Candida Albicans Cell Wall Research Articles

Combination immunotherapy of MAb B6.1 with fluconazole augments therapeutic effect to disseminated candidiasis

We recently reported that IgM MAb B6.1, specific for β-1, 2-mannotriose on the cell wall of Candida albicans, is therapeutic to disseminated candidiasis due to C. albicans. In the current study, we examined if MAbB6.1 enhances therapeutic effect of fluconazole (FLC) to the disseminated disease. To assess the combination effect, determination by the kidneys-colony forming unit and survival times were used. Results showed that the therapeutic effect of FLC on mice with disseminated candidiasis was dose-dependent, but a FLC dose at 0.8 mg/kg body weight of mice was ineffective. To determine combination effect, mice treated intraperitoneally with a combination of FLC plus MAb B6.1 at 1 h post-infection - a condition of developing partial therapeutic activity - enhanced survival times beyond the effect by only antibody (p < 0.05). The resulting MST (mean survival times) value from the combination-received mice was almost the same as MST value from 3.2 mg FLC dose-given animals (p < 0.05). Another combination of 1.6 mg FLC dose and B6.1 reduced severity of the disseminated disease at almost the same rate as combination efficacy of 0.8 mg FLC dose plus B6.1. This data indicates that B6.1 acts in concert with FLC and that this combination therapy augments protection, which suggests a possibility of reducing FLC dose. The augmentation response was specific because an irrelevant IgM MAb S9 was not effective to the disseminated disease. Thus, our present studies demonstrate that this combination immunotherapy may be a way of solving the problem of limited antifungal drug choices caused by drug-resistant C. albicans.

Synthesis and Immunogenicity of a Glycopolymer Conjugate

A protective β-mannan trisaccharide epitope from the Candida albicans cell wall phosphomannan has been synthesized and activated for copolymerization with acrylamide. The resulting glycopolymer displayed 33 trisaccharide haptens and was derivatized for conjugation to the immunogenic carrier protein, chicken serum albumin. The resulting conjugate achieves a high degree of oligosaccharide substitution while limiting the sites of substitution on the protein. The murine immune response against this conjugate was compared with the response to a trisaccharide-tetanus toxoid conjugate vaccine. The glycopolymer was shown to induce a more robust immune response with higher trisaccharide-specific antibody titers and with a significantly larger proportion of responding mice developing antibodies that bound the target, native cell wall antigen of C. albicans.

High polymeric IgA content facilitates recognition of microbial polysaccharide-natural serum antibody immune complexes by immobilized human galectin-1

Dextran-binding immunoglobulin (DIg) and anti-β-glucan antibody (ABG) are naturally occurring human serum antibodies specific to α- and β-glucoside epitopes respectively of polysaccharide antigens and heavily enriched in IgA. ABG and DIg are shown here to have much more of their IgA in polymeric form than does serum IgA in general. Cell wall β-glucans and glycoproteins of the widely consumed yeast (Saccharomyces cerevisiae) offered several hundred fold better ligands for ABG than did small β-glucosides. Candida albicans cell wall antigen (CCA), a commonly encountered polysaccharide-rich fungal antigen was recognized by normal human serum anti-carbohydrate antibodies to precipitate maximally at a definite stoichiometry typical of immune complexes (IC). IC formed in serum in vitro on addition of CCA contained a significantly higher percentage of IgA than did either naturally occurring IC or serum. Polymeric IgA was far better ligand than monomeric IgA for both anti-IgA antibody and the most widely expressed human tissue lectin galectin-1 which recognizes O-linked oligosaccharides characteristic of IgA, in contrast to N-linked oligosaccharides present in all immunoglobulins. Moreover, desialylation by neuraminidase, an enzyme released into circulation during many microbial infections and diabetes, increased lectin-binding activity of polymeric IgA much more than that of monomeric IgA. Human galectin-1 immobilized in active form in vitro sugar-specifically captured IgA and IgA-containing IC formed by CCA in serum but not IgG. Results suggest that while high IgA content especially in polymeric form may render polysaccharide IC more susceptible to tissue uptake, desialylation of IgA in IC could enhance the process.

CARD9 Syk-dependent and Raf-1 Syk-independent signaling pathways in target recognition of Candida albicans by Dectin-1

As a principle receptor to β-glucan in the Candida albicans cell wall, Dectin-1 performs recognition specifically and its mechanisms are diverse in different situations. With a view to developing certain antifungal targeted drugs, this review provides new insights into the novel immune signaling pathways induced by Dectin-1, especially the novel understandings of CARD9 in the Syk-dependent NF-κB pathway, and of Raf-1 in the Syk-independent NF-κB signaling pathway.

Efficacy of combination immunotherapy of IgM MAb B6.1 and amphotericin B against disseminated candidiasis

We previously reported that the IgM MAb B6.1, specific for β-1,2-mannotriose on the surface of the cell wall of Candida albicans, prevents mice from disseminated candidiasis. The preventive activity of the antibody is mediated by enhanced phagocytosis that caused killing of the fungus and involvement of the complement system. In the present study, MAb B6.1 was tested if the antibody enhances therapeutic efficacy of amphotericin B (Amp B) in a murine model of disseminated candidiasis due to C. albicans. Determination by the kidneys-cfu (colony forming unit) and survival times was used to assess treatment. Mice treated intraperitoneally with MAb B6.1 at 1 h post-infection with C. albicans (5 × 10 5 yeast cells/mouse) developed fewer 28% cfu and prolonged survival rates than negative controls, whereas administration of B6.1 to mice at 2 h post-infection was ineffective. Therapeutic effect of Amp B on mice with the disseminated disease was dose-dependent, but dosage of Amp B (0.5 mg/kg body weight of mice) was not effective. A combination of MAb B6.1given at 1 h post-infection plus Amp B (0.5 mg dose) enhanced survival times beyond the effect due to only antibody ( P < 0.05). The MST (mean survival times) value resulted from the combination therapy-received mice was as almost the same as MST value from 2 mg dose of Amp B-given animals ( P < 0.05). In case mice given a combination of Amp B (0.5 mg dose) plus MAb B6.1 at 2 h post-infection — a condition of developing no therapeutic efficacy, the combination also reduced disease severity. All these data indicate that MAb B6.1 acts in concert with Amp B, the antibody enhances therapeutic efficacy of Amp B, and this combination therapy augments protection which implies a possibility of reducing Amp B dose. The augmentation of the response appeared to be specific because an irrelevant IgM carbohydrate-specific MAb was not protective. In conclusion, these studies show that Amp B combined with MAb B6.1 may be an option of solving the problem of limited antifungal drug choices due to drug-resistant C. albicans

Candida albicans cell wall components and farnesol stimulate the expression of both inflammatory and regulatory cytokines in the murine RAW264.7 macrophage cell line

Candida albicans causes candidiasis, secretes farnesol, and switches from yeast to hyphae to escape from macrophages after phagocytosis. However, before escape, macrophages may respond to C. albicans' pathogen-associated molecular patterns (PAMPs) through toll-like receptor 2 (TLR2) and dectin-1 receptors by expressing cytokines involved in adaptive immunity, inflammation, and immune regulation. Therefore, macrophages and the RAW264.7 macrophage line were challenged with C. albicans preparations of live wild-type cells, heat-killed cells, a live mutant defective in hyphae formation, a live mutant producing less farnesol, or an isolate producing farnesoic acid instead of farnesol. Interleukin-6 (IL-6), and IL-1β, IL-10, and tumor necrosis factor-α (TNF-α) expression were evaluated by ELISA and/or qRT-PCR within 6 h after challenge. All viable strains producing farnesol, regardless of hyphae phenotype, induced IL-6, IL-1β, IL-10, and TNF-α. To determine which components of C. albicans induced IL-6, RAW264.7 cells were incubated with farnesol, farnesoic acid, with or without zymosan, a yeast cell wall preparation that contains PAMPs recognized by TLR2 and dectin-1. The highest expression of IL-6, TLR2, and dectin-1 occurred when RAW264.7 cells were stimulated with zymosan and farnesol together. Our results suggest that the rapid expression of cytokines from macrophages challenged with C. albicans is due to cell-wall PAMPs combined with farnesol.

Fusion expression of the extracellular carbohydrate recognition domain of mouse Dectin-1 and its recognition of β-glucans in the cell wall of Candida albicans

Objective To clone, express and purify the extracellular carbohydrate recognition do-main(CRD) of Dectin-1 in mouse peritoneal macrophages and to further investigate its ability to recognize and bind to β-glucans. Methods The Dectin-1 CRD gone was amplified by RT-PCR from RNA of mouse peritoneal macrophages and cloned into prokaryotic expression vector pET28a (+), the constructed pET-CRD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and the fusion protein was in-duced to express. After affinity purification and renaturation, the fusion protein was incubated with Candida albicans yeast and its ability to recognize and bind to β-glucans in the cell wall of fungi. Results The fu-sion protein could recognize β-glucans in the fungal cell wall. Conclusion The recombinant expression plasmid pET28a-CRD was successfully constructed and the fusion protein was induced. The fusion protein is able to recognize and bind to β-glucans in the fungal cell wall, thus laying a good foundation for fungal de-tection and the exploration of the biological role of β-glucans. Key words: Dectin-1; β-glucans; Fusion expression; Candida albicans

Fully deprotected β-(1→2)-mannotetraose forms a contorted α-helix in solution: convergent synthesis and conformational characterization by NMR and DFT

β-(1→2)-linked oligomannosides, found in the cell wall of Candida albicans, are promising structures for the development of C. albicans vaccines. Considerable effort in recent years has been devoted to the synthesis of these carbohydrate structures. As a result, several successful synthetic methodologies based on linear approaches have emerged. Here, we demonstrate that a fully deprotected β-(1→2)-linked mannotetrasaccharide can also be conveniently constructed by the convergent direct coupling of two appropriately protected disaccharides. This improved approach offers several advantages over previously published methods. The number of steps needed to reach larger oligosaccharides is decreased, while high selectivity is retained when the crucial glycosylation step is performed with two disaccharides, herein providing β-(1→2)-mannotetraose in 4.2% overall yield over 16 steps starting from D-mannose. Additionally, the complete structural characterization of the products by NMR spectroscopy is reported. In the conformational study of the final product, 2D-NOESY was used in combination with spectral simulations performed using PERCH software. The experimental results obtained confirm the contorted α-helical structure predicted earlier for these oligosaccharides in solution. As a culmination of the conformational study, a model was constructed by molecular modeling using DFT, and the minimum energy conformation was found to be in full agreement with the experimental results. This is the first study to date where the conformation of a fully deprotected mannotetraose has been reported.

Synthesis of 3,6-branched oligomannoside fragments of the mannan from Candida albicans cell wall corresponding to the antigenic factor 4

3-Aminopropyl glycosides of 3,6-branched penta- and hexamannoside fragments of the cell wall mannan from Candida albicans, corresponding to the antigenic factor 4, have been synthesized. Subsequent coupling of both oligosaccharides with BSA using the squarate procedure provided corresponding neoglycoconjugates.

Binding and activation of the human plasma kinin-forming system on the cell walls of Candida albicans and Candida tropicalis

Bacterial infections often upregulate the plasma kinin-forming cascade of the host (the 'contact system') which is triggered by adsorption of high molecular mass kininogen (HK), coagulation factor XII (FXII) and prekallikrein (pHPK) on the host or pathogen cell surfaces. A possible activation of the contact system upon infection of the human host by major fungal pathogens of Candida species has not been extensively explored until a recent report of tight binding of HK to the cell walls of these fungi. In the current study, the adsorption of the other contact system components to the cell surfaces of Candida albicans and Candida tropicalis was characterized. FXII was found to be tightly bound by Candida germ tube forms, to a level 5-fold higher than that for HK. In contrast, pHPK bound poorly but its additional amounts could dock to the cell wall through the surface-bound HK. It was also shown that within the complex of these proteins assembled on the cell walls of fungal hyphae, pHPK could be activated by FXIIa and the active HPK effectively produced kinins from HK. It is suggested that kinins, released at the Candida cell wall, can promote host colonization by the pathogen and the development of infection.

Monoclonal antibody‐mediated inhibition of adhesion of Candida albicans and Candida dubliniensis to human epithelial cells

The aim of this study was to assess the role of whole saliva, four saliva-derived preparations, and six monoclonal antibodies (mAbs), directed against components of the cell wall of Candida albicans, on the adhesion of C. albicans and Candida dubliniensis to human epithelial cells (HEC). C. albicans serotype A NCPF 3153 and C. albicans serotype B ATCC 90028 showed higher adhesion to HEC than C. dubliniensis NCPF 3949. Pooled whole saliva was more efficient than salivary secretory immunoglobulin A, partially purified by chromatography, at inhibiting the adhesion of C. albicans serotype A NCPF 3153 to HEC. Monoclonal antibodies C7, 14-8, and 26G7 were the most potent inhibitors of adhesion. Our results show that mAbs can mimic the inhibition of adhesion of C. albicans to HEC that is mediated by human saliva.

Covalently linked cell wall proteins ofCandida albicans and their role in fitness and virulence

The cell wall of Candida albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, containing c. 20 different protein species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat proteins vary widely in function. Many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell-cell aggregation. They further include iron acquisition proteins, superoxide dismutases, and yapsin-like aspartic proteases. In addition, several covalently linked carbohydrate-active enzymes are present, whose precise functions remain hitherto largely elusive. The expression levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary enormously. They depend on the mode of growth and the combined inputs of several signaling pathways that sense environmental conditions. This is reflected in the unusually long intergenic regions of most of these genes. Finally, the precise location of several covalently linked CWPs is temporally and spatially regulated. We conclude that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence and that their expression is tightly controlled.

The effects of Candida albicans cell wall protein fraction on dendritic cell maturation.

Candida albicans is a member of the normal human microflora. C. albicans cell wall is composed of several protein and carbohydrate components which have been shown to play a crucial role in C. albicans interaction with the host immune system. Major components of C. albican cell wall are carbohydrates such as mannans, beta glucans and chitins, and proteins that partially modulate the host immune responses. Dendritic cells (DC), as the most important antigen-presenting cells of the immune system, play a critical role in inducing immune responses against different pathogens. We investigated the effect of the cell wall protein fraction (CPF) of C. albicans on DC maturation. The CPF of C. albicans cells was extracted by a lysis buffer containing sodium dodecyl sulphate, 2-mercaptoethanol and phosphate-buffered saline. The extract was dialyzed and its protein pattern was evaluated by electrophoresis. Dendritic cells were purified from Balb/c mice spleens through a three-step method including mononuclear cell separation, as well as 2-h and overnight cultures. The purified CPF was added at different concentrations to DC. The purity and maturation status of DC were determined by flow cytometry using monoclonal antibodies against CD11c, MHC-II, CD40 and CD86. Treatment of DC with 10 microg/ml of CPF increased the expression of maturation markers including MHC-II, CD86 and CD40 on DC compared to the control group. In this study we used C. albicans CPF with the molecular weight of 40-45 kDa for pulsing and maturation of dendritic cells. Since according to our results CPF significantly increased the expression of maturation markers on DC, we suggest that CPF may act as an efficient immunomodulator, or may be used as a potential adjuvant to boost the host immune system against infections.

Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface

The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic, alpha-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.

Heterologous expression and characterization of a β-1,6-glucanase from Aspergillus fumigatus

The cell wall of Candida albicans is composed of mannoproteins associated to glycan polymers. Most of these proteins are retained in this compartment through a phosphodiester linkage between a remnant of their glycosylphosphatidylinositol anchor and the beta-1,6-glucan polymer. A pure beta-1,6-glucanase is thus required in order to release them. In this paper, we report the expression/secretion by the yeast Yarrowia lipolytica of an Aspergillus fumigatus enzyme homologous to previously described beta-1,6-glucanases. The coding sequence was expressed under the control of a strong promoter and the recombinant enzyme was targeted to the secretory pathway using the signal sequence of a well-known major secretory protein in this host. Addition of a FLAG epitope at the C-terminus allowed its efficient purification from culture supernatant following batch adsorption. The purified enzyme was characterized as a beta-1,6-glucanase and was shown to be active on C. albicans cell walls allowing the release of a previously described cell wall protein.

Effect of plagiochin E, an antifungal macrocyclic bis(bibenzyl), on cell wall chitin synthesis in Candida albicans

To investigate the effect of plagiochin E (PLE), an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L, on cell wall chitin synthesis in Candida albicans. The effect of PLE on chitin synthesis in Candida albicans was investigated at the cellular and molecular levels. First, the ultrastructural changes were observed under transmission electron microscopy (TEM). Second, the effects of PLE on chitin synthetase (Chs) activities in vitro were assayed using 6-O-dansyl-N-acetylglucosamine as a fluorescent substrate, and its effect on chitin synthesis in situ was assayed by spheroplast regeneration. Finally, real-time RT-PCR was performed to assay its effect on the expression of Chs genes (CHS). Observation under TEM showed that the structure of the cell wall in Candida albicans was seriously damaged, which suggested that the antifungal activity of PLE was associated with its effect on the cell wall. Enzymatic assays and spheroplast regeneration showed that PLE inhibited chitin synthesis in vitro and in situ. The results of the PCR showed that PLE significantly downregulated the expression of CHS1, and upregulated the expression of CHS2 and CHS3. Because different Chs is regulated at different stages of transcription and post-translation, the downregulation of CHS1 would decrease the level of Chs1 and inhibit its activity, and the inhibitory effects of PLE on Chs2 and Chs3 would be at the post-translational level or by the inhibition on the enzyme-active center. These results indicate that the antifungal activity of PLE would be attributed to its inhibitory effect on cell wall chitin synthesis in Candida albicans.

A study of the Candida albicans cell wall proteome

Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI motif in 19 of them. We also identified 66 "atypical" cell wall proteins that lack the above-mentioned characteristics. After tryptic removal of the most accessible proteins in the cell wall, several of the same expected GPI proteins and the most commonly found "atypical" wall proteins were identified. This result suggests that proteins are located not only at the cell wall surface, but are embedded within the cell wall itself. These results, which include new identified cell wall proteins, and comparison of proteins in blastospore and mycelial walls, will help to elucidate the C. albicans cell wall architecture.

Candida albicansCell Wall Proteins

The Candida albicans cell wall maintains the structural integrity of the organism in addition to providing a physical contact interface with the environment. The major components of the cell wall are fibrillar polysaccharides and proteins. The proteins of the cell wall are the focus of this review. Three classes of proteins are present in the candidal cell wall. One group of proteins attach to the cell wall via a glycophosphatidylinositol remnant or by an alkali-labile linkage. A second group of proteins with N-terminal signal sequences but no covalent attachment sequences are secreted by the classical secretory pathway. These proteins may end up in the cell wall or in the extracellular space. The third group of proteins lack a secretory signal, and the pathway(s) by which they become associated with the surface is unknown. Potential constituents of the first two classes have been predicted from analysis of genome sequences. Experimental analyses have identified members of all three classes. Some members of each class selected for consideration of confirmed or proposed function, phenotypic analysis of a mutant, and regulation by growth conditions and transcription factors are discussed in more detail.

Functional analysis of Candida albicans GPI-anchored proteins: Roles in cell wall integrity and caspofungin sensitivity

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity (SDS and Calcofluor White sensitivity) and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study.

Immunoproteomic analysis of the protective response obtained from vaccination with Candida albicans ecm33 cell wall mutant in mice

Systemic candidiasis remains a major cause of disease and death, particularly among immunocompromised patients. The cell wall of Candida albicans defines the interface between host and pathogen and surface proteins are major elicitors of host immune responses during candidiasis. The C. albicans ecm33 mutant (RML2U) presents an altered cell wall, which entails an increase in the outermost protein layer. Vaccination of BALB/c mice with RML2U mutant protected them from a subsequent lethal infection with virulent strain SC5314 in a systemic candidiasis model. Using immunoproteomics (2-DE followed by Immunoblotting) we detected 29 immunoreactive proteins specifically recognized by antibodies from vaccinated mice sera, six of which are described as immunogenic for the first time (Gnd1p, Cit1p, Rpl10Ep, Yst1p, Cys4p, Efb1p). Furthermore, identification of wild type and mutant cell surface proteome (surfome), confirmed us that the mutant surfome presented a larger number of proteins than the wild type. Interestingly, proteins exclusively identified in the mutant surfome (Met6p, Eft2p, Tkl1p, Rpl10Ep, Atp1p, Atp2p) were also detected as immunogenic, supporting the idea that their surface location enhances their immunoprotective capacity.