Identification of Candida albicans genes that induce Saccharomyces cerevisiae cell adhesion and morphogenesis

Morphogenesis and adhesion to host tissues and medical devices contribute to the virulence of Candida albicans, the most common fungal pathogen isolated from humans. However, identification of molecular mechanisms of C. albicans adhesion and morphogenesis has been impaired by the lack of effective molecular and genetic tools available for this organism. Saccharomyces cerevisiae provides an attractive model system for studying C. albicans adhesion and morphogenesis because of its well-characterized genetics and gene expression systems. To gain insight into the genetic mechanisms of C. albicans adhesion and morphogenesis, we used a parallel plate flow chamber to screen and quantitatively characterize attachment to polystyrene of an adhesion-deficient nonfilamentous flo8Delta S. cerevisiae strain expressing a C. albicans genomic library. We identified six C. albicans genes that are capable of promoting cell adhesion and pseudohyphal development in S. cerevisiae. We also analyzed the ability of these adhesion-promoting genes to regulate the expression of FLO11, which encodes an endogenous S. cerevisiae adhesin. One C. albicans gene, EAP1, appears to directly mediate adhesion and morphogenesis while the remaining five (EAP2, SWI1, MSB1, AAF1, and TEC1) upregulate expression of endogenous S. cerevisiae adhesins. These results suggest that S. cerevisiae is a useful system for molecular characterization of factors that regulate C. albicans adhesion and morphogenesis and that parallel plate flow chamber-based adhesion assays can be used in conjunction with genetic screens to identify molecular mechanisms regulating fungal cell adhesion.     

Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium

Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive to O-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigation.     

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.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB₄ induced a modest stimulation but LTC₄, LTD₄, LTE₄ as well as LXA₄ and LXB₄ were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin αIIbβ₃ and monoclonal antibodies against this integrin but not against α₅β₁, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

Immunochemical characterization of Candida albicans cell wall antigens: specific determinant of Candida albicans serotype A mannan

We investigated the chemical structure of the specific determinant in the mannan of Candida albicans M-1012 (serotype A) strain. Acetolysis of the mannan, obtained by alkali extraction and purified as the copper complex, gave mannose and six oligosaccharides (from di- to hexasaccharide) and a small amount of a heptasaccharide. We examined the inhibition by these oligosaccharides up to hexaose of the precipitin reaction between anti-factor 6 serum specific for serotype A and homologous mannan, and found that the mannohexaose was the most effective inhibitor. These, and results obtained by proton magnetic resonance (PMR) spectroscopy, methylation analysis, and other structural studies, suggest that the main component of this hexaose consists of one terminal alpha (1-3) linkage in addition to four alpha (1-2) linkages, and that this alpha (1-3)-containing mannohexaose may be responsible for the specificity of antigenic factor 6. Further results obtained by analyses of polarimetry, PMR spectroscopy, and chromium trioxide oxidation-methylation of C. albicans M-1012 mannan has a beta-linkage in addition to alpha-linkages, and that the mode of the beta-linkage is mainly (1-6) linkage. Further evidence obtained by Smith degradation-methylation analysis and by quantitative precipitin reactions of intact and acid-degraded mannan suggests that the antigenic determinant of antigenic factor 6 may be bound, via the beta (1-6) linkage, to C-6 of mannose residues involved in oligosaccharide side chains of serotype A mannan.     

Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity

Candida albicans is one of the most important opportunistic pathogenic fungi. Weakening of the defense mechanisms of the host, and the ability of the microorganism to adapt to the environment prevailing in the host tissues, turn the fungus from a rather harmless saprophyte into an aggressive pathogen. The disease, candidiasis, ranges from light superficial infections to deep processes that endanger the life of the patient. In the establishment of the pathogenic process, the cell wall of C. albicans (as in other pathogenic fungi) plays an important role. It is the outer structure that protects the fungus from the host defense mechanisms and initiates the direct contact with the host cells by adhering to their surface. The wall also contains important antigens and other compounds that affect the homeostatic equilibrium of the host in favor of the parasite. In this review, we discuss our present knowledge of the structure of the cell wall of C. albicans, the synthesis of its different components, and the mechanisms involved in their organization to give rise to a coherent composite. Furthermore, special emphasis has been placed on two further aspects: how the composition and structure of C. albicans cell wall compare with those from other fungi, and establishing the role of some specific wall components in pathogenesis. From the data presented here, it becomes clear that the composition, structure and synthesis of the cell wall of C. albicans display both subtle and important differences with the wall of different saprophytic fungi, and that some of these differences are of utmost importance for its pathogenic behavior.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis.

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB4 induced a modest stimulation but LTC4, LTD4, LTE4 as well as LXA4 and LXB4 were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin alpha IIb beta 3 and monoclonal antibodies against this integrin but not against alpha 5 beta 1, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Cholesterol increases adhesion of monocytes to endothelium by moving adhesion molecules out of caveolae

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs). We examined whether caveolae are involved in monocyte adhesion to ECs responding to a synergy of hypercholesterolemia and inflammation. Treating human umbilical vein ECs with cholesterol enhanced endotoxin lipopolysaccharide (LPS)-induced monocyte adhesion. Use of isolated caveolae-enriched membranes revealed that cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), co-localized with Cav-1 in caveolae. LPS upregulated CAMs expression and increased the co-localization. Cholesterol exposure decreased the level of CAMs in the caveolae. Co-immunoprecipitation and confocal microscopy revealed that ICAM-1 interacted with Cav-1. Electron microscopy showed that ICAM-1 was mainly located in caveolae. Cholesterol exposure decreased this interaction and drove ICAM-1 out of caveolae. Knockdown of Cav-1 reduced the synergistic effects of cholesterol and inflammation. In vivo, ICAM-1 and Cav-1 co-localization was lower in the aortic endothelium of ApoE^−/− mice than in that of wild-type controls. Cav-1 negatively regulates monocyte adhesion by the co-localization of CAMs in caveolae, which is disturbed by cholesterol. Thus, our study suggests a molecular basis underlying the synergistic effects of hypercholesterolemia and inflammation in atherogenesis.     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

Molecular and genetic aspects of resistance to azoles in Candida albicans

Fluconazole is one of the most useful drugs in the treatment of fungal systemic infections which frequently affect non immunocompetent individuals. However, the emergence of resistant strains in recent years may severely limit its usefulness in future. Although there are several described mechanisms involved in resistance to azoles, recent genetic studies demonstrate the role of specific genes in clinical resistance. Currently, the best characterized are the MDR1 and CDR1 genes, which code members of the MFS or ABC family of drug transporters, respectively. These proteins respond to the membrane potential (MFS) or hydrolyse ATP (ABC) thus promoting drug efflux and therefore reducing its intracellular accumulation. It has been shown that the mRNA from these genes is frequently increased in some Candida albicans resistant strains from patients receiving long term azole treatment. The development of molecular genetic tools in C. albicans is allowing characterization of their role in this and other important processes in the fungal cell.     

The study of adhesion of Candida albicans to the selected acrylic resins

The study has been performed on 3 acrylic resins used to fabricate removable dentures. Aim of the study was to detect possible differences in Candida albicans' adhesion within particular materials. Polished and non-polished samples were made, than these samples were sunk in precipitates containing Candida albicans material. Adhesion of Candida albicans to the surface of the materials occured within a concentration of 10. After 24 hours of incubation differences were found concerning the number of the plate cultures. Most of all plate cultures were observed on Lucitone 199, fewer on Zhermacryl, the poorest one was found on Palaxpress resin. Considerable number of plate cultures occured on non-polished samples relating to polished ones. After 48 hours of incubation further development of Candida albicans took place, with differences concerning various materials.     

Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells

Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.