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.

Hydrophobic cell wall protein glycosylation by the pathogenic fungus Candida albicans.

Cell surface hydrophobicity influences adhesion and virulence of the opportunistic fungal pathogen Candida albicans. Previous studies have shown that cell surface hydrophobicity is due to specific proteins that are exposed on hydrophobic cells but are masked by long fibrils on hydrophilic cells. This observation suggests that hydrophobic cell wall proteins may contain little or no mannosylation. In the present study, the glycosylation levels of three hydrophobic cell wall proteins (molecular mass range between 36 and 40 kDa) derived from yeast cells were examined. One hydrophilic protein (90 kDa) was also tested. Various endoglycosidases (endoglycosidase F-N-glycosidase F, O-glycosidase, beta-mannosidase, N-glycosidase F), an exoglycosidase (alpha-mannosidase), and trifluoromethane sulfonic acid were used to deglycosylate the proteins. All four proteins were reactive to the lectin concanavalin A, demonstrating that they were mannoproteins. However, gel electrophoresis of the control and treated proteins revealed that mannosyl groups of hydrophobic proteins were less than 2 kDa in size, while the mannosyl group of the hydrophilic protein had a molecular mass of approximately 20 kDa. These results suggest that unlike many hydrophilic proteins, hydrophobic proteins may have low levels of glycosylation. Changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface.

Expression of surface hydrophobic proteins by Candida albicans in vivo.

Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The polyclonal antiserum distinguished between hydrophobic and hydrophilic cell surfaces in vitro and gave similar staining patterns and intensities for C. albicans cells in vivo. Of the yeast forms present within tissue lesions, approximately half exhibited moderate to intense immunofluorescence with the antiserum. Immunoblot analysis indicated that antigens recognized by the antiserum are predominantly low-molecular-mass hydrophobic proteins that are expressed by different C. albicans isolates and are expressed regardless of growth temperature. Taken together, the immunofluorescence and immunoblot analyses of antigens indicate that C. albicans displays surface hydrophobic proteins during pathogenesis and these proteins are available for hydrophobic interactions with host tissues. The effect of hydrophobic protein exposure on the virulence of C. albicans is discussed.

Candida albicans- and Candida stellatoidea-specific DNA fragment.

DNA was isolated from whole cells of Candida albicans and digested with MspI restriction enzyme. In addition to the expected large number of low-molecular-weight DNA pieces resulting from the digestion, multiple high-molecular-weight (greater than 3.0 kilobase pairs) fragments were generated by this enzyme, which cleaves DNA at CCGG sequences. Some of these fragments appeared highly repeated. An MspI fragment which was similar in size to one of the repeat elements (2.9 kilobase pairs) was cloned into the ClaI site of the plasmid vector pBR322 and replicated in a suitable Escherichia coli host strain. The candidal fragment was radiolabeled and used to probe Southern blots of DNA from several Candida species, various other fungi, and mouse and human cells. Only DNA from C. albicans and a strain of Candida stellatoidea was found to contain sequences of significant homology for hybridization. The cloned fragment may possibly be of use as a DNA probe for detection of the presence of C. albicans.

Influence of cell surface hydrophobicity on attachment of Candida albicans to extracellular matrix proteins.

Cell surface hydrophobicity expression by Candida albicans facilitates a diffuse binding distribution of yeast cells to host tissues ex vivo. One possibility for the receptor site responsible for the binding pattern of hydrophobic cells is the extracellular matrix (ECM). In this study, we evaluated the interaction of hydrophobic and hydrophilic C. albicans with ECM proteins immobilized onto wells of microtitre tissue. Culture plates, and the ability of ECM proteins to block the binding of hydrophobic cells to splenic tissue ex vivo. Hydrophobic C. albicans bound in greater numbers than hydrophilic cells to the immobilized proteins, particularly fibrinogen, fibronectin, collagen type IV and laminin. Similar results were obtained regardless of C. albicans strains or of growth medium. Collagen and fibronectin blocked the binding of hydrophobic cells to the white pulp but not to the marginal zones in splenic tissues when tested with the ex vivo assay. These results suggests that the diffuse binding pattern of hydrophobic cells in the ex vivo assay may be due to their enhanced ability over hydrophilic cells to bind to ECM proteins, particularly fibronectin and collagen type IV.

Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing.

Hydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two-dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.

Presence of multiple laminin- and fibronectin-binding proteins in cell wall extract of Candida albicans: influence of dialysis.

Candida albicans has been reported to express only one to three proteins that bind extracellular matrix proteins, such as laminin and fibrinogen. In those reports, cell wall extracts were subjected to various processing steps, such as dialysis and lyophilization, prior to Western blot analysis. Here, we demonstrate that dialysis for only 2 h of cell wall protein extracts results in a substantial loss (40-60%) of protein. With overnight dialysis, the loss was increased further. After 2 h of dialysis, wall extracts contained fewer laminin- and fibronectin-reactive proteins. In addition, the number of wall proteins in the extracts detected by a polyclonal anti-human fibronectin receptor antiserum decreased after dialysis. These results demonstrate that the C. albicans yeast cell wall contains multiple proteins capable of binding laminin and fibronectin and many of these proteins are not functionally detectable following dialysis.

Methods for DNA extraction from Candida albicans.

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.

Peptide ligands that bind IgM antibodies and block interaction with antigen.

We have selected a peptide-display phage library on IgM Abs and identified a panel of phage-expressing peptides that bind to IgM Abs in general, but not to Abs of other classes. A synthetic peptide corresponding to one of the displayed peptide sequences also binds to IgM Abs. The peptides bind to both soluble pentameric Abs and to monomeric cell-surface IgM. The phage-displayed and synthetic peptides inhibit the binding of IgM Abs to Ag. These peptides may create confounding artifacts when IgM Abs are used for epitope mapping studies. Nonetheless, the peptides may have both experimental and therapeutic utility.

Peptides that mimic the group B streptococcal type III capsular polysaccharide antigen.

Microbial polysaccharides are notably poor immunogens. We have developed an alternate route for the production of Abs to important carbohydrate epitopes. mAb S9, a protective mAb against the type III capsular polysaccharide of group B streptococci (GBS), was used to select epitope analogues from a peptide display phage library. Depending upon desorption conditions, two populations of phage were identified with displayed sequences of WENWMMGNA and FDTGAFDPDWPA. ELISA results demonstrated that these phage bound to S9 and no other Abs. Phage blocked the binding of S9 to type III GBS, but did not block binding by another anti-GBS mAb. Phage displaying the latter peptide sequence showed greater inhibition. Ab S9 and other monoclonal and polyclonal anti-GBS type III antisera bound the synthetic peptide FDTGAFDPDWPAC. The binding of S9 to GBS was inhibited by the free peptide with an IC50 of 30 microg/ml. The binding of polyclonal anti-GBS antibodies to peptide could be blocked by intact GBS as well as purified capsular polysaccharide. The peptide was conjugated to three different carriers and was used to immunize mice. All mice produced a significant antibody response to GBS and to the purified capsular polysaccharide following a single immunization. These data demonstrate that a peptide mimetic of the GBS capsular polysaccharide is both antigenic and immunogenic. The incorporation of such peptides into vaccine preparations may enhance the efficacy of vaccines in inducing Ab responses to important carbohydrate epitopes.

Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow.

Adhesion interactions during hematogenous dissemination of Candida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1 beta activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.