Surface hydrophobicity changes of two Candida albicans serotype B mnn4delta mutants

Cell surface hydrophobicity (CSH) of Candida species enhances virulence by promoting adhesion to host tissues. Biochemical analysis of yeast cell walls has demonstrated that the most significant differences between hydrophobic and hydrophilic yeasts are found in the acid-labile fraction of Candida albicans phosphomannoprotein, suggesting that this fraction is important in the regulation of the CSH phenotype. The acid-labile fraction of C. albicans is unique among fungi, in that it is composed of an extended polymer of beta-1,2-mannose linked to the acid-stable region of the N-glycan by a phosphodiester bond. C. albicans serotype A and B strains both contain a beta-1,2-mannose acid-labile moiety, but only serotype A strains contain additional beta-1,2-mannose in the acid-stable region. A knockout of the C. albicans homolog of the Saccharomyces cerevisiae MNN4 gene was generated in two serotype B C. albicans patient isolates by using homologous gene replacement techniques, with the anticipation that they would be deficient in the acid-labile fraction and, therefore, demonstrate perturbed CSH. The resulting mnn4delta-deficient derivative has no detectable phosphate-linked beta-1,2-mannose in its cell wall, and hydrophobicity is increased significantly under conditions that promote the hydrophilic phenotype. The mnn4delta mutant also demonstrates an unanticipated perturbation in the acid-stable mannan fraction. The present study reports the first genetic knockout constructed in a serotype B C. albicans strain and represents an important step for dissecting the regulation of CSH.     

Surface hydrophobic and hydrophilic protein alterations in Candida albicans

Abstract Cell surface hydrophobicity influences pathogenesis of Candida albicans . Previous studies suggested that stationary-phase hydrophilic and hydrophobic cells, obtained by growth at 37 and 23°C, respectively, may have similar hydrophobic proteins. However, whether hydrophilic and hydrophobic surface proteins differ during the growth cycle at 37°C is unknown. Freeze-fracture analysis revealed surface fibrillar layer differences between hydrophobic late-lag and hydrophilic stationary-phase yeast cells grown at 37°C. Hydrophilic protein differences were also observed between these populations. However, similar hydrophobic proteins were detected among the late-lag and stationary phase cells grown at 37°C and hydrophobic stationary-phase cells grown at 23°C. These results suggest that hydrophobic proteins remain constant but hydrophilic proteins vary during growth. Thus, conversion from surface hydrophilicity to hydrophobicity by C. albicans may only require alterations in the hydrophilic fibrillar protein components.     

Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition

The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans.     

Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity

The opportunistic pathogenic yeast Candida albicans exhibits growth phase-dependent changes in cell surface hydrophobicity, which has been correlated with adhesion to host tissues. Cell wall proteins that might contribute to the cell surface hydrophobicity phenotype were released by limited glucanase digestion. These proteins were initially characterized by their rates of retention during hydrophobic interaction chromatography--high-performance liquid chromatography and used as immunogens for monoclonal antibody production. The present work describes the cloning and functional analysis of a C. albicans gene encoding a 38-kDa protein recognized by the monoclonal antibody 6C5-H4CA. The 6C5-H4CA antigen was resolved by two-dimensional electrophoresis, and a partial protein sequence was determined by mass spectrometry analysis of tryptic fragments. The obtained peptides were used to identify the gene sequence from the unannotated C. albicans DNA database. The antibody epitope was provisionally mapped by peptide display panning, and a peptide sequence matching the epitope was identified in the gene sequence. The gene sequence encodes a novel open reading frame (ORF) of unknown function that is highly similar to several other C. albicans ORFs and to a single Saccharomyces cerevisiae ORF. Knockout of the gene resulted in a decrease in measurable cell surface hydrophobicity and in adhesion of C. albicans to fibronectin. The results suggest that the 38-kDa protein is a hydrophobic surface protein that meditates binding to host target proteins.     

Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn(2+) as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca(2+) and Mn(2+) ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn(2+)-dependent mannosyltransferases. The Capmr1Delta null mutant was viable in vitro and had no growth defect even on media containing low Ca(2+)/Mn(2+) ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.     

The role of the Candida albicans histidine kinase [CHK1) gene in the regulation of cell wall mannan and glucan biosynthesis

The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.     

Mannosylation in Candida albicans: role in cell wall function and immune recognition

The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post‐translational modifications being the addition of O‐ and N‐linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O‐ and N‐linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host–fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease.     

Molecular and Phenotypic Analysis of CaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.     

Inhibition of Candida albicans attachment to extracellular matrix by antibodies which recognize hydrophobic cell wall proteins

Cell surface hydrophobicity influences the adhesive properties of the opportunistic fungal pathogen Candida albicans. Hydrophobic proteins are present in the C. albicans cell wall. These proteins were used to generate a polyclonal antiserum and monoclonal antibodies. We characterized three of these monoclonal antibodies (designated 6C5, 5F8 and 5D8) that recognize different hydrophobic cell wall proteins. Initial characterization of the three antigens, and assessment of their distribution among various Candida species was also carried out. Further, pretreatment of germ tube initials with the mAb inhibits binding of these cells to immobilized extracellular matrix. These results suggest that these hydrophobic proteins are involved in C. albicans adhesion events.     

Contact Angle Measurement and Cell Hydrophobicity of Granular Sludge from Upflow Anaerobic Sludge Bed Reactors

The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45(deg)) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45(deg)). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.     

Hydrophobicity as an Adhesion Mechanism of Benthic Cyanobacteria

The capacity of benthic cyanobacteria to adhere to solid substrates was examined in terms of their cell surface properties. By using a biphasic water-hydrocarbon test system, it was demonstrated that benthic cyanobacteria from divergent habitats were all hydrophobic, whereas all the planktonic cyanobacteria tested were hydrophilic. Divalent cations were found more efficient than monovalent cations in effecting the expression of hydrophobicity. Mechanical shearing of the cell surface, as well as chemical removal of the cell wall, demonstrated that the hydrophobicity was confined to the outer surface layers. The hydrophobic sites were distributed along the whole length of the cyanobacterial filament. Hydrophilic hormogonia of benthic cyanobacteria became hydrophobic within 48 h when grown in the light; chloramphenicol, 3(3,4-dichlorophenyl)1,1 dimethylurea, or incubation in the dark prevented this transition. Hydrophobicity of Phormidium filaments was masked in late stationary phase; this effect was removed by gentle washing.     

LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida

The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida.     

Determination of bacterial cell surface hydrophobicity of single cells in cultures and in wastewater in situ

Bacterial cell surface hydrophobicity is one of the most important factors that influence bacterial adhesion. A new method, microsphere adhesion to cells, for measuring bacterial cell surface hydrophobicity was developed. Microsphere adhesion to cells is based on microscopic enumeration of hydrophobic, fluorescent microspheres attaching to the bacterial surface. Cell surface hydrophobicity estimated by microsphere adhesion to cells correlates well with adhesion of bacteria to hydrocarbons or hydrophobic interaction chromatography for a set of hydrophilic and hydrophobic bacteria (linear correlation coefficients, R², were 0.845 and 0.981 respectively). We also used microsphere adhesion to cells to investigate the in situ properties of individual free-living bacteria directly in activated sludge. Results showed that the majority of the bacteria were hydrophilic, indicating the importance of cell surface hydrophobicity for bacterial adhesion in sludge, and for the overall success of the wastewater treatment process.     

Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy

MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.     

Agglutination of blastospores of Candida albicans by concanavalin A and its relationship with the distribution of mannan polymers and the ultrastructure of the cell wall.

Blastospores of Candida albicans were readily agglutinated by Concanavalin A (Con A) owing to the specific binding of this lectin to the mannan receptors of the cell surface. When mannan was extracted from the cell wall by neutral buffers, alkali and acid, the agglutination was decreased or lost depending on the degree of extraction. A relatively mild alkali treatment was sufficient to derange the multilayered wall organization and transform it into a uniform, medium-density structure having about the same thickness as the untreated wall. After a more drastic extraction, all the electron-dense components of the wall were lost, the residual, alkali-insoluble wall fabric being completely electron-transparent and of about the same thickness as the inner wall region of untreated cells. Thiol-reducing agents like mercaptoethanol or dithiothreitol also extracted wall materials, an effect which was enhanced by pronase. After dithiothreitol-pronase treatment, the outer wall layers were removed but the inner wall region was not apparently damaged and some electron-dense components remained. None of these treatments significantly affected blastospore agglutination by Con A--this was reduced (but not abolished) only by the sequential action of pronase and helicase, which led to sphaeroplast formation. These sphaeroplasts showed a varied amount of residual wall consisting of evenly distributed, fibrogranular components. Two main conclusions were drawn from these results: (i) mannan polymers extend throughout the wall of the blastospore of C. albicans; (ii) the layering of the wall, as seen by ordinary fixation and staining for electron microscopy, essentially reflects the distribution of the various alkali-soluble complexes, at different levels, both over and in the rigid, glucan-chitin matrix.     

Cell surface hydrophobicity-associated adherence of Candida dubliniensis to human buccal epithelial cells

Microbial adherence to mucosal surfaces is an important first step in the initiation of the pathogenic process in the oral cavity. Candida albicans, the most adherent and pathogenic Candida species, utilizes a variety of mechanisms to adhere to human tissues. Although the strongest mechanism of adherence involves mannoprotein adhesins on C. albicans, cell surface hydrophobicity (CSH) plays an important role in the adherence process by providing hydrophobic interactions that turn the initial attachment between the yeast and a surface into a strong bond. Recent cell wall analytical and comparative studies showed that, Candida dubliniensis, unlike C. albicans, possesses cell surface variations that allow it to be constantly hydrophobic, regardless of growth temperature. Based on these observations, the present study was designed to compare the adherence abilities of C. dubliniensis and C. albicans to pooled human buccal epithelial cells (BEC), in regards to their cell surface hydrophobicity. Ten C. albicans and nine C. dubliniensis isolates, as well as the C. albicans hydrophobic variant A9V10 were evaluated for adherence with BEC using visual aggregation in the wells of a microtiter plate and microscopic examination. All 11 C. albicans isolates failed to show adherence to BEC, visually or microscopically, when grown at 37 degrees C. The same isolates, however, showed significant increase in aggregation and microscopic adherence to BEC when grown at 25 degrees C. All C. dubliniensis isolates tested and the A9V10 C. albicans hydrophobic variant resulted in visual aggregation and adhered to BEC when grown at either temperature. The findings from this study show that, based on comparative adherence results and growth temperature changes, C. dubliniensis seems to have greater adherence to BEC than do typical C. albicans strains and that hydrophobic interactions seem to be the mechanism of adherence involved. Although many questions remain to be answered regarding the clinical implications of this observed in vitro enhanced adherence of C. dubliniensis to human BEC, these findings support the establishment of this novel species as a clinically significant yeast.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

A model vaccine exploiting fungal mannosylation to increase antigen immunogenicity

Ag mannosylation represents a promising strategy to augment vaccine immunogenicity by targeting Ag to mannose receptors (MRs) on dendritic cells. Because fungi naturally mannosylate proteins, we hypothesized that Ags engineered in fungi would have an enhanced capacity to stimulate T cell responses. Using the model Ag OVA, we generated proteins that differentially expressed N- and O-linked mannosylation in the yeast Pichia pastoris and compared them to their unglycosylated counterparts produced in Escherichia coli. We found that yeast-derived OVA proteins containing N-linkages, extensive O-linkages, or both were more potent than the unmannosylated Ags at inducing OVA-specific CD4+ T cell proliferation. This elevated response to fungal Ags was inhibited by mannan, suggesting involvement of MRs. However, the macrophage MR (CD206) was not essential, because macrophage MR-deficient dendritic cells were fully competent in presenting yeast-derived OVA Ags. Thus, the use of fungal glycosylation to provide N-linked and/or extensive O-linked mannosylation increased the capacity of the model Ag OVA to stimulate Ag-specific T cell responses in an MR-dependent manner. These data have implications for vaccine design by providing proof of principle that yeast-derived mannosylation can enhance immunogenicity.     

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

Nuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.