Daniel Prieto

The HOG pathway is critical for the colonization of the mouse gastrointestinal tract by Candida albicans.

The opportunistic pathogen Candida albicans is a frequent inhabitant of the human gastrointestinal tract where it usually behaves as a harmless commensal. In this particular niche, it needs to adapt to the different micro environments that challenge its survival within the host. In order to determine those factors involved in gut adaptation, we have used a gastrointestinal model of colonization in mouse to trace the behaviour of fungal cells. We have developed a genetic labelling system based on the complementary spectral properties of the fluorescent proteins GFP and a new C. albicans codon-adapted RFP (dTOM2) that allow a precise quantification of the fungal population in the gut via standard in vitro cultures or flow cytometry. This methodology has allowed us to determine the role of the three MAP kinase pathways of C. albicans (mediated by the MAPK Mkc1, Cek1 or Hog1) in mouse gut colonization via competitive assays with MAPK pathway mutants and their isogenic wild type strain. This approach reveals the signalling through HOG pathway as a critical factor influencing the establishment of C. albicans in the mouse gut. Less pronounced effects for mkc1 or cek1 mutants were found, only evident after 2–3 weeks of colonization. We have also seen that hog1 mutants is defective in adhesion to the gut mucosa and sensitive to bile salts. Finally, we have developed a genetic strategy for the in vivo excision (tetracycline-dependent) of any specific gene during the course of colonization in this particular niche, allowing the analysis of its role during gut colonization.

The Sko1 protein represses the yeast-to-hypha transition and regulates the oxidative stress response in Candida albicans

Cells respond to environmental changes triggering adaptive responses which are, in part, mediated by a transcriptional response. These responses are complex and are dependent on different transcription factors. The present work reports the implication of the Sko1 protein in several processes relevant to the physiology of Candida albicans. First, Sko1 acts as transcriptional repressor of genes involved in pathogenesis and hyphal formation, which results in increased expression of the hyphal related genes ECE1 and HWP1 without significant changes in the virulence using a mouse model of systemic infection. Second Sko1 is involved in the response to oxidative stress and sko1 mutants increase the sensitivity of hog1 to the myelomonocytic cell line HL-60. Genome-wide transcriptional analysis after hydrogen peroxide treatment revealed that sko1 mutants were able to generate an adaptive response similar to wild type strains, although important differences were detected in the magnitude of the transcriptional response. Collectively, these results implicate Sko1 as an important mediator of the oxidative stress response in C. albicans.

The role of the cell wall in fungal pathogenesis

Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.

Distinct stages during colonization of the mouse gastrointestinal tract by Candida albicans

Candida albicans is a member of the human microbiota, colonizing both the vaginal and gastrointestinal tracts. This yeast is devoid of a life style outside the human body and the mechanisms underlying the adaptation to the commensal status remain to be determined. Using a model of mouse gastrointestinal colonization, we show here that C. albicans stably colonizes the mouse gut in about 3 days starting from a dose as low as 100 cells, reaching steady levels of around 107 cells/g of stools. Using fluorescently labeled strains, we have assessed the competition between isogenic populations from different sources in cohoused animals. We show that long term (15 days) colonizing cells have increased fitness in the gut niche over those grown in vitro or residing in the gut for 1–3 days. Therefore, two distinct states, proliferation and adaptation, seem to exist in the adaptation of this fungus to the mouse gut, a result with potential significance in the prophylaxis and treatment of Candida infections.

The Pho4 transcription factor mediates the response to arsenate and arsenite in Candida albicans

Arsenate (As (V)) is the dominant form of the toxic metalloid arsenic (As). Microorganisms have consequently developed mechanisms to detoxify and tolerate this kind of compounds. In the present work, we have explored the arsenate sensing and signaling mechanisms in the pathogenic fungus Candida albicans. Although mutants impaired in the Hog1 or Mkc1-mediated pathways did not show significant sensitivity to this compound, both Hog1 and Mkc1 became phosphorylated upon addition of sodium arsenate to growing cells. Hog1 phosphorylation upon arsenate challenge was shown to be Ssk1-dependent. A screening designed for the identification of transcription factors involved in the arsenate response identified Pho4, a transcription factor of the myc-family, as pho4 mutants were susceptible to As (V). The expression of PHO4 was shortly induced in the presence of sodium arsenate in a Hog1-independent manner. Pho4 level affects Hog1 phosphorylation upon As (V) challenge, suggesting an indirect relationship between Pho4 activity and signaling in C. albicans. Pho4 also mediates the response to arsenite as revealed by the fact that pho4 defective mutants are sensitive to arsenite and Pho4 becomes phosphorylated upon sodium arsenite addition. Arsenite also triggers Hog1 phosphorylation by a process that is, in this case, independent of the Ssk1 kinase. These results indicate that the HOG pathway mediates the response to arsenate and arsenite in C. albicans and that the Pho4 transcription factor can differentiate among As (III), As (V) and Pi, triggering presumably specific responses.

CX3CR1+ mononuclear phagocytes control immunity to intestinal fungi

Phagocytes patrol intestinal fungi Maintaining a healthy balance of gut bacteria can promote good health. Leonardi et al. show that fungi can also interact with gut immune cells to maintain intestinal well-being. CX3CR1+ mononuclear phagocytes (MNPs) patrol the intestine and promote antifungal immunity. Genetic deletion of CX3CR1 in MNPs caused colitis-like symptoms in mice. CX3CR1 polymorphisms were detected in Crohns disease patients that were unable to produce antibodies against multiple fungal species. Thus, commensal fungi may be as important as bacteria in maintaining gut health, and antifungal therapy could hold promise for treating intestinal inflammation. Science, this issue p. 232 Phagocytes police the fungal microbiome. Intestinal fungi are an important component of the microbiota, and recent studies have unveiled their potential in modulating host immune homeostasis and inflammatory disease. Nonetheless, the mechanisms governing immunity to gut fungal communities (mycobiota) remain unknown. We identified CX3CR1+ mononuclear phagocytes (MNPs) as being essential for the initiation of innate and adaptive immune responses to intestinal fungi. CX3CR1+ MNPs express antifungal receptors and activate antifungal responses in a Syk-dependent manner. Genetic ablation of CX3CR1+ MNPs in mice led to changes in gut fungal communities and to severe colitis that was rescued by antifungal treatment. In Crohn’s disease patients, a missense mutation in the gene encoding CX3CR1 was identified and found to be associated with impaired antifungal responses. These results unravel a role of CX3CR1+ MNPs in mediating interactions between intestinal mycobiota and host immunity at steady state and during inflammatory disease.

Adaptation of Candida albicans to commensalism in the gut

Candida albicans is a common resident of the oral cavity, GI tract and vagina in healthy humans where it establishes a commensal relationship with the host. Colonization of the gut, which is an important niche for the microbe, may lead to systemic dissemination and disease upon alteration of host defences. Understanding the mechanisms responsible for the adaptation of C. albicans to the gut is therefore important for the design of new ways of combating fungal diseases. In this review we discuss the available models to study commensalism of this yeast, the main mechanisms controlling the establishment of the fungus, such as microbiota, mucus layer and antimicrobial peptides, and the gene regulatory circuits that ensure its survival in this niche.

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 Acidic Ribosomal Stalk Proteins Are Not Required for the Highly Specific Inactivation Exerted by α-Sarcin of the Eukaryotic Ribosome

The ribosomal sarcin/ricin loop (SRL) is the target of ribosome-inactivating proteins like the N-glycosidase ricin and the fungal ribotoxin α-sarcin. The eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the SRL. Here we tested this hypothesis for the ribotoxin α-sarcin. Experiments with isolated ribosomes, cell-free translation systems, and viability assays with Saccharomyces cerevisiae strains defective in acidic stalk proteins showed that the inactivation exerted by α-sarcin is independent of the composition of the ribosomal stalk. Therefore, α-sarcin, with the same ribosomal target as ricin, seems to access the SRL by a different pathway.

Complementary roles of the Cek1 and Cek2 MAP kinases in Candida albicans cell-wall biogenesis

AIMS To investigate whether Cek2 (as Cek1) is involved in the biogenesis of the fungal cell wall and to uncover differences and similitudes between both proteins. MATERIALS & METHODS We used molecular genetics to characterize the role of Cek2 in MTL-heterozygous cells. RESULTS Deletion of CEK2 (similar to CEK1) renders cells sensitive to cell-wall-interfering drugs and, when overexpressed, Cek2 can become phosphorylated upon the same stimuli that activate Cek1. This is dependent on elements of the sterile-vegetative growth (SVG) pathway. Cek2, contrary to Cek1, mediates a transcriptional activity in a C. albicans-adapted two-hybrid system that is essential for Cek1-Cek2 interaction. CONCLUSION Cek2 has a cryptic role in cell-wall biogenesis and its role is not entirely redundant to Cek1.