells we were able to show that hog1 mutants progress faster through the cell cycle under standard growth conditions in the absence of stress (YPD at 37°C). Consequently, hog1 mutants exhibited a smaller cell size. The altered cell cycle progression correlates with altered expression of the G1 cyclins Cln3 and Pcl2 in hog1 cells compared to the wild type strain. In addition, Hgc1 (a hypha-specific G1 cyclin) as well as Cln3 displayed a different kinetics of expression in the presence of hydrogen peroxide in hog1 mutants. Collectively, these results indicate that Hog1 regulates the expression of G1 cyclins not only in response to oxidative stress, but also under standard growth conditions. Hydrogen peroxide treated cells did not show fluctuations in the mRNA levels for SOL1, which are observed in untreated cells during cell cycle progression. In addition, treatment with hydrogen peroxide prevented degradation of Sol1, an effect which was enhanced in hog1 mutants. Therefore, in C. albicans, the MAPK Hog1 mediates cell cycle progression in response to oxidative stress, and further participates in the cell size checkpoint during vegetative growth.

The Mkk2 MAPKK Regulates Cell Wall Biogenesis in Cooperation with the Cek1-Pathway in Candida albicans.

The cell wall integrity pathway (CWI) plays an important role in the biogenesis of the cell wall in Candida albicans and other fungi. In the present work, the C. albicans MKK2 gene that encodes the putative MAPKK of this pathway was deleted in different backgrounds and the phenotypes of the resultant mutants were characterised. We show here that Mkk2 mediates the phosphorylation of the Mkc1 MAPK in response to cell wall assembly interfering agents such as zymolyase or tunicamycin and also to oxidative stress. Remarkably, mkk2 and mkc1 mutants display related but distinguishable- cell wall associated phenotypes and differ in the pattern of MAPK phosphorylation under different stress conditions. mkk2 and mkc1 mutants display an altered expression of GSC1, CEK1 and CRH11 genes at different temperatures. Combined deletion of MKK2 with HST7 supports a cooperative role for the Cek1-mediated and CWI pathways in regulating cell wall architecture under vegetative growth. However, and in contrast to Mkc1, Mkk2 does not seem to play a role in the virulence of C. albicans in the mouse systemic model or the Galleria mellonella model of infection.