Biomedical subjects

W L Chaffin

Publications and source records attributed to W L Chaffin.

At least 19 recordsLinked to original sources

PubMed1996

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

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.

Animals↗

PubMed1996

Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.

Animals↗

PubMed1996

Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family.

We have previously reported the isolation of several clones from a cDNA expression library from Candida albicans, one of which was associated with a constitutively expressed 70-kDa protein. The moiety was present in the beta-mercaptoethanol extracts of cell walls from both blastoconidia and germ tubes. The surface expression of this moiety was revealed by an indirect immunofluorescence assay using affinity-purified antibody to the fusion protein produced by the clone. The 0.68-kb cDNA insert was sequenced. A database search revealed extensive homology with the 70-kDa family of stress or heat shock proteins (hsps). The 77% homology with another C. albicans HSP70 sequence suggested that this fragment represented a second member of the HSP70 family in this organism. Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae HSP70 gene family. The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recognizing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to distinguish extracellular from cytosolic proteins. Biotinylated hsp70 was detected by Western blotting (immunoblotting) among the biotinylated components affinity purified by chromatography on streptavidin, thereby establishing its presence in the cell wall. Immunoelectron microscopy showed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through channels. Northern (RNA) blot analysis showed that the gene was expressed in yeast cells growing in yeast extract-peptone medium at both 25 and 37 degrees C and in Lee medium at 25 degrees C and during formation of germ tubes in Lee medium 37 degrees C. No obvious increase in the expression level was detected after the temperature shift. Members of the hsp70 family have been reported to be immunoreactive. The fusion protein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and translocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.

Amino Acid Sequence↗

PubMed1996

Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae.

Western blot (immunoblot) analysis of cell wall and cytosolic extracts obtained from parental and ssa1 and ssa2 single- and double-mutant strains of Saccharomyces cerevisiae showed that the heat shock protein 70 (Hsp70) products of these genes, previously thought to be restricted to the cell interior, are also present in the cell wall. A cell wall location was further confirmed by indirect immunofluorescence with intact cells and biotinylation of extracellular Hsp70. Hsp70s have been implicated in translocation across the membrane and as molecular chaperones, and changes in the profile of cell wall proteins suggested that these proteins may have a similar role in the cell wall.

Amino Acid Sequence↗

PubMed1995

Complex interaction between different proteinaceous components within the cell-wall structure of Candida albicans.

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface of Candida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to the C. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluorescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.

Antibodies, Fungal↗

PubMed1995

Adherence of germ tubes of Candida albicans to tissues from immunocompromised mice.

The influence of immune status of the host on binding of germ tubes of Candida albicans to murine tissue sections in an ex vivo assay was examined. Generally, germ tubes appeared randomly adhered to the tissues examined and binding was unaffected by immunodeficiency induced by treatment with cyclophosphamide and cortisone acetate. Adherence was somewhat reduced in spleen and kidney sections or increased in liver sections and unchanged in lymph node sections from treated mice compared to sections from control animals. Scanning electron micrographs showed organisms appeared to be loosely or tightly bound to the surface or partially embedded in spleen sections from both control and treated mice. These observations suggested that qualitative and quantitative difference in adhesion of germ tubes to various tissues may contribute little to the susceptibility of the immunodeficient animal to candidal infection.

Animals↗

PubMed1995

Comparative study of the C3d receptor and 58-kilodalton fibrinogen-binding mannoproteins of Candida albicans.

Using polyclonal antibodies (PAbs) raised against the Candida albicans C3d receptor (CR2; PAb anti-CR2) and the 58-kDa fibrinogen-binding mannoprotein (mp58; PAb anti-mp58) as well as ligand interactions, we have studied the relationship between these two receptors. In an indirect immunofluorescence assay with germ tubes, greater intensity was observed on the mother blastoconidium when PAb anti-CR2 was used, whereas greater intensity was localized to the hyphal extension when PAb anti-mp58 or binding of soluble fibrinogen was used. No competition or change in the fluorescence pattern was observed in dual-labeling experiments with PAb anti-CR2 and either fibrinogen or PAb anti-mp58. Binding competition also was not observed in an enzyme-linked immunosorbent assay using the components present in a beta-mercaptoethanol extract from the cell wall of germ tubes. In immunoblots, PAb anti-CR2 recognized three different discrete bands with apparent molecular masses of 21, 40, and 66 kDa in the beta-mercaptoethanol extracts from the cell wall, whereas a different, single, broader band with an apparent molecular mass of 58 kDa was detected with PAb anti-mp58. However, when nondenaturing conditions were used to separate the materials present in the cell wall extracts, no reactivity could be detected on Western blots (immunoblots) with PAb anti-mp58. When PAb anti-CR2 was used for analysis, a single band migrating in the area corresponding to approximately 40 kDa was detected. These observations suggest a higher molecular weight for mp58 and one or more of the components detected with PAb anti-CR2 in their native state.

Adhesins, Bacterial↗

PubMed1995

Adhesion of Candida albicans to brain tissue of Macaca mulata in an ex vivo assay.

An ex vivo adhesion assay was used to examine adhesion of Candida albicans yeast cells to brain tissue of the primate Macaca mulata. Tissues from frontal lobes and striatum (caudate, putamen, and portions of the globus pallidus) were used in the assay. Yeast cells adhered to gray matter at about six times the level of adhesion to white matter. The fungus was able to bind to different cell types within the cortex, basal ganglia, and white matter. Binding to neurons, small neurons or glia, endothelial cells, and neuropil was observed.

Adhesiveness↗

PubMed1994

Coordination of germ tube formation and surface antigen expression in Candida albicans.

If the determinants of shape and cell wall topography are independently regulated and induced in germ tube formation in Candida albicans, these processes may be separable in a non-germ tube forming strain. The expression of several preferentially expressed hyphal surface components in a parental, non-germ tube forming variant, and a germ tube forming revertant strain were examined by indirect immunofluorescence. The proportion of germ tubes expressing the determinants and the morphological localization of expression was similar. Few yeast cells in germ tube cultures bound probes and there was no increase in binding by yeast cells of the variant strain. Extraction with beta-mercaptoethanol prior to analysis had little effect on probe binding and the shape of yeast cells were similar. These observations suggest the ability to promote apical expansion in germ tube formation and surface expression of certain markers were coordinately regulated.

Antigens, Fungal↗

PubMed1994

Murine tissues exposed to cytotoxic drugs display altered patterns of Candida albicans adhesion.

An ex vivo adhesion assay was used to examine the binding of Candida albicans yeast cells to tissues from mice treated with cytotoxic drugs such as lipopolysaccharide and the clinically used anticancer drugs doxorubicin, cisplatin, and vincristine. No major differences were observed in binding of the fungal cells to liver and kidney tissues from treated or untreated animals. All drug-treated spleens displayed altered patterns of C. albicans adhesion compared with the control group, with yeast cells bound not only to the marginal zone but also to the white and red pulp. Immunostaining for macrophages, which are proposed as the site of normal adhesion, showed no apparent differences between the control and the experimental spleens that could account for the change in adhesion patterns. Scanning electron microscopy images suggested that yeast binding to the white pulp of treated tissue is mediated through fibers, perhaps extracellular matrix components exposed as result of the cytotoxic treatment. Exposure of new attachment sites for C. albicans in treated tissues may facilitate initiation of infection.

Adhesiveness↗

PubMed1994

Binding of the extracellular matrix component entactin to Candida albicans.

We have investigated the interaction between Candida albicans and entactin, a recently characterized glycoprotein present in the extracellular matrix, especially in the basement membrane. Organisms of both the yeast and the hyphal morphologies of the fungus had the ability to bind recombinant entactin, as detected by an indirect immunofluorescence assay. Material present in the 2-mercaptoethanol cell wall extracts from both C. albicans growth forms was capable of binding to immobilized recombinant entactin in a dose-dependent manner. Binding to entactin was approximately twice that observed for laminin. Binding of an extract component(s) to entactin was partially inhibited by an Arg-Gly-Asp-Ser peptide. A polyclonal antientactin antiserum, as well as a pooled antiserum preparation raised against components present in different C. albicans cell wall extracts, completely or almost completely abolished binding. The existence of morphology-specific receptor-like molecules which bind to different domains of the entactin molecule was ruled out in a competition binding assay. The entactin-binding material(s) in the cell wall also displayed some ability to bind laminin and fibronectin, since preadsorption in the presence of these extracellular matrix components resulted in reduction of binding to entactin. Moieties with a molecular mass of approximately 25, 44, and 65 kDa present in the 2-mercaptoethanol cell wall extracts from both blastoconidia and germ tubes were detected in a ligand affinity blotting experiment as having the ability to bind entactin. Interactions between C. albicans and entactin could be important in mediating adhesion of the fungus to the host tissues and may play a role in the establishment of the disseminated form of the disease.

Adhesiveness↗

PubMed1994

Heterogeneous surface distribution of the fibrinogen-binding protein on Candida albicans.

As detected by indirect immunofluorescence and confocal microscopy, fibrinogen binding was heterogeneously distributed on the surface of Candida albicans. A low level of binding was generally observed homogeneously distributed on some yeast and most hyphal extensions of germ tubes. However, on most hyphal extensions, there were randomly distributed areas of increased expression, as revealed by patches of greater fluorescence intensity.

Candida albicans↗

PubMed1994

Adherence of Candida albicans germ tubes to murine tissues in an ex vivo assay.

Adhesion of Candida albicans germ tubes to murine tissues was examined. An ex vivo assay previously employed to examine adhesion of yeast cells of C. albicans was adapted for use with germ tubes. Binding of germ tubes to kidney, liver, spleen, and lymph node tissues was found to occur throughout the tissue section, with little morphologic specificity. In general, more organisms adhered to spleen and lymph node tissues than to kidney and liver tissues. Observation of adhesion with scanning organisms adhered to spleen and lymph node tissues than to kidney and liver tissues. Observation of adhesion with scanning electron microscopy showed three germ tube-tissue interactions described as loose, tight, or embedded.

Animals↗

PubMed1993

Characterization of mutant strains of Candida albicans deficient in expression of a surface determinant.

Monoclonal antibody (MAb) 17E4 reacts with a surface carbohydrate determinant and agglutinates cells of Candida albicans. Using this MAb, we have isolated N-methyl-N'-nitro-N-nitrosoguanidine-induced nonagglutinating mutants. Eleven of these were characterized for the presence and expression of the surface antigen recognized by MAb 17E4 by immunoblot analysis of whole cells and by fluorescence flow cytometry. Soluble cell wall extracts from five mutant strains were negative by immunoblot analysis. The reactivities of the strains with several other MAbs and commercial antisera (Candida Check; Iatron Laboratories, Tokyo, Japan) which also recognize carbohydrate determinants were examined by immunoblot analysis of whole cells. Mutant strains showed no or reduced expression of the MAb 17E4 antigen and could be placed into at least two distinct phenotypic classes. Recognition by the other MAbs tested showed a similar pattern, while recognition by the commercial antisera was unchanged in the mutant strains. 1H and 13C nuclear magnetic resonance spectral analysis of mannan prepared from the wild type and nonexpressing mutant-strain 4A showed that the spectra from the mutant strain were simpler than those of the wild type. Most of the beta-1,2 linkages and all of the C-1 phosphate linkages were absent in the 4A mannan spectra, which suggested that the mutant mannan lacked the phosphate-bound beta-1,2-linked mannooligosaccharides. The effect of the surface defect on the ability of mutant strain 4A to adhere and to invade host tissue was examined in two murine models. In ex vivo binding assays, strain 4A showed reduced binding to the marginal zone and increased binding to the white pulp of splenic tissue, decreased binding to kidney tissue, and no change in binding to liver tissue compared with the wild type. In vivo, no difference was observed in translocation of the wild type or strain 4A to liver following immuno-compromising treatment of infant mice which had been challenged with either strain by the oral-intragastric route.

Animals↗

PubMed1991

Identification of germ tube cell wall antigens of Candida albicans.

The reactivity of affinity-purified antibody to two components of germ tube cell wall extracts of Candida albicans showed that the components shared a common determinant(s). Surface expression of at least one of these determinants was demonstrated by indirect immunofluorescence where antibody binding was observed only on the hyphal extension of the organism.

Antigens, Fungal↗

PubMed1991

Cell wall glycoproteins of Candida albicans as released by different methods.

Different methods of extraction frequently used in other studies were used to release glycoproteins from both intact cells and isolated cell walls of yeast and hyphal forms of Candida albicans. Extracts were obtained from whole cells by treatment (i) with 2-mercaptoethanol (beta ME) at pH 8.6 and 37 C degrees and (ii) with zymolyase after treatment with beta ME. Extracts were obtained from isolated and washed cell walls (i) by boiling with beta ME and sodium dodecyl sulphate (SDS), (ii) by boiling with SDS and (iii) by treatment with zymolyase after SDS. The extracts were separated by SDS-polyacrylamide gel electrophoresis and analysed by Western blotting with four reagents. Analysis with concanavalin A (ConA) revealed different glycoprotein populations depending on the treatment. Three possible germ-tube-specific constituents were observed; and 80 kDa component released by beta ME from both intact cells and cell walls, and 47 kDa and 43 kDa moieties released by zymolyase only from intact cells. MAb 4C12, specific for the protein portion of a large germ tube constituent, recognized polydisperse material which just entered the gel in beta ME extracts and in the region extending up from 200 kDa to near the top of the gel in zymolyase extracts. MAb 24.17, specific for a carbohydrate determinant of yeast phase cells, reacted with disperse material in the region from the top of the gel to one-third to two-thirds the distance to the 220 kDa mass marker. Antiserum specific for the serotype A determinant of mannan reacted with large disperse component(s) migrating in the region from the top of the gel to about two-thirds the distance to the 220 kDa mass marker and with a 180 kDa component. The components recognized by MAb 4C12, but not those recognised by MAb 24.17 and serotype A antiserum, were effected by treatment with endo-beta-N-acetylglucosamidase H. The various analyses revealed that the method of extraction affected the composition and size of the constituents recognized by the reagents.

Antibodies, Monoclonal↗

PubMed1991

Phosphate-containing proteins and glycoproteins of the cell wall of Candida albicans.

The distribution of phosphate, carbohydrate, and protein in the cell wall components extracted from intact yeast cells of Candida albicans by beta-mercaptoethanol (beta ME) at pH 8.6 was examined by analysis of the material separated by DEAE-cellulose chromatography. All protein peaks did not coincide with peaks of both carbohydrate and phosphate. Subsequent analysis was performed on material obtained from yeast cells and germ tubes which were grown in medium containing [32P]phosphate. Two extracts were obtained by treating cells with beta ME or with zymolyase following beta ME. The extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. beta ME-extracted material contained high-molecular-mass (HMM), greater than or equal to 200 kDa, polydisperse material and a major and minor band of 19 to 20 kDa, Zymolyase extracts contained (i) three components of less than or equal to 40 kDa, one of which may correspond to the major beta ME band; (ii) four bands within the HMM region which may correspond to previously reported bands; and (iii) one band of 100 to 120 kDa. After longer exposures, additional midrange bands were detected in the zymolyase extract. In extracts treated with endo-beta-N-acetylglucosaminidase H, the HMM polydisperse material increased in mobility although retaining sufficient radiolabel for detection. Western immunoblot analysis of extracts with germ tube-specific antiserum and a germ tube-specific monoclonal antibody and concanavalin A showed that not all components contained detectable phosphate, not all glycoproteins contained detectable phosphate, and at least one 19- to 20-kDa protein may be phosphorylated in the absence of carbohydrate.

Acetylglucosaminidase↗

PubMed1990

Fab fragments from a monoclonal antibody against a germ tube mannoprotein block the yeast-to-mycelium transition in Candida albicans.

Fab fragments prepared from the immunoglobulin G monoclonal antibody (MAb) 4C12, which reacts with a determinant expressed on the hyphal extension of germ tubes of Candida albicans, inhibited germ tube formation, but intact MAb 4C12 did not. Indirect immunofluorescence showed a punctate binding pattern on cells incubated with Fab fragments but a confluent binding on cells incubated with intact MAb 4C12.

Antibodies, Monoclonal↗