Candida albicans Hexokinase 2 Challenges the Saccharomyces cerevisiae Moonlight Protein Model

Survival of the pathogenic yeast Candida albicans depends upon assimilation of fermentable and non-fermentable carbon sources detected in host microenvironments. Among the various carbon sources encountered in a human body, glucose is the primary source of energy. Its effective detection, metabolism and prioritization via glucose repression are primordial for the metabolic adaptation of the pathogen. In C. albicans, glucose phosphorylation is mainly performed by the hexokinase 2 (CaHxk2). In addition, in the presence of glucose, CaHxK2 migrates in the nucleus and contributes to the glucose repression signaling pathway. Based on the known dual function of the Saccharomyces cerevisiae hexokinase 2 (ScHxk2), we intended to explore the impact of both enzymatic and regulatory functions of CaHxk2 on virulence, using a site-directed mutagenesis approach. We show that the conserved aspartate residue at position 210, implicated in the interaction with glucose, is essential for enzymatic and glucose repression functions but also for filamentation and virulence in macrophages. Point mutations and deletion into the N-terminal region known to specifically affect glucose repression in ScHxk2 proved to be ineffective in CaHxk2. These results clearly show that enzymatic and regulatory functions of the hexokinase 2 cannot be unlinked in C. albicans

Hexokinase and Glucokinases Are Essential for Fitness and Virulence in the Pathogenic Yeast Candida albicans

The pathogenic yeast Candida albicans is both a powerful commensal and a pathogen of humans that can infect wide range of organs and body sites. Metabolic flexibility promotes infection and commensal colonization by this opportunistic pathogen. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low levels in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient detection and metabolism are fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase (CaHxk2) and two glucokinases (CaGlk1 and CaGlk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly performed by CaHxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, CaHxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, CaHxk2 participates in oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans. By contributing to metabolic flexibility, stress response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans

Development of an In Vitro Model for the Multi-Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes

La mort cellulaire par incompatibilité (caractérisation d'un facteur de transcription bZIP induit lors de la réaction d'incompatibilité chez Podospora anserina)

Chez le champignon filamenteux Podospora anserina, la co-expression de gènes incompatibles (gènes het) dans la même cellule conduit à une réaction de mort cellulaire, l'incompatibilité végétative. Des gènes sont induits lors de l'incompatibilité (gènes idi). Le gène idi-4 code pour un facteur de transcription à motif bZIP, induit en condition d'incompatibilité mais aussi en condition de carence nutritionnelle, en présence de rapamycine, et par différents stress. L'inactivation du gène idi-4 ne supprime pas la réaction de mort cellulaire. Par contre, sa surexpression conduit à une réaction de mort cellulaire apparentée à la réaction de mort celulaire par incompatibilité tant au niveau cytologique qu'au niveau de l'induction des gènes idi. La protéine IDI-4 fixe in vitro une séquence qui ressemble à celle décrite pour d'autres facteurs bZIP. Les promoteurs de certains gènes idi contiennent cette séquence consensus, ils sont donc probablement des cibles directes d'IDI-4.In the filamentous fungus Podospora anserina, co-expression of incompatible genes (het genes) in the same cell leads to a cell death reaction; the vegetative incompatibility. Some genes are induced during incompatibility (idi genes). The idi-4 gene encodes a bZIP transcription factor induced during the incompatibility reaction, but also by nutrient starvation, rapamycin and other stresses. The gene disruption of idi-4 does not suppress the cell death reaction. Conversely, over-expression of idi-4 leads to induction of idi genes and to a cell death reaction similar to cell death by incompatibility at the cytological level. The IDI-4 protein binds in vitro a sequence close to the one described for other bZIP factors. The promoters of some idi genes contain this sequence and so they probably are direct targets of IDI-4.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Nonallelic Interactions Between het-c and a Polymorphic Locus, pin-c, Are Essential for Nonself Recognition and Programmed Cell Death in Neurospora crassa

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic nonallelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain; predicted proteins containing HET domains are frequent in filamentous ascomycete genomes. These data suggest that nonallelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions

Analysis of human neutrophil interaction with live yeast cells in stationary phase at different MOIs over 5-hour time course experiments.

<p>The diagram shows the flow cytometer analysis of the neutrophils. The horizontal bar represents the neutrophil survival, indicated as a percentage on the left side of the bar. The white part of the bar represents the percentage of non-phagocytosing neutrophils, the shaded tones part represents the percentage of phagocytosing neutrophils. Note that more neutrophils died when infected with <i>C. albicans</i> than with <i>C. glabrata</i> and <i>C. lusitaniae</i>. Each condition was performed in quintuplet per experiment. Each bar is the average of three independent experiments ± standard error.</p

Host-pathogen interaction and signaling molecule secretion are modified in the dpp3 knockout mutant of Candida lusitaniae.

International audienceCandida lusitaniae is an emerging opportunistic yeast and an attractive model to discover new virulence factors in Candida species by reverse genetics. Our goal was to create a dpp3Δ knockout mutant and to characterize the effects of this gene inactivation on yeast in vitro and in vivo interaction with the host. The secretion of two signaling molecules in Candida species, phenethyl alcohol (PEA) and tyrosol, but not of farnesol was surprisingly altered in the dpp3Δ knockout mutant. NO and reactive oxygen species (ROS) production as well as tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) secretion were also modified in macrophages infected with this mutant. Interestingly, we found that the wild-type (WT) strain induced an increase in IL-10 secretion by zymosan-activated macrophages without the need for physical contact, whereas the dpp3Δ knockout mutant lost this ability. We further showed a striking role of PEA and tyrosol in this modulation. Last, the DPP3 gene was found to be an essential contributor to virulence in mice models, leading to an increase in TNF-α secretion and brain colonization. Although reinsertion of a WT DPP3 copy in the dpp3Δ knockout mutant was not sufficient to restore the WT phenotypes in vitro, it allowed a restoration of those observed in vivo. These data support the hypothesis that some of the phenotypes observed following DPP3 gene inactivation may be directly dependent on DPP3, while others may be the indirect consequence of another genetic modification that systematically arises when the DPP3 gene is inactivated