Effect of Candida albicans cell wall components on the adhesion of the fungus to human and murine vaginal mucosa

In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance.Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions.Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory.     

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.     

DNA homology between Candida albicans strains: Evidence to justify the synonymous status of C. stellatoidea

Genetic relatedness between strains of C. albicans and C. stellatoidea was studied by measuring G + C content and overall sequence homology. G + C contents determined by high-performance liquid chromatography were 32.6 to 34.2% for 26 strains of C. albicans and 33.0 to 33.9% for eight strains of C. stellatoidea. DNA-DNA hybridization with two C. albicans and two C. stellatoidea probes revealed that all 34 test strains formed a single cluster in which the extents of hybridization with the heterologous probes ranged between 77.9 and 105.6% of those with the homologous probes. These results give support to the unification of C. albicans and C. stellatoidea into a single species.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

Non-glucan Attached Proteins of Candida albicans Biofilm Formed on Various Surfaces

Non-glucan attached proteins of the cell surface and extracellular matrix of Candida albicans biofilms formed on two catheter surfaces and denture acrylic were examined. The SDS-PAGE protein profiles of these proteins compared with that obtained from planktonic yeast cells and germ tubes were generally similar. This observation suggested that this class of biofilm surface proteins is not composed of a unique set of extracellular proteins or that one or a few proteins dominate the non-glucan attached proteins of biofilm. However, differences were observed in the proteins obtained from biofilm formed on one catheter surface and two proteins, Grp2p and ORF19.822p, identified by mass spectrometry following two-dimensional separation. These proteins have previously been associated with drug resistance and their presence or abundance appeared to be influenced by the surface on which the biofilm was formed.     

Recombinagenicity of caffeine for Candida albicans

Caffeine at concentrations of 0.5 × 10^–2 M or higher inhibited cell replication and induced gene segregations in Candida albicans cultured on defined complete medium. Both responses increased incrementally with increasing caffeine concentrations, and were more severe during incubation at 37 °C than 25 °C; at 37 °C, caffeine levels above 1.5 × 10^–2 M caused cellular inactivation. Caffeine effects occurred only under conditions permitting cell growth, and their magnitudes were greater for unbudded than budding cells, were influenced by cellular genetic backgrounds, and were unaffected by the presence of adenine in the medium. Evaluations of segregations for recessive auxotrophic markers of a four member linkage group carried heterozygously in a cis arrangement in treated cells established that induced segregants arise through either reciprocal or nonreciprocal recombinations. The frequency distributions of classes of reciprocal and nonreciprocal recombinants for these markers conformed with those previously obtained following induction by ultraviolet radiation, indicating that the probabilities of recombinational events within the chromosomal regions defined by the markers are not biased by the differences in kinds of initial DNA lesions caused by the two recombinagens. A panel of four protoplast fusion hybrids considered deficient for DNA repair because of enhanced susceptibilities to UV induced cellular inactivation and mitotic recombination exhibited corresponding increased sensitivities to caffeine, signifying that DNA damage induced by caffeine is subject to repair. Caffeine did not affect behavior of a variant strain exhibiting high frequency phenotypic switching between minute smooth and large rough colonial forms, and no evidence for mutagenicity of the drug was obtained with systems for detection of forward or reverse mutations. The mechanism of caffeine's recombinagenicity, and the implications of that property for genetic studies of C. albicans are discussed.     

Immune responses elicited by vaccinations with Candida albicans ribosomes in cyclophosphamide treated animals

This study describes humoral and cell mediated immune (CMI) responses detected in cyclophosphamide (CY) treated animals who were vaccinated with Candida albicans ribosomes and were protected against systemic candidiasis (previous study).Mice treated with CY and vaccinated with C. albicans ribosomes revealed CMI responses towards the ribosomes as measured in vivo by the foot pad swelling test and in vitro by the lymphocyte transformation assay. Both reactions were higher in CY treated and ribosome vaccinated mice than in controls (mice that were only vaccinated). Humoral immune responses were measured by the enzyme linked immunosorbent assay (ELISA). Anti ribosomal antibody titer contrary to the CMI responses was lower in CY treated animals than in non treated controls.These data point to a possible explanation of the mechanisms underlying the ribosomal vaccinations in CY treated hosts, and show the potential of such vaccinations in compromised individuals.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

The attachment to human buccal epithelial cells by Candida albicans: An in vitro kinetic study using concanavalin A

The early in vitro kinetics of Candida albicans attachment to human buccal epithelial cells was studied with the aid of an adhesion assay and solutions of concanavalin A (Con A), a lectin which is capable of inhibiting yeast adhesion. Various saccharides and putative receptor analogues were also tested. Solutions of each single reagent were added to tubes containing aliquots of mucosal cells and germinated yeasts at the beginning of a 1-hour incubation period (time O) or at 10 minute intervals during the assay. The number of yeasts attached to 200 mucosal cells was subsequently determined microscopically. Yeast adhesion remained constant following addition of phosphate-buffered saline (PBS) at time 0 or at any time thereafter. However, addition of Con A at 0, 10 or 20 minutes of incubation decreased adhesion significantly to 38%, 45% and 63% of control values. This inhibitory effect dwindled as time of incubation prior to lectin addition increased and Con A could not inhibit adhesion significantly after twenty minutes. Results obtained with Con A using live germinated yeasts were similar to those obtained with formalin-killed C. albicans. The other reagents tested failed to decrease adhesion significantly. These included the putative receptor analogues fibronectin, N-acetyl-d-glucosamine and d-galactose, and several non-specific saccharides such as -d-methylglucopyranoside, d-ribose and d-xylose. It is suggested that in vitro attachment to human mucosal cells by C. albicans is inhibitable up to a defined point in time by a lectin with affinity for mannosecontaining surface moieties, but becomes non-reversible thereafter. This experimentally-observed irreversibility is independent of yeast cell viability.     

Detection of Candida albicans in disseminated candidosis by immunofluorescence staining

An indirect immunofluorescence (IF) method using rabbit anti-Candida albicans was used to detect C. albicans in blood samples of 12 patients with systemic candidosis defined clinically, histologically and by blood cultures. Positive staining of C. albicans could be detected in all of the patients. The findings suggest that IF-method offers a more rapid method in the diagnosis of disseminated candidosis.     

Activity of North Jordan soil streptomycete isolates against Candida albicans

Eighteen percent of 116 different isolates of Streptomyces recovered from soils of northern Jordan showed activity against Candida albicans. The recovered isolates were distributed into three groups according to the diameter of the inhibition zone on the agar plate: group 1 (5–10 mm, slightly active); group 2 (11–15 mm, moderately active); and group 3 (16–35 mm, highly active). Isolates of group 3 were further grouped into four sub-groups and were culturally and morphologically identified. The u.v. spectra of the fermentation broth for the isolates in sub-group 4 were determined, and showed absorbance peaks ranging between 230 and 300 nm.     

Fungispecificity of fluconazole against Candida albicans

Candida albicans is an opportunistic pathogen of human mucosal surfaces. Colonization of oral and vaginal mucosa by this yeast is antagonized by the resident normal bacterial population. However, antibacterial therapy can alter the normal flora to allow fungal cells to attach, grow and invade host tissues. We studied the antimicrobic activity of fluconazole against clinical isolates of oral and vaginal bacteria and Candida albicans in vitro and in vivo by scanning and transmission electron microscopy; we also compared the bactericidal activity of fluconazole with clotrimazole in vitro by microbiologie assay. Fluconazole lysed fungi but did not change the ultrastructure of bacteria. Clotrimazole, but not fluconazole, was bactericidal against lactobacillus and streptococcus, the principal species of the oral and vaginal cavities. We conclude that Candida albicans, but not oral and vaginal bacteria, is susceptible to fluconazole. These observations help explain the antimycotic specificity of fluconazole and its efficacy against candidiasis in humans.     

Genotypic Differences of Candida albicans and C. dubliniensis Isolates Related to Ethnic/Racial Differences within the Same Geographic Area

Candida albicans and C. dubliniensis genotype differences among Israeli ethnic groups were assessed. Isolates from Jews (51), Arabs (35) and Druze (25) were genotyped. The distributions among ethnic groups were not different, however they differed (p = 0.002) from global populations. Therefore, C. albicans and C. dubliniensis genotype distribution differences in Israel are related to changes in all ethnic groups.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

Oxidative stress by antimicrobial peptide pleurocidin triggers apoptosis in Candida albicans

Pleurocidin (GWGSFFKKAAHVGKHVGKAALTHYL-NH₂), found in skin mucous secretions of the winter flounder Pleuronectes americanus, is known to possess a high potency and broad-spectrum antimicrobial peptide without cytotoxicity. In this study, to investigate the impact of pleurocidin on apoptotic progress, we observed morphological and physiological changes in Candida albicans. In cells exposed to pleurocidin, intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased, and hydroxyl radicals were especially a large part of ROS. The increase of ROS induced oxidative stress and mitochondrial membrane depolarization which causes release of pro-apoptotic factors. Using FITC-VAD-FMK staining, we confirmed activation of yeast metacaspases which lead to apoptosis and phosphatidylserine externalization at early stage apoptosis was observed using annexin V FITC. In addition, pleurocidin induced-apoptotic cells underwent apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC) in flow cytometry dot plots. Under the influence of oxidative stress, DNA and nuclei were fragmented and condensed in cells, and they were visualized by 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These apoptotic phenomena represent that oxidative stress by inducing pleurocidin must be an important factor of the apoptotic process in C. albicans.