Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

Differential Interaction of the Two Related Fungal Species <i>Candida albicans</i> and <i>Candida dubliniensis</i> with Human Neutrophils

Abstract Candida albicans, the most common facultative human pathogenic fungus is of major medical importance, whereas the closely related species Candida dubliniensis is less virulent and rarely causes life-threatening, systemic infections. Little is known, however, about the reasons for this difference in pathogenicity, and especially on the interactions of C. dubliniensis with the human immune system. Because innate immunity and, in particular, neutrophil granulocytes play a major role in host antifungal defense, we studied the responses of human neutrophils to clinical isolates of both C. albicans and C. dubliniensis. C. dubliniensis was found to support neutrophil migration and fungal cell uptake to a greater extent in comparison with C. albicans, whereas inducing less neutrophil damage and extracellular trap formation. The production of antimicrobial reactive oxygen species, myeloperoxidase, and lactoferrin, as well as the inflammatory chemokine IL-8 by neutrophils was increased when stimulated with C. dubliniensis as compared with C. albicans. However, most of the analyzed macrophage-derived inflammatory and regulatory cytokines and chemokines, such as IL-1α, IL-1β, IL-1ra, TNF-α, IL-10, G-CSF, and GM-CSF, were less induced by C. dubliniensis. Similarly, the amounts of the antifungal immunity-related IL-17A produced by PBMCs was significantly lower when challenged with C. dubliniensis than with C. albicans. These data indicate that C. dubliniensis triggers stronger early neutrophil responses than C. albicans, thus providing insight into the differential virulence of these two closely related fungal species, and suggest that this is, in part, due to their differential capacity to form hyphae.</jats:p