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.

Comparative Analysis of the Fitness of Candida albicans Strains During Colonization of the Mice Gastrointestinal Tract.

Candida albicans populations present in the mammalian gastrointestinal tract are a major source of candidemia and subsequent severe invasive candidiasis in those individuals with acquired or congenital immune defects. Understanding the mechanisms used by this fungus to colonize this niche is, therefore, of primary importance to develop new therapeutic options that could lead to control its proliferation in the host. The recent popularization of models of commensalism in mice combined with the already powerful tools in C. albicans genetics allows to analyze the role of specific genes during colonization. Fitness can be analyzed for a specific C. albicans strain (test strain) by comparing its growth in vivo with an otherwise isogenic control strain via the analysis of the luminal content of the mouse gastrointestinal tract using flow cytometry, qPCR, or viable fungal cell counting. While all these procedures have limitations, they can be used to estimate the degree of adaptation of the test strain to the mammalian tract by determining its relative abundance with an internal control strain. By using specific genetically engineered C. albicans and mouse strains, antibiotic regimes, or even germ-free mice, this methodology allows to determine the role of the host immunological status, the bacterial microbiota, or individual fungal features (e.g., dimorphism) in the process of colonization of C. albicans of the mammalian gut.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Mycobiota-induced IgA antibodies regulate fungal commensalism in the gut and are dysregulated in Crohn's disease.

Secretory immunoglobulin A (sIgA) plays an important role in gut barrier protection by shaping the resident microbiota community, restricting the growth of bacterial pathogens and enhancing host protective immunity via immunological exclusion. Here, we found that a portion of the microbiota-driven sIgA response is induced by and directed towards intestinal fungi. Analysis of the human gut mycobiota bound by sIgA revealed a preference for hyphae, a fungal morphotype associated with virulence. Candida albicans was a potent inducer of IgA class-switch recombination among plasma cells, via an interaction dependent on intestinal phagocytes and hyphal programming. Characterization of sIgA affinity and polyreactivity showed that hyphae-associated virulence factors were bound by these antibodies and that sIgA influenced C. albicans morphotypes in the murine gut. Furthermore, an increase in granular hyphal morphologies in patients with Crohn's disease compared with healthy controls correlated with a decrease in antifungal sIgA antibody titre with affinity to two hyphae-associated virulence factors. Thus, in addition to its importance in gut bacterial regulation, sIgA targets the uniquely fungal phenomenon of hyphal formation. Our findings indicate that antifungal sIgA produced in the gut can play a role in regulating intestinal fungal commensalism by coating fungal morphotypes linked to virulence, thereby providing a protective mechanism that might be dysregulated in patients with Crohn's disease.

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.

The Glyoxylate Cycle Is Involved in White-Opaque Switching in Candida albicans.

Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans. The master regulator of the white-opaque transition WOR1 has been implicated in the adaptation to this commensal status. A proteomic analysis of cells overexpressing this transcription factor (WOR1OE) suggested an altered metabolism of carbon sources and a phenotypic analysis confirmed this alteration. The WOR1OE cells are deficient in using trehalose and xylose and are unable to use 2C sources, which is consistent with a reduction in the amount of Icl1, the isocitrate lyase enzyme. The icl1Δ/Δ mutants overexpressing WOR1 are deficient in the production of phloxine B positive cells, a main characteristic of opaque cells, a phenotype also observed in mating type hemizygous mtla1Δ icl1Δ/Δ cells, suggesting the involvement of Icl1 in the adaptation to the commensal state. In fact, icl1Δ/Δ cells have reduced fitness in mouse gastrointestinal tract as compared with essentially isogenic heterozygous ICL1/icl1Δ, but overproduction of WOR1 in an icl1Δ/Δ mutant does not restore fitness. These results implicate the glyoxylate shunt in the adaptation to commensalism of C. albicans by mechanisms that are partially independent of WOR1.

The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

Adaptation to Endoplasmic Reticulum Stress in Candida albicans Relies on the Activity of the Hog1 Mitogen-Activated Protein Kinase.

Adaptation to ER stress is linked to the pathogenicity of C. albicans. The fungus responds to ER stress primarily by activating the conserved Ire1-Hac1-dependent unfolded protein response (UPR) pathway. Subsequently, when ER homeostasis is re-established, the UPR is attenuated in a timely manner, a facet that is unexplored in C. albicans. Here, we show that C. albicans licenses the HOG (high-osmolarity glycerol) MAPK pathway for abating ER stress as evidenced by activation and translocation of Hog1 to the nucleus during tunicamycin-induced ER stress. We find that, once activated, Hog1 attenuates the activity of Ire1-dependent UPR, thus facilitating adaptation to ER stress. We use the previously established assay, where the disappearance of the UPR-induced spliced HAC1 mRNA correlates with the re-establishment of ER homeostasis, to investigate attenuation of the UPR in C. albicans. hog1Δ/Δ cells retain spliced HAC1 mRNA levels for longer duration reflecting the delay in attenuating Ire1-dependent UPR. Conversely, compromising the expression of Ire1 (ire1 DX mutant strain) results in diminished levels of phosphorylated Hog1, restating the cross-talk between Ire1 and HOG pathways. Phosphorylation signal to Hog1 MAP kinase is relayed through Ssk1 in response to ER stress as inactivation of Ssk1 abrogates Hog1 phosphorylation in C. albicans. Additionally, Hog1 depends on its cytosolic as well as nuclear activity for mediating ER stress-specific responses in the fungus. Our results show that HOG pathway serves as a point of cross-talk with the UPR pathway, thus extending the role of this signaling pathway in promoting adaptation to ER stress in C. albicans. Additionally, this study integrates this MAPK pathway into the little known frame of ER stress adaptation pathways in C. albicans.

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.

Characterization of a Candida albicans Mutant Defective in All MAPKs Highlights the Major Role of Hog1 in the MAPK Signaling Network.

The success of Candida albicans as a pathogen relies on its ability to adapt and proliferate in different environmental niches. Pathways regulated by mitogen-activated protein kinases (MAPKs) are involved in sensing environmental conditions and developing an accurate adaptive response. Given the frequent cooperative roles of these routes in cellular functions, we have generated mutants defective in all combinations of the four described MAPKs in C. albicans and characterized its phenotype regarding sensitiveness to specific drugs, morphogenesis and interaction with host immune cells. We demonstrate that all MAPKs are dispensable in this yeast as a mutant defective in Cek1, Cek2, Mkc1 and Hog1 is viable although highly sensitive to oxidative and osmotic stress, displaying a specific pattern of sensitivity to antifungals. By comparing its phenotype with single, double and triple combinations of MAPK-deletion mutants we were able to unveil a Cek1-independent mechanism for Hog1 resistance to Congo red, and confirm the predominant effect of Hog1 on oxidative and osmotic adaptation. The quadruple mutant produces filaments under non-inducing conditions, but is unable to develop chlamydospores. Furthermore, cek1 cek2 mkc1 hog1 cells switch to the opaque state at high frequency, which is blocked by the ectopic expression of HOG1 suggesting a role of this kinase for phenotypic switching.

The HOG MAPK pathway in Candida albicans: more than an osmosensing pathway.

In 1993, Brewster and Gustin described the existence of a kinase whose activity was essential for Saccharomyces cerevisiae to grow in environments with high osmolarity. This led to the discovery of the HOG pathway, a MAP kinase (MAPK) pathway that has been revealed to be crucial to respond to a wide range of stress conditions frequently encountered by fungi in their common habitats. MAPK signaling is initiated at the plasma membrane, where triggering stimuli lead to a phosphorylation cascade that ultimately activates transcription factors to ensure an appropriate adaptive response. In pathogenic fungi, the HOG pathway gains special significance as it is involved in traits related to pathogenicity; these include biofilm formation, adhesion to surfaces, and morphogenetic and epigenetic transitions. It also plays a role in controlling both the pathogen and the commensal state program. Understanding the signals leading to its activation, the elements of the pathways and the targets of the pathway are therefore of primary importance in the design of novel antifungals.

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.

Ifu5, a WW domain-containing protein interacts with Efg1 to achieve coordination of normoxic and hypoxic functions to influence pathogenicity traits in Candida albicans.

Hypoxic adaptation pathways, essential for Candida albicans pathogenesis, are tied to its transition from a commensal to a pathogen. Herein, we identify a WW domain-containing protein, Ifu5, as a determinant of hypoxic adaptation that also impacts normoxic responses in this fungus. Ifu5 activity supports glycosylation homeostasis via the Cek1 mitogen-activated protein kinase-dependent up-regulation of PMT1, under normoxia. Transcriptome analysis of ifu5Δ/Δ under normoxia shows a significant up-regulation of the hypoxic regulator EFG1 and EFG1-dependent genes. We demonstrate physical interaction between Ifu5 by virtue of its WW domain and Efg1 that represses EFG1 expression under normoxia. This interaction is lost under hypoxic growth conditions, relieving EFG1 repression. Hypoxic adaptation processes such as filamentation and biofilm formation are affected in ifu5Δ/Δ cells revealing the role of Ifu5 in hypoxic signalling and modulating pathogenicity traits of C. albicans under varied oxygen conditions. Additionally, the WW domain of Ifu5 facilitates its role in hypoxic adaptation, revealing the importance of this domain in providing a platform to integrate various cellular processes. These data forge a relationship between Efg1 and Ifu5 that fosters the role of Ifu5 in hypoxic adaptation thus illuminating novel strategies to undermine the growth of C. albicans.

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.