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Candida albicans Ras1 Inactivation Increases Resistance to Phagosomal Killing by Human Neutrophils.

ABSTRACT: Host phagocytic cells are crucial players in initial defense against Candida albicans infection. C. albicans utilizes MAP kinases and Ras1 stress response signaling pathways to protect itself from killing by immune cells. In this study, we tested the importance of these pathways in C. albicans phagocytosis by neutrophils and subsequent phagosomal survival. Phagocytosis was influenced by C. albicans morphology, so hyphal length of >10 μm reduced the phagocytic index (PI) 2- to 3-fold in human neutrophils. Primary human neutrophils killed 81% of phagocytosed C. albicans, while primary mouse neutrophils killed 63% of yeasts. We found that both the C. albicans Cek1 and Hog1 pathways were required for survival of phagocytosed yeast, whereas deletion of C. albicans RAS1 resulted in an 84% increase in survival within neutrophils compared to that of the wild type (WT). The absence of Ras1 did not alter reactive oxygen species (ROS) production by C. albicans; however, phagocytosed C. albicans Δ/Δras1 cells reduced ROS release by neutrophils by 86%. Moreover, C. albicans Δ/Δras1 cells had increased resistance to hydrogen peroxide as a result of high levels of catalase activity. This phenotype was specific to Ras1, since these effects were not observed in the absence of its partner Cyr1 or with its downstream target Efg1. In addition, C. albicans Δ/Δras1 cells had a significantly increased resistance to nonoxidative killing by human neutrophil peptide 1 (HNP-1) that was reversed by restoring cellular cAMP levels. These data show that C. albicans Ras1 inactivation leads to fungal resistance to both oxidative and nonoxidative mechanisms of neutrophil phagosomal killing.

SUBMITTER: Salvatori O

PROVIDER: S-EPMC6246910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7? mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7? cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1? lsp1?), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7? in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161?, rvs167?, and arp2? arp3?). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2? mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7? cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

Project description:An important feature associated with Candida albicans pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 has an elongated shape and limited conformational flexibility and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays disclosed that CaRas1-activation by CaCdc25 is highly efficient, with activities up to 2,000-fold higher than reported for human GEFs. The crystal structure of the CaCdc25 catalytic region revealed an active conformation for the α-helical hairpin, critical for CaRas1-activation, unveiling a specific region exclusive to CTG-clade species. Structural studies on CaRas1/CaCdc25 complexes also revealed an interaction surface clearly distinct from that of homologous human complexes. Furthermore, we identified an inhibitory synthetic peptide, prompting the proposal of a key regulatory mechanism for CaCdc25. To our knowledge, this is the first report of specific inhibition of the CaRas1-activation via targeting its GEF. This, together with their unique pathogen-structural features, disclose a set of novel strategies to specifically block this important virulence-related mechanism. IMPORTANCE Candida albicans is the main causative agent of candidiasis, the commonest fungal infection in humans. The eukaryotic nature of C. albicans and the rapid emergence of antifungal resistance raise the challenge of identifying novel drug targets to battle this prevalent and life-threatening disease. CaRas1 and CaCdc25 are key players in the activation of signaling pathways triggering multiple virulence traits, including the yeast-to-hypha interconversion. The structural similarity of the conserved G-domain of CaRas1 to those of human homologs and the lack of structural information on CaCdc25 has impeded progress in targeting these proteins. The unique structural and functional features for CaRas1 and CaCdc25 presented here, together with the identification of a synthetic peptide capable of specifically inhibiting the GEF activity of CaCdc25, open new possibilities to uncover new antifungal drug targets against C. albicans virulence.

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Candida albicans Ras1 Inactivation Increases Resistance to Phagosomal Killing by Human Neutrophils.

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