Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance

Mammalian innate immune cells produce reactive oxygen species (ROS) in the oxidative burst reaction to destroy invading microbial pathogens. Using quantitative real-time ROS assays, we show here that both yeast and filamentous forms of the opportunistic human fungal pathogen Candida albicans trigger ROS production in primary innate immune cells such as macrophages and dendritic cells. Through a reverse genetic approach, we demonstrate that coculture of macrophages or myeloid dendritic cells with C. albicans cells lacking the superoxide dismutase (SOD) Sod5 leads to massive extracellular ROS accumulation in vitro . ROS accumulation was further increased in coculture with fungal cells devoid of both Sod4 and Sod5. Survival experiments show that C. albicans mutants lacking Sod5 and Sod4 exhibit a severe loss of viability in the presence of macrophages in vitro . The reduced viability of sod5 Δ/Δ and sod4 Δ/Δ sod5 Δ/Δ mutants relative to wild type is not evident with macrophages from gp91phox ^{−/ −} mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans , and perhaps many other microbial pathogens, can evade host immune surveillance in vivo .     

Candida albicans infection inhibits macrophage cell division and proliferation

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen.     

HIV aspartyl protease inhibitors as promising compounds against Candida albicans André Luis Souza dos Santos

Cells of Candida albicans (C. albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. In this context, both the host immune status and the ability of C. albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance; in this last case, culminating in the establishment of successful infection known as candidiasis. C. albicans possesses a potent armamentarium consisting of several virulence molecules that help the fungal cells to escape of the host immune responses. There is no doubt that the secretion of aspartyl-type proteases, designated as Saps, are one of the major virulence attributes produced by C. albicans cells, since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions. For these reasons, Saps clearly hold promise as new potential drug targets. Corroborating this hypothesis, the introduction of new anti-human immunodeficiency virus drugs of the aspartyl protease inhibitor-type (HIV PIs) have emerged as new agents for the inhibition of Saps. The introduction of HIV PIs has revolutionized the treatment of HIV disease, reducing opportunistic infections, especially candidiasis. The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status, but also as a result of direct inhibition of C. albicans Saps. In this article, we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C. albicans, focusing on the effects of these compounds on Sap activity, growth behavior, morphological architecture, cellular differentiation, fungal adhesion to animal cells and abiotic materials, modulation of virulence factors, experimental candidiasis infection, and their synergistic actions with classical antifungal agents.     

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.     

Nitric oxide and nitrosative stress tolerance in yeast

The opportunistic human fungal pathogen Candida albicans encounters diverse environmental stresses when it is in contact with its host. When colonizing and invading human tissues, C. albicans is exposed to ROS (reactive oxygen species) and RNIs (reactive nitrogen intermediates). ROS and RNIs are generated in the first line of host defence by phagocytic cells such as macrophages and neutrophils. In order to escape these host-induced oxidative and nitrosative stresses, C. albicans has developed various detoxification mechanisms. One such mechanism is the detoxification of NO (nitric oxide) to nitrate by the flavohaemoglobin enzyme CaYhb1. Members of the haemoglobin superfamily are highly conserved and are found in archaea, eukaryotes and bacteria. Flavohaemoglobins have a dioxygenase activity [NOD (NO dioxygenase domain)] and contain three domains: a globin domain, an FAD-binding domain and an NAD(P)-binding domain. In the present paper, we examine the nitrosative stress response in three fungal models: the pathogenic yeast C. albicans, the benign budding yeast Saccharomyces cerevisiae and the benign fission yeast Schizosaccharomyces pombe. We compare their enzymatic and non-enzymatic NO and RNI detoxification mechanisms and summarize fungal responses to nitrosative stress.     

First human model of in vitro Candida albicans persistence within granuloma for the reliable study of host-fungi interactions

BACKGOUND: The balance between human innate immune system and Candida albicans virulence signaling mechanisms ultimately dictates the outcome of fungal invasiveness and its pathology. To better understand the pathophysiology and to identify fungal virulence-associated factors in the context of persistence in humans, complex models are indispensable. Although fungal virulence factors have been extensively studied in vitro and in vivo using different immune cell subsets and cell lines, it is unclear how C. albicans survives inside complex tissue granulomas. METHODOLOGY/PRINCIPAL FINDING: We developed an original model of in vitro human granuloma, reproducing the natural granulomatous response to C. albicans. Persistent granulomas were obtained when the ratio of phagocytes to fungi was high. This in vitro fungal granuloma mimics natural granulomas, with infected macrophages surrounded by helper and cytotoxic T lymphocytes. A small proportion of granulomas exhibited C. albicans hyphae. Histological and time-lapse analysis showed that C. albicans blastoconidia were located within the granulomas before hyphae formation. Using staining techniques, fungal load calculations, as well as confocal and scanning electron microscopy, we describe the kinetics of fungal granuloma formation. We provide the first direct evidence that C. albicans are not eliminated by immunocompetent cells inside in vitro human granulomas. In fact, after an initial candicidal period, the remaining yeast proliferate and persist under very complex immune responses. CONCLUSIONS/SIGNIFICANCE: Using an original in vitro model of human fungal granuloma, we herein present the evidence that C. albicans persist and grow into immunocompetent granulomatous structures. These results will guide us towards a better understanding of fungal invasiveness and, henceforth, will also help in the development of better strategies for its control in human physiological conditions.     

Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth

Candida albicans is a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. Although Candida can cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature of Candida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation of C. albicans filamentation in vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growth in vivo.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.     

Conventional dendritic cells mount a type I IFN response against Candida spp. requiring novel phagosomal TLR7-mediated IFN-β signaling

Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-β response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/β receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-β release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-β and the β-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-β release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-β signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata.     

Interaction of Candida albicans with macrophages

Study of the in vitro interaction of mouse peritoneal macrophages with C. albicans has revealed that these macrophages, though easily phagocytizing C. albicans blastospores, are incapable of destroying the fungus. The phagocytic, but not candidostatic, activity of these macrophages has been found to depend on the conditions on their cultivation, as well as on the age and viability of fungal cells. The representatives of 7 C. albicans strains, obtained from various sources and stored at the museum for different spans of time, have shown practically no difference in the character of their interaction with mouse peritoneal macrophages.     

Differential phagocytosis of white versus opaque Candida albicans by Drosophila and mouse phagocytes

The human fungal pathogen Candida albicans resides asymptomatically in the gut of most healthy people but causes serious invasive diseases in immunocompromised patients. Many C. albicans strains have the ability to stochastically switch between distinct white and opaque cell types, but it is not known with certainty what role this switching plays in the physiology of the organism. Here, we report a previously undescribed difference between white and opaque cells, namely their interaction with host phagocytic cells. We show that both Drosophila hemocyte-derived S2 cells and mouse macrophage-derived RAW264.7 cells preferentially phagocytose white cells over opaque cells. This difference is seen both in the overall percentage of cultured cells that phagocytose white versus opaque C. albicans and in the average number of C. albicans taken up by each phagocytic cell. We conclude that susceptibility to phagocytosis by cells of the innate immune system is an important distinction between white and opaque C. albicans, and propose that one role of switching from the prevalent white form into the rarer opaque form may be to allow C. albicans to escape phagocytosis.     

Identification of pH-regulated antigen 1 released from Candida albicans as the major ligand for leukocyte integrin alphaMbeta2

Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungal disease in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is of critical importance in host defense and the prime task of cells of the innate immune system. We previously demonstrated that the integrin alpha(M)beta(2) (CD11b/CD18) is the major leukocyte receptor involved in C. albicans recognition, mediating both adhesive and migratory responses to the fungus. In the present study, we demonstrate that various C. albicans strains release a protease-sensitive activity into their conditioned medium that supports alpha(M)beta(2)-mediated cell adhesion and migration. The isolation and characterization of this protein was undertaken by two independent approaches: 1) immunoaffinity purification on a mAb raised to conditioned medium which blocked alpha(M)beta(2)-dependent adhesion and migration; and 2) affinity chromatography on purified alpha(M)beta(2). Each approach led to the isolation of the same protein, which was unequivocally identified as pH-regulated Ag 1 (Pra1p), based on mass spectrometry and amino acid sequence analyses. C. albicans mutant strains lacking Pra1p were unable to support leukocyte adhesion or migration. In a neutrophil-mediated fungal killing assay, such mutant strains were resistant to killing and/or phagocytosis. Addition of purified Pra1p or reagents that block alpha(M)beta(2) function prevented killing of Pra1p-expressing but not Pra1p-deficient strains of C. albicans. Together, these data indicate that Pra1p is a ligand of alpha(M)beta(2) on C. albicans and that the soluble form of Pra1p may assist the fungus in escaping host surveillance.     

TLR2, but not TLR4, triggers cytokine production by murine cells in response to Candida albicans yeasts and hyphae

Toll-like receptors (TLRs) function as sensors for infection that induce the activation of the immune responses. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans. Although both TLR2 and TLR4 have been implicated in the host interaction with C. albicans, their specific role during infection has not been unequivocally established, as conflicting results have been reported. In this review, we summarize and discuss our own and others' key findings about the specific role of TLR2 and TLR4 in murine resistance to candidiasis, and in triggering cytokine secretion by murine cells in response to C. albicans yeasts and hyphae.     

Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes . InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans . Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans , we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans , like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.     

Promoter regulation in Candida albicans and related species

Regulation of gene expression has been studied extensively in Saccharomyces cerevisiae and Schizosaccharomyces pombe . Some, but by far not all, of the findings are also applicable to Candida albicans , an important ascomycete fungal pathogen of humans. Areas of research in C. albicans include the influence of key signal transduction cascades on morphology, and the response to host-generated influences, such as host immune effector cells, blood, pH or elevated carbon dioxide. The resistance to antifungal agents and response to stress are also well researched. Conditional gene expression and reporter genes adapted to the codon usage of C. albicans are now widely used in C. albicans . Here we present a comprehensive overview of the current techniques used to investigate regulation mechanisms for promoters in C. albicans and other Candida species. In addition, we discuss reporter genes used for the study of gene expression.     

Lipase of Candida albicans induces activation of NADPH oxidase and l-arginine pathways on resting and activated macrophages

Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (M?) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on M? functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting M?s and M?s primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.     

抗系统性白念珠菌感染的先天性免疫

白念珠菌是引起浅部、深部真菌感染常见的病原菌.先天免疫反应在宿主抗系统性白念珠菌感染中起主导作用.介导宿主抗念珠菌感染的先天性免疫包括一系列真菌识别受体及免疫效应细胞.宿主对系统性白念珠菌感染的免疫反应是决定患者预后的关键.本文就宿主抗系统性白念珠菌感染的先天性免疫机制进行综述.     

Effects of interferon-γ and granulocyte colony-stimulating factor on antifungal activity of human polymorphonuclear neutrophils against Candida albicans grown as biofilms or planktonic cells

Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system.