Isolation of hydrophobic and hydrophilic variants of Candida albicans

We have previously demonstrated that most isolates of C. albicans are hydrophobic when grown at room temperature (RT, ca. 22-24 degrees C) and hydrophilic when grown at 37 degrees C. Variants of our standard strain LGH1095 were isolated that are hydrophobic at 37 degrees C and hydrophilic at RT. After repeated phase partitioning with cyclohexane-water cell populations that were 6-16% hydrophobic at RT and 66-80% hydrophobic at 37 degrees C were obtained. Subsequent limiting dilution experiments provided clones which were more hydrophobic at RT or hydrophilic at 37 degrees C. These were then recloned until the resultant populations were consistently under 5% cell surface hydrophobicity (CSH) at RT or over 95% at 37 degrees C. Treatment with several detergents as well as sugars did not decrease the CSH of these cells. Lipase and several proteases also had no effect. When treated with trypsin at a concentration twice that used to lower CSH of normal cells to less than 5%, the hydrophobic variant only decreased in CSH by 50%. Both variants were capable of germinating, although at different levels depending on prior growth temperature. Sensitivity to the germination inhibitor morphogenic autoregulatory substance (MARS) was similar to that of the parent strain.     

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.     

Cell wall anchoring to cytoplasmic membrane of Candida albicans

Abstract Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane.     

Identification of a 49-kDa hydrophobic cell wall mannoprotein present in velum yeast which may be implicated in velum formation

Analysis of velum-forming yeast cell wall components released by beta-1,3-glucanase treatment were compared with those of a non velum-forming yeast. SDS-PAGE electrophoresis and Western blotting with ConA-peroxidase staining of mannoproteins allowed us to identify a 49-kDa mannoprotein present in the cell wall of the velum-forming yeast and hardly visible in the control. The cell wall nature of this protein was confirmed by labelling with the non-permeable sulfosuccinimydiyl-6-(biotinamido)hexanoate reagent. A partial purification of this mannoprotein by anion exchange HPLC followed by surface hydrophobicity determination revealed that the fraction containing the 49-kDa mannoprotein was the most hydrophobic. Since cell surface hydrophobicity plays an important role in aggregate formation, it is likely that this mannoprotein is involved in velum formation.     

Expression of the fibrinogen binding mannoprotein and the laminin receptor of Candida albicans in vitro and in infected tissues

Abstract We have previously reported a 37 kDa laminin-binding protein (p37) and a 58 kDa fibrinogen-binding mannoprotein (mp58) on the surface of Candida albicans . A few yeast cells expressed both functional receptors at the surface while germ tubes expressed a functional mp58 fibrinogen but not a functional p37 laminin receptor. These receptors were heterogeneously dispersed at the surface as shown by binding of rabbit antiserum to mp58 (PAb anti-mp58) and antiserum to the human high affinity laminin receptor. In this report we have used a dual fluorescence technique to determine if the two receptors colocalize, perhaps as part of a receptor complex. Fibrinogen was used as a probe for mp58 and polyclonal antiserum generated to the p37 (PAb anti-p37) was used as a probe for the 37 kDa laminin-binding protein. Both receptors were heterogeneously distributed, but the receptors were not colocalized as the areas of concentration of each receptor were different. Immunohistochemical analysis of tissue sections from patients with disseminated and superficial candidiasis with PAb anti-p37 and PAb anti-mp58 revealed that both receptors were also expressed in infected tissues. The patterns of morphological expression were similar to the in vitro patterns detected by immunofluorescence.     

Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans

The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E-I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans.     

Proteomic analysis of Candida albicans yeast and hyphal cell wall and associated proteins

Candida albicans is an important human pathogen that causes systemic infections, predominantly among populations with weakened immune systems. The morphological transition from the yeast to the hyphal state is one of the key factors in C. albicans pathogenesis. Owing to their location at the host-pathogen interface, the cell wall and associated proteins are of interest, especially with respect to the yeast to hyphal transition. This study entailed the proteomic analysis of differentially regulated proteins involved in this transition. The protein profiles of C. albicans DTT/SDS-extractible proteins and the cyanogen bromide (CNBr)/trypsin-extractable proteins of a cell wall-enriched fraction from yeast and hyphae were compared. In total, 107 spots were identified from the DTT/SDS-extractible cell wall-enriched fraction, corresponding to 82 unique proteins. Of these DTT/SDS-extractible proteins, 14 proteins were upregulated and 10 were downregulated in response to hyphal induction. Approximately 6-9% of total cell wall-protein-enriched fraction was found to be resistant to DTT/SDS extraction. Analysis of the DTT/SDS-resistant fraction using a CNBr/trypsin extraction resulted in the identification of 29 proteins. Of these, 17 were identified only in the hyphae, four were identified only in the yeast, and eight were identified in both the yeast and hyphae.     

Ultrastructural and biochemical studies on Candida albicans after prolonged incubation in sea water

Abstract Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.     

Influence of fluconazole at subinhibitory concentrations on cell surface hydrophobicity and phagocytosis of Candida albicans

Cell surface hydrophobicity (CSH) status influences virulence of Candida albicans and decreases the susceptibility of yeast cells to phagocytic killing. We tested whether subinhibitory concentrations of fluconazole, which is widely used in the treatment and prophylaxis of candidiasis, affect CSH and the susceptibility of C. albicans to enzymatic digestion by glucanase and to phagocytic killing. Treatment of yeast cells with subinhibitory fluconazole concentrations resulted in greater phagocytosis. This effect was independent of CSH but may be related to increased cell wall porosity resulting from alterations in the cell envelope. The use of subinhibitory concentrations of fluconazole in patients with competent phagocytes may contribute to resistance to candidiasis regardless of yeast CSH status.     

白念珠菌破壁方法的研究应用进展

细胞壁作为真菌中特殊和必须的细胞结构,相对于哺乳动物的细胞膜更加坚硬,难以用简单的方法使其充分破碎。因此,达到理想的破壁效果是白念珠菌研究中的关键步骤之一。在提取白念珠菌RNA、DNA和蛋白质等细胞组分的过程中,为获得足量和稳定的实验样品。该文对多种白念珠菌破壁方法作一综述,以便为白念珠菌相关研究提供适用、高效的破壁方案。     

Candida albicans RHO1 is required for cell viability in vitro and in vivo

In Saccharomyces cerevisiae, Rho1p plays an important role in cell wall integrity by regulating beta-1,3-glucan synthase, Pkc1p and the actin cytoskeleton. To determine the physiological role of Rho1p in the dimorphic fungus Candida albicans, the major human fungal pathogen, we constructed mutants that conditionally express Rho1p from the glucose-repressible phosphoenolpyruvate carboxykinase promoter (pPCK1). We examined the growth of these cells in a range of conditions. Depletion of Rho1p from yeast cells resulted in cell death, lysis, and aggregation. The Rho1p conditional mutant was inviable on 10% serum indicating that Rho1p was also required for hyphal viability. Furthermore, in a mouse model of systemic candidiasis, strains dependent on pPCK1-driven RHO1 expression failed to colonise the kidneys and establish disease, suggesting that the level of glucose in serum was sufficient to repress the pPCK1 and that Rho1p-depleted strains were inviable within the host. Therefore, Rho1p is essential for the viability of C. albicans in vitro and in vivo.     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

Identification and immunochemical characterization of a germ tube specific antigen of Candida albicans

Abstract Germ tube specific fractions of the dimorphic pathogenic fungus Candida albicans were fractionated according to their ability to link fibrinogen. These fibrinogen binding factors were used as immunogens to prepare monoclonal antibodies (mAbs) with BALB/c mice. Among the resulting mAbs, one (mAb 3D9.3) was shown by indirect immunofluorescence to be specific to the surface of the mycelial phase of the C. albicans species. No labelling of the cell wall of any other Candida species was observed. This morphological shape specificity was confirmed by immunoblotting where a polydispersed high molecular mass component was identified. The molecular mass varied with the extraction procedure used; over 210 kDa with EDTA-2ME treatment, and ranging from 110 to 220 kDa after Zymolyase digestion. This phase-specific epitope was sensitive to proteolysis with pronase E, proteinase K and trypsin, but not to periodate treatment. Further purification of this material would allow further development of new serodiagnostic assays that might be more specific for invasive disease than currently available tests.     

Structural mannoproteins released by β-elimination from Candida albicans cell walls

Abstract Mild alkaline solutions (β-elimination), after removing the non-covalently bonded wall materials by hot SDS, released 13% and 26% of remaining wall proteins from mycelial and yeast cells of Candida albicans , respectively. When the β-elimination was carried out after digestion of the walls with chitinase, four-fold more proteinaceous materials were released from mycelium and a similar amount in yeast walls. The solubilized materials were shown to be highly polydisperse, and endo-glycosidase H reduced their polydispersity and molecular masses, revealing different electrophoretic patterns in yeast and mycelial cell walls. The solubilized mycelial proteins carried N-glycosidic sugar chains and the epitopes recognized by two monoclonal antibodies were preserved, although showing a different behaviour in yeast walls. These results are consistent with the idea that significant amounts of intrinsic O-glycosylated mannoproteins are interconnected in the walls of C. albicans .     

Cell surface shaving of Candida albicans biofilms, hyphae, and yeast form cells

We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.     

Dimorphism-associated changes in amino acid transport of Candida albicans

Abstract Amino acid uptake was followed during pH-regulated dimorphism of Candida albicans . It was observed that transport activities of various amino acids differed with the morphological phenotype. The uptake rates of l-alanine , l -phenylalanine and of l -lysine were lower and those of l -methionine were higher in elongated hypha (germ tube), while the rates of glycine, l -glutamic acid and l -proline were similar in bud and hyphal phenotypes. Minimum threshold of amino acids transport activity is required at the time of phenotypic commitment in a diverging population of Candida albicans .     

Azole sensitivity in Candida albicans

Abstract In the present study, we assessed the influence of three culture media on the susceptibility 'in vitro' of twenty four clinical strains belonging to Candida albicans against three imidazole-derivatives and also, we investigated the situation of azole sensitivity in three of these strains.