Role of cell surface hydrophobicity in Candida albicans biofilm

Overall cell surface hydrophobicity (CSH) is predicted to play an important role during biofilm formation in Candida albicans but is the result of many expressed proteins. This study compares the CSH status and CSH1 gene expression in C. albicans planktonic cells, sessile biofilm, and dispersal cells. Greater percentages of hydrophobic cells were found in non-adhered (1.5 h) and dispersal forms (24 or 48 h) (41.34±4.17% and 39.52±7.45%, respectively), compared with overnight planktonic cultures (21.69±3.60%). Results from quantitative real-time PCR confirmed greater up-regulation of the CSH1 gene in sessile biofilm compared with both planktonic culture and dispersal cells. Up-regulation was also greater in dispersal cells compared with planktonic culture. The markedly increased CSH found both in C. albicans biofilm, and in cells released during biofilm formation could provide an advantage to dispersing cells building new biofilm.     

Experimental methodology to quantify Candida albicans cell surface hydrophobicity

A new method of formation of yeast cell lawns for contact angle measurement (with water, formamide and 1-bromonaphthalene) is described. The cell lawns were formed on agar layers avoiding liquid penetration. The method was validated by comparing the hydrophobicity of Candida albicans grown at different temperatures and the hydrophobicity of bacterial cell lawns built on agar layers and obtained by the usual filtration method.     

Variation of cell surface hydrophobicity and biofilm formation among genotypes of Candida albicans and Candida dubliniensis under antifungal treatment

Candida infections are frequently associated with formation of biofilms on artificial medical devices. This work studied variation of cell surface hydrophobicity (CSH) and formation of biofilm in relation to Candida albicans and Candida dubliniensis genotypes and an effect of some conventional antifungal agents on both CSH and biofilm. The 50 isolates of C. albicans and C. dubliniensis were classified into genotypes A, B, C, and D, genotype D being exclusively represented by C. dubliniensis. No significant differences between CSH of genotypes A and B and B and C were observed with respect to cultivation temperature 25 or 37 degrees C. Candida dubliniensis showed increased CSH in comparison with other C. albicans genotypes (p < 0.001) regardless of temperature used. Using XTT reduction assay and dry masses, genotypes B and C showed reduced ability to form biofilm in comparison with genotype A (p < 0.05) and C. dubliniensis (p < 0.001). Fluconazole reduced biofilm in C. albicans genotypes A, B, and C (p < 0.05) but not CSH. The opposite effect was observed in C. dubliniensis. Voriconazole effectively reduced both biofilm formation and CSH in all tested genotypes of C. albicans and C. dubliniensis (p < 0.05).     

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.     

Differential phagocytosis of white versus opaque Candida albicans by Drosophila and mouse phagocytes

The human fungal pathogen Candida albicans resides asymptomatically in the gut of most healthy people but causes serious invasive diseases in immunocompromised patients. Many C. albicans strains have the ability to stochastically switch between distinct white and opaque cell types, but it is not known with certainty what role this switching plays in the physiology of the organism. Here, we report a previously undescribed difference between white and opaque cells, namely their interaction with host phagocytic cells. We show that both Drosophila hemocyte-derived S2 cells and mouse macrophage-derived RAW264.7 cells preferentially phagocytose white cells over opaque cells. This difference is seen both in the overall percentage of cultured cells that phagocytose white versus opaque C. albicans and in the average number of C. albicans taken up by each phagocytic cell. We conclude that susceptibility to phagocytosis by cells of the innate immune system is an important distinction between white and opaque C. albicans, and propose that one role of switching from the prevalent white form into the rarer opaque form may be to allow C. albicans to escape phagocytosis.     

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.     

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.     

A polystyrene microsphere assay for detecting surface hydrophobicity variations within Candida albicans populations

Adherence of microbial pathogens to host cell surfaces may involve hydrophobic interactions. Here, we describe the development of an assay for detecting cell surface hydrophobicity of populations and individual cells of the opportunistic fungal pathogen Candida albicans. The assay involves mixing polystyrene latex microspheres with cells and subsequent enumeration of cell-attached microspheres. Similar levels of hydrophobicity within a population of yeast cells were obtained with the microsphere assay and with a commonly used aqueous-hydrocarbon biphasic partitioning assay. Various buffers were found to support detection of surface hydrophobicity with the microsphere assay. Complex fungal growth media did not. Serum in test media prevented microsphere attachment. A unique advantage of the assay compared to others is that individual cells can be assessed for surface hydrophobicity. Within a population of C. albicans yeast cells, strongly, moderately and weakly hydrophobic cells were observed. Within some pairs of mother-daughter cells, only one cell was hydrophobic. Germ tbes and hyphae were hydrophobic regardless of the hydrophobic status of the parent cell. These results indicate that the microsphere assay is a useful test evaluating cell surface hydrophobicity of C. albicans.     

The impact of polyene,azole, and DNA analogue antimycotics on the cell surface hydrophobicity of Candida albicans and Candida tropicalis in HIV infection

Oral candidiasis is the most common opportunistic infection in individuals infected with the human immunodeficiency virus. Though Candida albicans is the major aetiological agent, non-albicans species such Candida tropicalis are now emerging as important agents of such infection. The Candida cell surface hydrophobicity (CSH) is considered a critical factor contributing to its colonization potential and virulence. It is also known that brief exposure to sub-cidal concentrations of antifungal agents is a likely scenario in the oral environment where the administered drugs are diluted continuously due to the flushing action of saliva. Hence the objective of the present study was to compare the CSH of 10 isolates each of C. albicans and C. tropicalis from HIV-infected individuals following brief exposure (1hour) of isolates to sub-therapeutic concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine. The CSH was assessed by a previously described biphasic aqueous-hydrocarbon assay. The mean percentage reduction of CSH of C. albicans following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was27.33 (p < 0.001), 21.34 (p < 0.05), 11.74 (p > 0.05), 18.4 (p > 0.05) and 14.64 (p > 0.05) respectively. The mean percentage reduction of CSH of C. tropicalis following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was 33.81 (p < 0.01), 28.88 (p < 0.01), 12.6 (p > 0.05), 21.53(p > 0.05) and 17.68 (p > 0.05) respectively. A significant inter-species variation in CSH was observed for nystatin and amphoterecin B. Overall the results reveal that the CSH of C. albicans is affected to a significantly lesser degree compared with C. tropicalis when exposed to the antifungals. These data further illustrate another mode of action of antifungals on Candida leading to a reduction in the CSH and thereby the yeast adherence to host tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Generating cell surface diversity in Candida albicans and other fungal pathogens

The fungal cell surface contributes to pathogenesis by mediating interactions with host cells and eliciting host immune responses. This review focuses on the cell wall proteome of the major fungal pathogen Candida albicans and discusses how diversity at the cell surface can be introduced by altering the expression and structure of cell wall proteins. Remodelling the cell wall architecture is critical to maintain cellular integrity in response to different environments and stresses including challenge with antifungal drugs. In addition, the dynamic nature of the cell surface alters the physical properties of the fungal interface with host cells and thereby influences adhesion to the host and recognition by components of the host's immune system. Examples of the role of cell surface diversity in the pathogenesis of a number of microorganisms are described.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

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.     

Cell surface hydrophobicity of Candida albicans isolated from elder patients undergoing denture‐related candidosis

Background: The virulence potential of Candida albicans strains enrolled in denture‐related candidosis still remains uncertain. Candida albicans cells with higher cell surface hydrophobicity (CSH) rates, so‐called hydrophobic, present higher adhesion success in different host tissues than cells with lower rates, or even hydrophilic. Objective: The proposition of this study was to evaluate the differences in the CSH of strains isolated from denture users with and without denture‐related candidosis. Material and methods: The strains were obtained from two paired groups of patients living a same retirement house. Fungal cells were submitted to CSH evaluation by the hydrocarbon partition test using xylene. Results: The measures revealed that the yeasts from patients with candidosis had CSH values ranging from 4.52% to 12.24%, with an average of 8.22 ± 2.92%. In the countergroup, the CSH ranged from 3.86% to 14.36%, with an average of 8.38 ± 3.76%. The difference between the groups were considered not relevant (p = 0.997). Conclusion: The results let to the inference that natural populations of C. albicans from patients with and without clinical manifestation denture‐related candidosis do not differ one from the other regarding to CSH.     

Contribution of cell surface hydrophobicity protein 1 (Csh1p) to virulence of hydrophobic Candida albicans serotype A cells

The CSH1 gene product is the first protein implicated to affect the phenotype of cell surface hydrophobicity in Candida albicans. Ablation of expression of CSH1 resulted in a 75% loss of the cell surface hydrophobicity (CSH) phenotype. When the C. albicans csh1 knockout derivative was cultured from frozen stocks, it had reacquired CSH levels similar to the parent strain and isogenic reintegrant in the absence of Csh1p re-expression through an unknown mechanism. Prior to reacquisition of CSH, the knockout was less adherent to fibronectin than the parent. Comparison of the csh1 knockout and CSH1 reintegrant in a hematogenous dissemination model allows analysis of Csh1p contribution to virulence using matched strains with similar levels of CSH. No statistical significance between the knockout and reintegrant was found in virulence based on median day of survival, although a reproducible delay in onset of lethal infection for the knockout was observed. A modest difference in mucosal colonization in a vaginal infection model was also observed between the knockout and reintegrant. The present study demonstrates that Csh1p contributes to virulence of C. albicans in mice, but other gene products also contribute to the CSH phenotype and virulence.