Stage Specific Assessment of Candida albicans Phagocytosis by Macrophages Identifies Cell Wall Composition and Morphogenesis as Key Determinants

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Subunits of the vacuolar H+-ATPase complex,Vma4 and Vma10, are essential for virulence and represent potential drug targets in Candida albicans

Hyphal morphogenesis of Candida albicans is important for its pathogenesis. Here, we showed that the filamentous growth of C. albicans requires vacuolar H⁺-ATPase function. Results showed that levels of Vma4 and Vma10 increased in cells undergoing hyphal growth compared to those undergoing yeast growth. Deleting VMA4 or VMA10 abolished vacuolar functions and hyphal morphogenesis. These deletion mutants were also characterized as avirulent in a mouse model of systemic infection. Furthermore, VMA4 and VMA10 deletion strains showed hypersensitivity to fluconazole, terbinafine, and amphotericin B. Based on these findings, Vma4 and Vma10 are not only involved in vacuole biogenesis and hyphal formation, but also are good targets for antifungal drug development in C. albicans.     

Inhibition of yeast-to-hypha transition in Candida albicans by phorbasin H isolated from Phorbas sp.

Phorbasin H is a diterpene acid of a bisabolane-related skeletal class isolated from the marine sponge Phorbas sp. In this study, we examined whether phorbasin H acted as a yeast-to-hypha transition inhibitor of Candida albicans. Growth experiments suggest that this compound does not inhibit yeast cell growth but inhibits filamentous growth in C. albicans. Northern blot analysis of signaling pathway components indicated that phorbasin H inhibited the expression of mRNAs related to cAMP–Efg1 pathway. The exogenous addition of db-cAMP to C. albicans cells had no influence on the frequency of hyphal formation. The expression of hypha-specific HWP1 and ALS3 mRNAs, both of which are positively regulated by the important regulator of cell wall dynamics Efg1, was significantly inhibited by the addition of phorbasin H. This compound also reduced the ability of C. albicans cells to adhere in a dose-dependent manner. Our findings suggest that phorbasin H impacts the activity of the cAMP–Efg1 pathway, thus leading to an alteration of C. albicans morphology.     

Acetaldehyde inhibits the yeast-to-hypha conversion and biofilm formation in <Emphasis Type="Italic">Candida albicans</Emphasis>

In Candida albicans, alcohol metabolism is implicated in biofilm formation. The alcohol dehydrogenase gene (ADH1) is involved in the conversion of acetaldehyde to ethanol and reported to be downregulated during biofilm formation. C. albicans produces acetaldehyde under both in vivo and in vitro conditions. Mutations in ADH genes result in increased acetaldehyde production in vitro, but studies are lacking on the morphogenetic role(s) of acetaldehyde in C. albicans. We report here that acetaldehyde at a concentration of 7 mM was able to inhibit the conversion from yeast to hyphal forms induced by four standard inducers at 37°C. The hyphal inhibitory concentrations did not adversely affect the growth and viability of C. albicans cells. The same concentration of acetaldehyde also significantly inhibited biofilm development, and only adhered yeast cells were found. We hypothesize that acetaldehyde produced by C. albicans may exert a morphogenetic regulatory role influencing yeast-to-hypha conversion, biofilm formation, dissemination and establishment of infection.     

Candida albicans PHO81 is required for the inhibition of hyphal development by farnesoic acid

Farnesoic acid is a signaling molecule that inhibits the transition from budding yeast to filament formation in Candida albicans, but the molecular mechanism regulated by this substance is unknown. In this study, we analyzed the function of CaPHO81, which is induced by farnesoic acid. The pho81Δ mutant cells existed exclusively as filaments under favorable yeast growth conditions. Furthermore, the inhibition of hyphal growth and repression of CPH1, EFG1, HWP1, and GAP1 mRNA expression in response to farnesoic acid were defective in pho81Δ mutant cells. These data suggest a role for CaPHO81 in the inhibition of hyphal development by farnesoic acid.     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

Background

Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus.

Methodology/Principal Findings

Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Δ mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Δ cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Δ cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Δ mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p.

Conclusions/Significance

The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Δ cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Δ cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Δ cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target.     

CDC42 is required for polarized growth in human pathogen Candida albicans

Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42p^G12V or CaCdc42p^D118A ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p^G12V was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.     

Adaptations of the Secretome of Candida albicans in Response to Host-Related Environmental Conditions

The wall proteome and the secretome of the fungal pathogen Candida albicans help it to thrive in multiple niches of the human body. Mass spectrometry has allowed researchers to study the dynamics of both subproteomes. Here, we discuss some major responses of the secretome to host-related environmental conditions. Three β-1,3-glucan-modifying enzymes, Mp65, Sun41, and Tos1, are consistently found in large amounts in culture supernatants, suggesting that they are needed for construction and expansion of the cell wall β-1,3-glucan layer and thus correlate with growth and might serve as diagnostic biomarkers. The genes ENG1, CHT3, and SCW11, which encode an endoglucanase, the major chitinase, and a β-1,3-glucan-modifying enzyme, respectively, are periodically expressed and peak in M/G₁. The corresponding protein abundances in the medium correlate with the degree of cell separation during single-yeast-cell, pseudohyphal, and hyphal growth. We also discuss the observation that cells treated with fluconazole, or other agents causing cell surface stress, form pseudohyphal aggregates. Fluconazole-treated cells secrete abundant amounts of the transglucosylase Phr1, which is involved in the accumulation of β-1,3-glucan in biofilms, raising the question whether this is a general response to cell surface stress. Other abundant secretome proteins also contribute to biofilm formation, emphasizing the important role of secretome proteins in this mode of growth. Finally, we discuss the relevance of these observations to therapeutic intervention. Together, these data illustrate that C. albicans actively adapts its secretome to environmental conditions, thus promoting its survival in widely divergent niches of the human body.     

Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.     

Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis

Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8 were only detected in the hyphal cells. Genetic analysis of these sites revealed allele-specific effects on growth or hyphae formation at 42 °C. Colony morphology, which is normally wrinkled or crenellated at 37 °C, reverted to smooth in several mutants, but this colony morphology phenotype was unrelated to cellular morphology. Two mutants exhibited a mild increase in sensitivity to the cell wall-active compounds caspofungin and calcofluor white. We suggest that this analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence. The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client proteins, and that modification of these sites can play cellular regulatory roles, consistent with the “chaperone code” hypothesis.     

Roles of Ca2+ in hyphal and yeast-form growth inCandida albicans. Growth regulation by altered extracellular and intracellular free Ca2+ concentrations

The dimorphic fungusCandida albicans has both a yeast form and a hyphal form. When yeast-form cells were starved and then transferred to aN-acetylglucosamine medium, the formation of true hyphae from the unbudded yeast-form cells was induced. Removal of Ca²⁺ from the medium with EGTA inhibited hyphal formation by 50%, resulting in only thin and short hyphae. Externally applied excess Ca²⁺ (>10⁻²M) also affected the hyphal formation, resulting in formation of pseudohyphae. This effect required a high concentration of Ca²⁺ but was Ca²⁺-specific. Deprivation of Ca²⁺ also inhibited yeast-form growth. Interestingly, such cells had abnormally wide bud necks and became defective in cell separation. To measure cytosolic free Ca²⁺, fura-2 was introduced into hyphal cells by electroporation. Its normal value was estimated to be about 100 nM. The electroporation caused transient elevation of cytosolic free Ca²⁺ concentration and transient cessation of hyphal growth. There was a close correlation between the timing of recovery of Ca²⁺ concentration and that of the resumption of hyphal growth. Our results demonstrate the importance of extracellular and intracellular free Ca²⁺ for the growth ofC. albicans.     

Intestinal Cell Tight Junctions Limit Invasion of Candida albicans through Active Penetration and Endocytosis in the Early Stages of the Interaction of the Fungus with the Intestinal Barrier

C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ) formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin), we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ.     

The antibacterial agent,moxifloxacin inhibits virulence factors of <Emphasis Type="Italic">Candida albicans</Emphasis> through multitargeting

Fluoroquinolines are broad spectrum fourth generation antibiotics. Some of the Fluoroquinolines exhibit antifungal activity. We are reporting the potential mechanism of action of a fluoroquinoline antibiotic, moxifloxacin on the growth, morphogenesis and biofilm formation of the human pathogen Candida albicans. Moxifloxacin was found to be Candidacidal in nature. Moxifloxacin seems to inhibit the yeast to Hyphal morphogenesis by affecting signaling pathways. It arrested the cell cycle of C. albicans at S phase. Docking of moxifloxacin with predicted structure of C. albicans DNA Topoisomerase II suggests that moxifloxacin may bind and inhibit the activity of DNA Topoisomerase II in C. albicans. Moxifloxacin could be used as a dual purpose antibiotic for treating mixed infections caused by bacteria as well as C. albicans. In addition chances of developing moxifloxacin resistance in C. albicans are less considering the fact that moxifloxacin may target multiple steps in yeast to hyphal transition in C. albicans.