The role of Candida albicans oxidative stress responses in triggering filament formation and macrophage escape following phagocytosis

Abstract

Candida albicans is an important opportunistic fungal pathogen which causes life threatening systemic diseases in immunocompromised people. An important virulence attribute of C. albicans is morphological plasticity. Yeasts engulfed by macrophages can transition to a hyphal filamentous morphology which either induce pyroptosis or rupture the phagosomal membrane of the macrophage and evade the toxic reactive oxygen species (ROS) generated by the NADPH oxidase complex of the phagocyte. Recent studies revealed that ROS causes cell cycle arrest resulting in hyperpolarised bud formation in C. albicans. Moreover, the ability of this pathogen to mount robust transcriptional responses to ROS is an essential pre-requisite for hyphal filament formation following phagocytosis. Thus, it was hypothesised that macrophage-derived reactive oxygen species (ROS) can inhibit C. albicans growth, which is more pronounced in ROS-sensitive mutants resulting in impaired filament formation following phagocytosis. Hence, the aim of this study was to decipher why C. albicans responses to ROS are important for filament formation in the phagolysosomal environment of the macrophage. In vitro, it was examined whether hydrogen peroxide (H2O2)-treatment could inhibit serum-induced hyphae formation and whether this was more pronounced in cells lacking the key Cap1 oxidative-stress responsive transcription factor, and its regulators Ybp1 and Gpx3. The results showed that exposure to H2O2 inhibited serum-induced hyphae formation. In addition, the inhibition of filament formation was more sustained in the cap1Δ, ybp1Δ and gpx3Δ mutant cells compared to the wild-type strain. A concurrent analysis of cell survival indicated that the mutant cells displayed a longer H2O2-induced growth arrest compared to wild-type cells. Hence, to identify regulators of hyperpolarised bud formation and, determine if ROS-resistance is a global requirement for filament formation following phagocytosis, a library of transcription factor mutants was screened for sensitivity to different forms of ROS. The most sensitive mutants; cap1Δ, efg1Δ, skn7Δ, ndt80Δ, and gzf3Δ were examined for ability to survive and form filaments following phagocytosis using confocal video microscopy. Only cap1Δ and efg1Δ cells failed to form filaments following phagocytosis. However, the three remaining mutants effectively formed filaments inside the phagosomal environment. Collectively, these data show that whilst Cap1 is essential for filament formation inside the macrophage, wild-type levels of oxidative stress resistance are not a necessary pre-requisite for this morphological switch following phagocytosis.