Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid beta-oxidation pathway. Here, we asked whether fatty acid beta-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Delta/pex5Delta mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Delta/fox2Delta mutant, which lacks the second enzyme of the beta-oxidation pathway. Both mutant strains had strongly reduced beta-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Delta/fox2Delta mutant, and not the pex5Delta/pex5Delta mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Delta/fox2Delta mutant was comparable to that of the icl1Delta/icl1Delta mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid beta-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Delta/fox2Delta mutant is largely due to a dysfunctional glyoxylate cycle.     

Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans

Candida albicans is an opportunistic human fungal pathogen which causes disease mainly in immunocompromised patients. Activity of hydrolytic enzymes is essential for virulence of C. albicans and so is the capacity of these cells to undergo transition from yeast to mycelial form of growth. Ocimum sanctum is cultivated worldwide for its essential oil which exhibits medicinal properties. This work evaluates the anti-virulence activity of O. sanctum essential oil (OSEO) on 22 strains of C. albicans (including a standard strain ATCC 90028) isolated from both HIV positive and HIV negative patients. Candida isolates were exposed to sub-MICs of OSEO. In vitro secretion of proteinases and phospholipases was evaluated by plate assay containing BSA and egg yolk respectively. Morphological transition from yeast to filamentous form was monitored microscopically in LSM. For genetic analysis, respective genes associated with morphological transition (HWP1), proteinase (SAP1) and phospholipase (PLB2) were also investigated by Real Time PCR (qRT-PCR). Results were analyzed using Student's t-test. OSEO inhibits morphological transition in C. albicans and had a significant inhibitory effect on extracellular secretion of proteinases and phospholipases. Expression profile of respective selected genes associated with C. albicans virulence by qRT-PCR showed a reduced expression of HWP1, SAP1 and PLB2 genes in cells treated with sub-inhibitory concentrations of OSEO. This work suggests that OSEO inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as down regulates the associated genes. Further studies will assess the clinical application of OSEO and its constituents in the treatment of fungal infections.     

Spindle assembly checkpoint component CaMad2p is indispensable for Candida albicans survival and virulence in mice

Here, we report an indispensable role for spindle assembly checkpoint (SAC) component CaMad2p in the survival and virulence of Candida albicans in mice. We hypothesized that cell cycle checkpoint functions, especially those monitoring the integrity of DNA and chromosome segregation, might be required for the pathogen to repair damage caused by host defence. To test this idea, we created SAC-defective mutants by deleting the CaMAD2 gene that encodes a key component of the SAC pathway. The CaMAD2 mutant appears normal in morphology, growth rate and growth mode switch in unperturbed conditions. However, it quickly loses viability when treated with nocodazole, which causes disassembly of mitotic spindles. The mutant also exhibits increased frequency of chromosome loss. The virulence of the mutant is greatly reduced in mice, presumably because of the inability of the mutant cells to stop the cell cycle when the host defence damages cellular components important for chromosome segregation. Supporting this hypothesis, unlike the wild-type cells that can proliferate within and eventually grow out of macrophages, most of the CaMAD2 null mutant cells are unable to survive. This study suggests that SAC is required for survival of C. albicans in the host and could thus be targeted for anti-C. albicans therapies.     

Calcineurin A of Candida albicans: involvement in antifungal tolerance,cell morphogenesis and virulence

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.     

Chromatin-mediated Candida albicans virulence

Candida albicans is the most prevalent human fungal pathogen. To successfully propagate an infection, this organism relies on the ability to change morphology, express virulence-associated genes and resist DNA damage caused by the host immune system. Many of these events involve chromatin alterations that are crucial for virulence. This review will focus on the studies that have been conducted on how chromatin function affects pathogenicity of C. albicans and other fungi. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.     

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.     

RNA triphosphatase is essential in Schizosaccharomyces pombe and Candida albicans

Background

The first two steps in the capping of cellular mRNAs are catalyzed by the enzymes RNA triphosphatase and RNA guanylyltransferase. Although structural and mechanistic differences between fungal and mammalian RNA triphosphatases recommend this enzyme as a potential antifungal target, it has not been determined if RNA triphosphatase is essential for the growth of fungal species that cause human disease.

Results

We show by classical genetic methods that the triphosphatase (Pct1) and guanylyltransferase (Pce1) components of the capping apparatus in the fission yeast Schizosaccharomyces pombe are essential for growth. We were unable to disrupt both alleles of the Candida albicans RNA triphosphatase gene CaCET1, implying that the RNA triphosphatase enzyme is also essential for growth of C. albicans, a human fungal pathogen.

Conclusions

Our results provide the first genetic evidence that cap synthesis is essential for growth of an organism other than Saccharomyces cerevisiae and they validate RNA triphosphatase as a target for antifungal drug discovery.     

Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1Δ/Δ, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulent in a mouse model of systemic candidiasis. The cho1Δ/Δ mutant exhibits defects in cell wall integrity, mitochondrial function, filamentous growth, and is auxotrophic for ethanolamine. PS is a precursor for de novo PE biosynthesis. A psd1Δ/Δ psd2Δ/Δ double mutant, which lacks the PS decarboxylase enzymes that convert PS to PE in the de novo pathway, has diminished PE levels like those of the cho1Δ/Δ mutant. The psd1Δ/Δ psd2Δ/Δ mutant exhibits phenotypes similar to those of the cho1Δ/Δ mutant; however, it is slightly more virulent and has less of a cell wall defect. The virulence losses exhibited by the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutants appear to be related to their cell wall defects which are due to loss of de novo PE biosynthesis, but are exacerbated by loss of PS itself. Cho1p is conserved in fungi, but not mammals, so fungal PS synthase is a potential novel antifungal drug target.     

Pga26 mediates filamentation and biofilm formation and is required for virulence in Candida albicans

The Candida albicans gene PGA26 encodes a small cell wall protein and is upregulated during de novo wall synthesis in protoplasts. Disruption of PGA26 caused hypersensitivity to cell wall-perturbing compounds (Calcofluor white and Congo red) and to zymolyase, which degrades the cell wall β-1,3-glucan network. However, susceptibility to caspofungin, an inhibitor of β-1,3-glucan synthesis, was decreased. In addition, pga26Δ mutants show increased susceptibility to antifungals (fluconazol, posaconazol or amphotericin B) that target the plasma membrane and have altered sensitivities to environmental (heat, osmotic and oxidative) stresses. Except for a threefold increase in β-1,6-glucan and a slightly widened outer mannoprotein layer, the cell wall composition and structure was largely unaltered. Therefore, Pga26 is important for proper cell wall integrity, but does not seem to be directly involved in the synthesis of cell wall components. Deletion of PGA26 further leads to hyperfilamentation, increased biofilm formation and reduced virulence in a mouse model of disseminated candidiasis. We propose that deletion of PGA26 may cause an imbalance in the morphological switching ability of Candida, leading to attenuated dissemination and infection.     

Dbf2 is essential for cytokinesis and correct mitotic spindle formation in Candida albicans

We have characterized the DBF2 gene, encoding a protein kinase of the NDR family in Candida albicans, and demonstrate that this gene is essential for cell viability. Conditional mutants were constructed by using the MET3 promoter to analyse the phenotype of cells lacking this kinase. The absence of Dbf2 resulted in cells arrested as large-budded pairs that failed to contract the actomyosin ring, a function similar to that described for its Saccharomyces cerevisiae orthologue. In addition to its role in cytokinesis, Dbf2 regulates mitotic spindle organization and nuclear segregation as Dbf2-depleted cells have abnormal microtubules and severe defects in nuclear migration to the daughter cell, which results in a cell cycle block during mitosis. Taken together, these results imply that Dbf2 performs several functions during exit from mitosis and cytokinesis. Consistent with a role in spindle organization, the protein localizes to the mitotic spindle during anaphase, and it interacts physically with tubulin, as indicated by immunoprecipitation experiments. Finally, DBF2 depletion also resulted in impaired true hyphal growth.     

CDK-dependent phosphorylation of Mob2 is essential for hyphal development in Candida albicans

Nuclear Dbf2-related (NDR) protein kinases are essential components of regulatory pathways involved in cell morphogenesis, cell cycle control, and viability in eukaryotic cells. For their activity and function, these kinases require interaction with Mob proteins. However, little is known about how the Mob proteins are regulated. In Candida albicans, the cyclin-dependent kinase (CDK) Cdc28 and the NDR kinase Cbk1 are required for hyphal growth. Here we demonstrate that Mob2, the Cbk1 activator, undergoes a Cdc28-dependent differential phosphorylation on hyphal induction. Mutations in the four CDK consensus sites in Mob2 to Ala significantly impaired hyphal development. The mutant cells produced short hyphae with enlarged tips that displayed an illicit activation of cell separation. We also show that Cdc28 phosphorylation of Mob2 is essential for the maintenance of polarisome components at hyphal tips but not at bud tips during yeast growth. Thus we have found a novel signaling pathway by which Cdc28 controls Cbk1 through the regulatory phosphorylation of Mob2, which is crucial for normal hyphal development.     

Requirement for Candida albicans Sun41 in biofilm formation and virulence

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41, Orf19.5412, Orf19.1277, MSB2, Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41Delta/sun41Delta deletion mutants and sun41Delta/sun41Delta+pSUN41-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41Delta/sun41Delta mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41Delta/sun41Delta cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.     

Candida albicans adhesins: Biochemical aspects and virulence

The recognition of host cells by the pathogenic yeast, Candida albicans, is probably an essential step in the pathogenesis of disease development. The interaction of yeast and hyphal mannoproteins with host cell receptors has been studied by a number of laboratories. C. albicans recognizes a variety of host cells as well as host cell extracellular matrix proteins. This observation is not unexpected given the number of sites within and on the body which can be colonized and infected by the organism. Indeed, it would appear that C. albicans has evolved a number of ways in which it recognizes the host. This statement is made with the qualification that the organism uses other processes to infect, such as morphogenesis, phenotypic switching and the production of invasive enzymes, including secreted aspartyl proteases and phosholipases. Recognition of epithelial cells is accomplished through cell surface mannoproteins (adhesins) which bind to carbohydrate-containing receptors. The number of such mannoproteins is not known; pro adhesins exist. The organism also binds to keratinocytes, endothelial cells and matrix proteins, such as fibronectin, laminin, collagen and entactin, and, as such, appears to have a integrin-like cell surface adhesin. In most cases, the adhesin for each of these host proteins is a mannoprotein. The biochemistry of the candidal adhesins has been extensively studied. However, molecular analyses of the encoding genes is only now being studied. Thus, until clean, genetic analyses are complete and strains lacking an adhesin function are constructed, a direct role for the adhesins in pathogenesis can only be inferred. At present, spontaneous, non-adhering strains of the organism have been described which are avirulent in animal models of candidiasis. However, these data only suggest a role for adherence; future studies should be directed towards resolving questions about the role of these proteins in pathogenesis.     

Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.     

Toll-like receptor-2 is essential in murine defenses against Candida albicans infections

In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity of i.p. infected TLR2-/- mice. In contrast, in vitro phagocytosis of yeasts and production of reactive oxygen intermediates (ROI) were not affected in macrophages from TLR2-/- animals. Our data indicate that TLR2 plays a major role in the response of macrophages to C. albicans, triggering cytokine and chemokine expression, and it is essential for in vivo protection against infection.     

Calcineurin is required for virulence of Cryptococcus neoformans.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.