Involvement of alpha(v)beta3 integrin-like receptor and glycosaminoglycans in Candida albicans germ tube adhesion to vitronectin and to a human endothelial cell line

The present study was undertaken to investigate the expression of alpha(v)beta3 and alpha(v)beta5 integrin-like vitronectin receptors (VNRs) on Candida albicans germ tube and their involvement in its adhesion to vitronectin (VN) and human endothelial cells. By immunofluorescence and FACS analysis, several monoclonal antibodies directed against human alpha(v) or beta3 integrin subunit or alpha(v)beta3 and alpha(v)beta5 heterodimers, positively stained C. albicans germ tubes. C. albicans germ tubes specifically adhered (45-50%) to VN and this adhesion was markedly inhibited by RGD-, but not RGE-containing peptides. Adhesion of C. albicans germ tubes to VN was strongly inhibited by anti-alphav, anti-beta3 or anti-alpha(v)beta3, but not by alpha(v)beta5 monoclonal antibody. C. albicans germ tube adhesion to VN was also inhibited by glycosaminoglycans (GAGs) such as heparin or chondroitin sulphate. Finally, we show that C. albicans germ tubes adhere to the human EA.hy 926 endothelial cell line. This adhesion is markedly blocked by anti-beta3 monoclonal antibody, GRGDSP peptide or heparin, and is completely abolished by their combination. Overall these results indicate that C. albicans germ tube adherence to VN and to a human endothelial cell line is mediated by alpha(v)beta3, but not by alpha(v)beta5-like integrin, and depends on GAGs which may act by regulating alpha(v)beta3 integrin-like/VN adhesive interaction.     

Platelet interactions with Candida albicans.

The interaction of human platelets and Candida albicans was studied. Platelet-rich plasma was obtained from freshly drawn blood or outdated platelet concentrates. From the platelet-rich plasma, a platelet extract was derived which stimulated germ tube formation by C. albicans when incubated with yeast cells at 37 degrees C. The active component(s) was heat stable, trypsin sensitive, and ribonuclease and deoxyribonuclease insensitive, and possessed cationic properties since it readily attached to carboxymethyl-Sephadex. The active component(s) seemed to bind to heparin also, since germ tube-promoting activity was eluted from a heparin-cyanogen bromide-activated Sepharose 4B column. In addition, platelet-derived growth factor (Collaborative Research, Inc.) stimulated germination when incubated with low amounts (0.4% final concentration) of bovine calf serum. The aggregation of platelets, prepared as platelet-rich plasma by C. albicans cell wall or alkali-extracted cell wall fractions, was also studied. Aggregation of platelets was observed when cell wall or cell wall fractions were incubated with platelet-poor plasma at 37 degrees C for 20 min and then added to platelet-rich plasma. The component of platelet-poor plasma which promoted aggregation of platelets by C. albicans cell wall or alkali-extracted fractions was inactivated at 56 degrees C (30 min) and by cobra venom factor, indicating a role for the alternate complement pathway in the aggregation response.     

Germ-tube formation by oral strains of Candida tropicalis.

Candida species isolated from the mouths of healthy children and of patients with denture stomatitis included strains of Candida tropicalis that formed germ tubes when incubated in serum. Twenty-six germ-tube-forming strains of C. albicans and of C. tropicalis were subcultured weekly for 9 wk on blood agar and on Sabouraud's agar and the ability of each subculture to form germ tubes was measured. All the strains of C. albicans formed almost as many germ tubes after nine weekly subcultures as they did when first isolated. By contrast, although all 26 strains of C. tropicalis formed germ tubes when first isolated, all had lost the ability to do so after six serial weekly subcultures. Germ-tube formation should not be the sole criterion for the identification of oral C. albicans strains.     

Trehalose hydrolysis is not required for human serum-induced dimorphic transition in Candida albicans: evidence from a tps1/tps1 mutant deficient in trehalose synthesis

Exponential yeast-like cells of a Candida albicans wild-type strain exhibited strong capacity for germ tube formation in a glucose-containing medium (YPD) after induction with human serum at 37 degrees C, whereas the isogenic double disruptant tps1/tps1 mutant, which is deficient in trehalose synthesis, failed to produce germ tubes. In a medium without glucose (YP), the morphological transition fraction was roughly equivalent in both strains. Substitution of glucose by galactose or glycerol increased the number of wild-type proliferating cells able to enter the dimorphic program with no noticeable change in their trehalose content, while stationary cells, which accumulate a large amount of trehalose, did not form germ tubes. When fresh medium was added, a high proportion of these resting cells recovered their ability to carry out dimorphic transition. The tps1/tps1 mutant followed the same pattern of hyphae formation, despite the fact that it was unable to accumulate trehalose either during dimorphism induction or after several stress challenges. Furthermore, trehalose-6-phosphate synthase activity was barely detectable in the mutant. These results strongly suggest that serum-induced dimorphic transition does not require trehalose mobilization; they also support the idea that TPS1 is the only activity involved in trehalose biosynthesis in C. albicans.     

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

In vitro effect of fibrinogen on Candida albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.     

Antigenic differences in the surface mannoproteins of Candida albicans as revealed by monoclonal antibodies.

Monoclonal antibodies to Candida albicans were prepared with blastoconidia bearing germ tubes used as the immunogen. Four antibodies reacted by immunofluorescence with surfaces of C. albicans as well as Candida stellatoidea, Candida tropicalis, and several strains of C. albicans, but not with Torulopsis glabrata. One antibody reacted with Saccharomyces cerevisiae. In addition, the monoclonal antibodies precipitated material of approximately 200 kilodaltons when tested against metabolically labeled blastoconidia digests. The monoclonal antibodies exhibited heterogeneous staining of C. albicans surfaces, as shown by immunofluorescence. None of the monoclonal antibodies were specific to germ tubes. More importantly, however, two of the monoclonal antibodies reacted with the mannoprotein precipitin arc of C. albicans that was produced by reference rabbit polyclonal antisera by crossed immunoelectrophoresis, thus linking the heterogeneity seen by immunofluorescence to the heterogeneity in mannoproteins. Finally, three of the monoclonal antibodies reacted with a glycan fraction of cell digests, indicating their reactivity with the carbohydrate portion of the mannoprotein.     

The Role of BCG/PPD-Activated Macrophages in Resistance against Systemic Candidiasis in Mice

The main conclusions of this study are that BCG/PPD-activated macrophages, in contrast to macrophages from control mice, exhibit an increased PMA-induced production of H2O2, kill about one-third of the phagocytosed Candida albicans, and cause more than 50% inhibition of the intracellular formation of germ tubes by C. albicans. Peritoneal macrophages from mice that were colonized post-natally with C. albicans do not show increased production of H2O2 upon stimulation with PMA and the intracellular outgrowth of germ tubes is inhibited to only a limited degree. These macrophages are capable of killing about 20% of the ingested C. albicans. In vivo, the number of Candida in the kidney, spleen and liver after intravenous injection of Candida albicans is significantly lower in BCG-treated mice than in control mice. Post-natal colonization with C. albicans has only a limited effect on the outgrowth of intravenously injected C. albicans in the spleen and liver but does not influence growth in the kidney. These results indicate that acquired immunity against a systemic Candida infection involves both oxidative and non-oxidative mechanisms of intracellular killing and that these mechanisms may have different effects on the yeast and hyphal forms of C. albicans.     

Electric and chemical fusions for the production of monoclonal antibodies reacting with the in-vivo growth phase of Candida albicans

To obtain monoclonal antibodies (MAbs) directed preferentially against the pathogenic phase of Candida albicans, mice were immunised with germ tubes of C. albicans serotype A, strain VW.32, killed by exposure to ultraviolet (UV) irradiation. Fusions were performed either by the standard chemical procedure with polyethylene glycol, or by electric discharge following linkage of the myeloma and lymphocyte cells with a Concanavalin A-mannoprotein bridge. The preliminary characteristics of one MAb obtained from each of these fusions are described. An IgM antibody (3B7) obtained from the chemical fusion reacted with a polysaccharide antigen that was heterogeneously distributed on both in-vitro and in-vivo forms of C. albicans. This MAb agglutinated different strains of C. albicans irrespective of their serotype. An IgG1 antibody (3G6) that had been obtained from the electric fusion was found to react in vitro with a proteinaceous antigen located only on the germ tubes of strain VW.32. However, MAb 3G6 displayed strong reactivity against all growth forms of C. albicans in vivo and reactivity extended to other strains.     

Comparison of cream of rice agar and horse serum for differentiating germ tubes of Candida albicans from filaments of Candida tropicalis.

Simple cream of rice agar was superior to horse serum for the demonstration of germ tubes by Candida albicans and in the differentiation of pseudohyphae of Candida tropicalis from germ tubes at 37 degrees C. Mycelium and chlamydospores were also produced on this medium.     

Immunodetection of CD45 epitopes on the surface of Candida albicans cells in culture and infected human tissues

Candida albicans is a leading cause of disseminated fungal infection in immunocompromised patients. Candida-host cell interactions are mediated at the cell surface. Since blood-group I epitopes have been detected on the surface of C albicans cells, we investigated whether CD45, the molecule that carries the I antigen on human lymphocytes, is present on the C albicans cell surface, in culture and in human tissue specimens of human candidiasis. By using monoclonal antibodies to CD45, CD45RO, and CD45RA, we found a strong immunoreactivity at the cell surface of blastoconidia bearing germ tubes but weak or no immunostaining of the germ tubes themselves. In human tissues, immunostaining of C albicans yeast cells was detected, whereas pseudohyphae were mostly negative. CD45 epitopes on the surface of C albicans might have a role in tissue invasion and dissemination of the fungus. On the other hand, its detection may disturb quantitative non-morphology-based determinations of lymphoid cell populations in infected tissues.     

Hormonal factors in vaginal candidiasis in rats.

The hormonal status of rats affected vaginal infection with Candida albicans. Four hours after infection viable counts were higher and germ tubes were longer in those animals in estrous than in other animals. However, the infection was not maintained with the change in epithelial cell type which occurred as part of the estrous cycle. Estrogen dosing following ovariectomy predisposed toward infection, while progesterone dosing did not. In rats injected with progesterone, germ tube clumping was seen, leukocytes were present, and elimination occurred before hyphal growth was evident. In rats injected with estrogen, however, infection was maintained, with hyphal growth extending throughout the cornified epithelial layer. Vaginal washings from rats dosed with estrogen promoted elongation of germ tubes in vitro to a greater extent than washings from other rats. Preincubation of blastospores in progesterone and subsequent infection of rats in pseudoestrous promoted clumping of germ tubes in the vagina. Strains of C. albicans varied in their virulence, which correlated with their ability to produce germ tubes in vitro. Loss of virulence occurred on subculture of a clinical isolate.     

Assessment of Germ Tube Dispersion Activity of Serum from Experimental Candidiasis: a New Procedure for Serodiagnosis

Germ tubes of Candida albicans and C. stellatoidea clump in normal serum but disperse in serum from animals infected with either species of Candida. A new procedure for the assessment of grades of germ tube dispersion activity of serum is presented; this procedure is to count the number of freely dispersed germ tubes in test serum into which a definite number of yeast-type C. albicans has been inoculated. The relationship between the serum activity and macroscopic lesions caused by candidal infection is observed, indicating the possibility of applying the phenomenon to the serodiagnosis of deep-seated candidiasis. The specificity and sensitivity of the test are also examined.     

Evidence for expression of the C3d receptor of Candida albicans in vitro and in vivo obtained by immunofluorescence and immunoelectron microscopy.

The complement conversion product C3d binds to a receptor on the cell surface of Candida albicans. While the function of this receptor is still uncertain, we investigated whether it is expressed during a murine infection. Rabbit antiserum raised against purified receptor was used in conjunction with immunofluorescence microscopy and immunocolloidal gold electron microscopy to examine kidney tissue and peritoneal lavages from infected mice for receptor expression by C. albicans in vivo. Specificity of the antiserum was indicated by reactivity with purified receptor (55 to 60 kDa) and with a protein of similar molecular mass from whole hyphal extracts in Western blots (immunoblots). In vitro analysis by immunofluorescence microscopy showed that the antiserum reacted with both yeast and pseudohyphal forms of the organism, but reactivity was strongest with pseudohyphae. Immunocolloidal gold electron microscopy of fungal cells from peritoneal lavages revealed intense staining of mother cells of germinative forms, germ tubes, and pseudohyphae. Staining of the mother cells was heaviest at the innermost layers of the cell wall but only scant on the cell surface. In contrast, staining was observed throughout the cell walls of germ tubes and pseudohyphae. In kidney, expression of the C3d receptor was found primarily on the cell walls of hyphae and pseudohyphae, although some staining was observed in the cytoplasm. These data support that the C3d receptor of C. albicans is expressed in vivo.     

Cell extracts of Candida albicans block adherence of the organisms to endothelial cells.

Cell extracts of Candida albicans were fractionated by concanavalin A affinity chromatography. Eluted mannosylated proteins (fraction II) and nonbinding, nonmannosylated proteins (fraction I) were collected and assayed directly for inhibition of adherence of C. albicans to endothelium. Fraction II blocked blastospore adherence to endothelial cells. Fraction I blocked both blastospore and germ tube adherence to endothelial cells. Monoclonal antibody OKM-1 (anti-CR3) and an anti-C. albicans monoclonal antibody, CA-A (anti-CR2), reacted in Western blots with proteins from fraction I, suggesting the presence of the CR2- and CR3-like proteins that have been previously identified on C. albicans germ tubes.     

Adherence of Candida albicans germ tubes to plastic: ultrastructural and molecular studies of fibrillar adhesins.

Germ tubes of Candida albicans produced an additional fibrillar surface layer responsible for enhanced adherence to plastic. The correlation between germination of C. albicans and adherence of germ tubes to a plastic matrix led us to consider the existence of germ tube-specific adhesive components involved in the attachment process. Using concanavalin A-sensitized latex microspheres, we first detected extracellular molecules on the plastic surface after removal of the adherent germ tubes. Electron microscopy confirmed that fibrils of the germ tube involved in cell-substratum interconnections were retained on the plastic surface. Cytochemistry with concanavalin A-gold labeling demonstrated that these fibrillar structures contained mannoproteins. Dithiothreitol and iodoacetamide treatment of washed plastic allowed us to further characterize these fibrillar adhesins. Through analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two components with molecular weights (MWs) of 68,000 and 60,000 were detected on the plastic surface. The 68,000-MW component appeared to be one of the major constituents of the germ tube surface layers. Biosynthetic labeling experiments performed with L-[35S]methionine revealed two additional proteins: a high-MW component (greater than 200,000), and a 200,000-MW component. These four proteins, strongly labeled on the plastic surface and on the germ tube cell wall layers, were in contrast slightly labeled or even nonidentified in the culture supernatant, suggesting their involvement in germ tube adherence.     

Enzymatic release of germ tube-specific antigens from cell walls of Candida albicans.

The differences between blastospores and germ tubes of Candida albicans, as previously shown by immunofluorescence, were further studied by comparing digests of cell walls of both growth forms. Organisms were surface labeled with 125I, and cell walls were digested enzymatically. When Zymolase digests were treated with polyclonal, polyspecific antiserum to C. albicans 441B, which stains only germ tubes in immunofluorescence assays, components of molecular weights 200,000 and 155,000 were immunoprecipitated from digests of germ tubes of strain B311, but nothing was recovered from blastospores. Whereas the 200,000-molecular-weight component was found in the three strains tested, the 155,000-molecular-weight component was found only in strain B311. When Zymolase digests were treated with unadsorbed antiserum, which stains both blastospores and germ tubes in immunofluorescence assays, an additional component was precipitated from digests of both growth forms with a molecular weight greater than the 200,000-molecular-weight marker. All three antigens were mannoproteins, as was shown by their abilities to bind concanavalin A and to be labeled by 125I. Also, all antigens were located on the cell surface, as was shown by the following criteria: adsorption of antisera with live organisms removed antibody to these components, and antibody eluted from surfaces of whole organisms precipitated all components. Components common to both growth forms, as well as germ tube-specific components, were detected in trypsin and chymotrypsin digests, but their molecular weights differed from those of Zymolase digests. Thus, germ tube-specific surface determinants as well as determinants common to both growth forms were detected on enzymatically released cell wall components.     

Antigenic differences between mannoproteins of germ tubes and blastospores of Candida albicans.

To determine the nature of germ tube-specific antigens of Candida albicans, procedures for intrinsically labeling cell wall antigens metabolically were developed. Blastospores or germ tubes labeled either in their proteins with L-[35S]methionine or in mannose-containing carbohydrates with D[2-3H]mannose contained surface components similar to those found previously with 125I-labeled organisms. Germ tube-specific determinants, were found on a 200-kilodalton protein in digests from germ tubes, whereas a component of similar molecular size in blastospore extracts reacted weakly or not at all with germ tube-specific antibody. In addition, a glycan fraction prepared from germ tubes reacted with the unadsorbed anti-C. albicans polyvalent antibody but not with the germ tube-specific antibody, suggesting that the germ tube-specific determinants are not carbohydrates.     

Germination of Candida albicans induced by proline.

Blastospores of Candida albicans germinated in proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glatamate, asparate, and pyruvate families also fostered germinaton but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of L-proline inhibited proline-stimualted germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts.     

Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans.

The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined. Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration [MIC]) to 0.004% (1/128 MIC). GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls. At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01). A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5%. Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum. Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01). GTF by C. albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO. This may have therapeutic implications.