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The Cek1?mediated MAP kinase pathway regulates exposure of ??1,2 and ??1,2?mannosides in the cell wall of Candida albicans modulating immune recognition.

ABSTRACT: The Cek1 MAP kinase (MAPK) mediates vegetative growth and cell wall biogenesis in the fungal pathogen Candida albicans. Alterations in the fungal cell wall caused by a defective Cek1?mediated signaling pathway leads to increased ??1,3?glucan exposure influencing dectin?1 fungal recognition by immune cells. We show here that cek1 cells also display an increased exposure of ??1,2 and ??1,2?mannosides (??M and ??M), a phenotype shared by strains defective in the activating MAPKK Hst7, suggesting a general defect in cell wall assembly. cek1 cells display walls with loosely bound material as revealed by transmission electron microscopy and are sensitive to tunicamycin, an inhibitor of N?glycosylation. Transcriptomal analysis of tunicamycin treated cells revealed a differential pattern between cek1 and wild type cells which involved mainly cell wall and stress related genes. Mapping ??M and ??M epitopes in the mannoproteins of different cell wall fractions (CWMP) revealed an important shift in the molecular weight of the mannan derived from mutants defective in this MAPK pathway. We have also assessed the role of galectin?3, a member of a ??galactoside?binding protein family shown to bind to and kill C. albicans through ??M recognition, in the infection caused by cek1 mutants. Increased binding of cek1 to murine macrophages was shown to be partially blocked by lactose. Galectin-3(-/-) mice showed increased resistance to fungal infection, although galectin-3 did not account for the reduced virulence of cek1 mutants in a mouse model of systemic infection. All these data support a role for the Cek1?mediated pathway in fungal cell wall maintenance, virulence and antifungal discovery.

SUBMITTER: Roman E

PROVIDER: S-EPMC5038168 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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The Cek1‑mediated MAP kinase pathway regulates exposure of α‑1,2 and β‑1,2‑mannosides in the cell wall of Candida albicans modulating immune recognition.

Román E E Correia I I Salazin A A Fradin C C Jouault T T Poulain D D Liu F-T FT Pla J J

Virulence 20160518 5

The Cek1 MAP kinase (MAPK) mediates vegetative growth and cell wall biogenesis in the fungal pathogen Candida albicans. Alterations in the fungal cell wall caused by a defective Cek1‑mediated signaling pathway leads to increased β‑1,3‑glucan exposure influencing dectin‑1 fungal recognition by immune cells. We show here that cek1 cells also display an increased exposure of α‑1,2 and β‑1,2‑mannosides (α‑M and β‑M), a phenotype shared by strains defective in the activating MAPKK Hst7, suggesting a ...[more]

PMID: 27191378

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Project description:Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide ? (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans We previously reported that hyperactivation of the Cek1 mitogen-activated protein (MAP) kinase pathway promotes ? (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase-activating protein (GAP) that has been suggested to inhibit the GTPase Rho1. We found that disruption of LRG1 causes Cek1 hyperactivation and ? (1,3)-glucan unmasking. However, when GTPase activation was measured for a panel of GTPases, the lrg1?? mutant exhibited increased activation of Cdc42 and Ras1 but not Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1?? mutant can be blocked by inhibition of the Ste11 MAP kinase kinase kinase (MAPKKK), indicating that the lrg1?? mutant acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyperactivation specifically impacts virulence, a doxycycline-repressible hyperactive STE11?N467 allele was expressed in C. albicans In the absence of doxycycline, this allele overexpressed STE11?N467 , which induced production of proinflammatory tumor necrosis factor alpha (TNF-?) from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11?N467 causes unmasking was explored with RNA sequencing (RNA-Seq) analysis. Overexpression of Ste11?N467 caused upregulation of the Cph1 transcription factor and of a group of cell wall-modifying proteins which are predicted to impact cell wall architecture.IMPORTANCE Candida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer ? (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenicity. In this study, we identified a novel regulator of the Cek1 pathway called Lrg1. Lrg1 is a predicted GTPase-activating protein (GAP) that represses Cek1 activity by downregulating the GTPase Cdc42 and its downstream MAPKKK, Ste11. Upregulation of Cek1 activity diminished fungal virulence in the mouse model of infection, and this correlates with increased cytokine responses from macrophages. We also analyzed the transcriptional profile determined during ? (1,3)-glucan exposure driven by Cek1 hyperactivation. Our report provides a model where Cek1 hyperactivation causes ? (1,3)-glucan exposure by upregulation of cell wall proteins and leads to more robust immune detection in vivo, promoting more effective clearance.

Project description:We investigated the relationships of the Cek1 and Cek2 mitogen-activated protein (MAP) kinases and the putative MAP kinase phosphatase Cpp1 in the mating process of Candida albicans Mutants of the CPP1 gene are hyperresponsive to pheromone, generating large halos, high levels of projections, and an increase in pheromone-responsive gene expression. Mating-type-homozygous opaque cells that lack both kinases are sterile, consistent with previous observations, although several lines of evidence show that the two kinases do not simply provide redundant functions in the mating process. Loss of CEK1 reduces mating significantly, to about 0.3% of wild-type strains, and also reduces projection formation and pheromone-mediated gene expression. In contrast, loss of CEK2 has less of an effect, reducing mating to approximately one-third that of the wild-type strain and moderately reducing projection formation but having little influence on the induction of gene expression. However, loss of Cek2 function reduces adaptation to pheromone-mediated arrest. The mutation enhances pheromone response halos to a level similar to that of cpp1 mutants, although the cpp1 mutants are considerably more mating defective than the cek2 mutant. The double cek2 cpp1 mutant shows enhanced responsiveness relative to either single mutant in terms of gene expression and halo formation, suggesting the kinase and phosphatase roles in the adaptation process are independent. Analysis of protein phosphorylation shows that Cek1 undergoes pheromone-mediated phosphorylation of the activation loop, and this phosphorylation is enhanced in cells lacking either the Cpp1 phosphatase or the Cek2 kinase. In addition, Cek1-GFP shows enhanced nuclear localization in response to pheromone treatment. In contrast, Cek2 shows no evidence for pheromone-mediated phosphorylation or pheromone-mediated nuclear localization. Intriguingly, however, deletion of CPP1 enhances both the phosphorylation state and the nuclear localization of Cek2-GFP. Overall, these results identify a complex interaction among the MAP kinases and MAP kinase phosphatase that function in the C. albicans mating pathway.IMPORTANCE MAP kinases and their regulators are critical components of eukaryotic signaling pathways implicated in normal cell behavior as well as abnormal behaviors linked to diseases such as cancer. The mating pathway of the yeast Saccharomyces cerevisiae was central in establishing the MAP kinase paradigm. Here we investigate the mating pathway in a different ascomycete, the fungal pathogen C. albicans In this dimorphic fungus MAP kinases are also implicated in the mating response, with two MAP kinases apparently playing redundant roles in the mating process. This work establishes that while some level of mating can occur in the presence of a single kinase, the Cek1 kinase is most important for mating, while the Cek2 kinase is involved in adaptation to signaling. While both kinases appear to be themselves regulated by dephosphorylation through the action of the Cpp1 phosphatase, this process appears important for mating only in the case of Cek1.

Dataset Information

The Cek1?mediated MAP kinase pathway regulates exposure of ??1,2 and ??1,2?mannosides in the cell wall of Candida albicans modulating immune recognition.

Publications

The Cek1‑mediated MAP kinase pathway regulates exposure of α‑1,2 and β‑1,2‑mannosides in the cell wall of Candida albicans modulating immune recognition.

Similar Datasets

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