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.     

Changes in internal and external pH accompanying growth of Candida albicans: studies of non-dimorphic variants

Non-dimorphic variants of Candida albicans, which were unable to form germ tubes or mature hyphae in media containing amino acids, glucose and salts or N-acetylglucosamine or serum, were prepared from two hyphal positive laboratory strains using a physical separation method. The hyphal-minus phenotype was stable and may be due to mutations or phenotypic variation. The variant strains maintained their internal pH within narrower bounds as compared to their parental wild-types. When exposed to conditions that normally supported the induction of germ tubes the cytoplasmic pH of the wild type strains increased from 6.8 to over pH 8.0 within 5 min while in the variants the rise in internal pH was only about 0.3 pH units. The wild type strains acidified the growth medium more rapidly than the variants. The results suggest that the control of internal pH is directly or indirectly associated with the regulation of dimorphism. The variants had unaltered cell volumes and specific growth rates. The hyphal-minus phenotype was however fully reversible since revertants occurred spontaneously on serum containing agar.     

Characterization of a major cell wall antigen and potential adhesin in three strains of Candida albicans

Selected strains of Candida albicans were examined to reveal the surface antigenicity and biochemical nature of major cell wall proteins that also were shown to serve as cellular adhesins on human buccal epithelial cells. Confirmation of the adhesive properties of these cells was made by scanning electron microscopy and immunofluorescence microscopy. Particular attention was directed at the clinical isolate KM-302. By means of indirect immunofluorescence staining, the KM-302 blastoconidia absorbed rabbit anti-C. albicans ATCC-32354 serum, revealing specific localization of surface antigens on germ tubes and pseudohyphae. Extracellular polymeric material and the cell wall extract of C. albicans KM-302 blastoconidia were found to contain a major surface antigen of 49 kDa that exhibited 42% adhesion inhibition in vitro. Of considerable significance is that immunogold localization by electron microscopy showed the antigen to be almost exclusively cell wall bound. This major antigen, identified in affinity and gel filtration chromatography fractions, was composed of 4% carbohydrate and 95.7% protein and had an isoelectric point of 6.1. The major antigen also showed a high level of similarity with that of C. albicans strain SC-5314 inasmuch as the major antigen of that strain had carbohydrate and protein compositions of 4 and 95.5%, respectively. Both of these strains also possessed the same percent of adhesion inhibition of human buccal epithelial cells.Abbreviations BECs buccal epithelial cells - CWE cell wall extract - EPP extracellular polymers and proteins - FITC fluorescein isothiocyanate - mAg major antigen - OD ₆₀₀ optical density at 600 nm - PBS phosphate buffered saline - TEM transmission electron microscopy - YNB yeast nitrogen base     

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.     

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37°C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.     

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.     

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.     

An electron microscopy study of wall expansion during Candida albicans yeast and mycelial growth using concanavalin A-ferritin labelling of mannoproteins

Depending upon growth temperature, Candida albicans can exhibit two different morphologies, a budding yeast or a mycelium. By studying the distribution of concanavalin A-ferritin particles on the cell wall surface during bud and germ tube formation, we have elucidated the way cell wall extension occurs. Both processes initially require the localized lysis of the wall in order to allow the incorporation of the newly synthesized material. Later on, the cell wall behaves as an elastic structure, allowing extension by an intosusception process and, as a consequence, cell growth.Abbreviation Con A concanavalin A     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

The influence of post-filtration washing on the in vitro assay ofCandida albicans adherence to human buccal epithelial cells

A simple in vitro assay technique was used to determine the effect of post-filtration washing on the adherence ofC. albicans (NCPF 3736) to human buccal epithelial cells (BEC). Washing was carried out with a range of volumes of phosphate buffered saline (PBS), viz. 0, 5, 10 and 20 ml, at a standard flow rate. Both the number ofC. albicans adherent to BEC and the percentage of BEC with adherentC. albicans were significantly decreased (p<0.001 for each of these measures) after washing with 5 ml PBS. Further increases in the volume of PBS did not significantly decrease either measure of adherence. These data indicate that only a small volume of PBS, 5 ml, is required to achieve the removal of non-adherentC. albicans from the surface of BEC. The result of the adherence assay is not significantly affected by increasing the volume of PBS used. It is concluded that considerable savings in time may be made through using only a small (5 ml) volume of washing buffer at a standard flow rate.Abbreviations BEC buccal epithelial cells - PBS phosphate buffered saline - MEM Eagle's minimum essential medium - NCPF National collection of pathogenic fungi     

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.     

Antibody formation in experimental immunizations with Candida albicans ribosomal fractions

Sera of mice immunized with ribosomal fractions of Candida albicans showed the presence of anti-C. albicans antibodies, detected by the gel-immunodiffusion, agglutination and immune adherence tests.Candida infections are among the most prevalent opportunistic yeast infections, attacking debilitated individuals, and against which there is no effective prophylactic treatment currently available (1, 2, 3, 4, 5). In view of the succes reported in experimental immunizations with ribosomal fractions from various bacteria and some fungi, as summarized by Youmans and Tewari (7, 8), a similar approach for immunization in experimental candidiasis appears reasonable. The present work describes preliminary results on circulating antibodies elicited in the course of immunizations with ribosomal fractions of Candida albicans.Ribosomal preparations were obtained from mechanically disrupted cell-pellets of C. albicans by differential centrifugation and purification in a 15% sucrose and 5% ammonium sulfate solution (in sodium-magnesium-Tris buffer), using a modification of the procedure described by Rubin (6). Concentration of ribosomal-RNA was determined by the absorbance at 260 nm; ribosomal-protein concentration by the Lowry reaction; and purity of the ribosomal preparation checked by the ratio of absorbance at 260 nm to 280, and at 260 to 235 nm. Mice (ICR strain) were immunized with these ribosomal preparations in amounts of 50–100 g ribosomalprotein/mouse, by 2–3 subcutaneous inoculations with Frend's adjuvant, with a 10–21 day interval between the inoculations.This work constitutes part of Ruth Levy's research study towards the Ph.D. degree.     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

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.     

Selective inactivation of heterokaryons of Candida albicans by anaerobiosis

Heterokaryons (hets), but not monokaryons of Candida albicans die when grown anaerobically on minimal medium. Their rates of inactivation increase with decreases in growth temperatures from 37°C to 25°C. At 10°C, however, anaerobiosis is not lethal and suppresses the inactivation which normally occurs among hets cultured aerobically at that temperature. Killing of hets by anaerobiosis can be altered significantly by certain exogenously provided amino acids or intermediates of oxidative respiration. Aspartic acid alone promotes inactivation whereas alanine, glutamic acid or lysine individually have no effects. However, glutamate and lysine combined afford slight protection against inactivation while aspartate and glutamate combined, with or without lysine, are highly protective: the activity of the aspartate-glutamate combination is completely negated by the addition of alanine. Other common amino acids have no effects on het responses to anaerobiosis other than the ability, when combined, to relieve the antagonism of alanine for the aspartate-glutamate combination. Anaerobic survivals are also enhanced by oxalacetic acid or -ketoglutaric acid, and even more so by a combination of these two intermediates. The resistances to inactivation elicited by the oxalacetate -ketoglutarate or aspartate-glutamate combinations are not additive. These relationships are interpreted to signify that inactivation of hets by anaerobic growth is largely, if not exclusively, due to depletion of their oxalacetic acid and -ketoglutaric acid contents for amino acid biosyntheses, and the unique inability of het cells to replenish those keto acids upon subsequent return to aerobic conditions. The observations are consistent with previous indications that mitochondria formed by hets are functionally abnormal.     

Anaerobically induced production of hybrid monokaryons by heterokaryons of Candida albicans

Summary During aerobic replication, balanced heterokaryons (hets) of Candida albicans produced by fusing protoplasts of complementing auxotrophic strains characteristically segregate low frequencies of prototrophic monokaryons bearing hybrid nuclei formed either through karyogamy or unidirectional internuclear genetic transfers within het cells. Anaerobic growth causes exponential inactivation of hets and induces their production of hybrid monokaryons. Both responses are functions of heterokaryosis as such and not the genetic backgrounds of hets. Evidence is presented that (i) the nuclei of anaerobically generated hybrids arise through induction in hets of karyogamy not internuclear genetic transfers and that (ii) the events underlying that induction are different from those responsible for inactivation of the cells.     

Mannan and D-arabinitol concentrations in serum from a patient with Candida albicans endocarditis

In an attempt to clarify the comparative values of serological and microbiological examinations for the early diagnosis of systemic candidiasis, antibodies against Candida albicans, serum mannan, and the D-arabinitol creatinine ratio were investigated in a patient with aortic valve endocarditis associated with carcinoma of the bile duct. Candida precipitins and the antibody titer against Candida cell wall mannan were examined by an immunodiffusion technique and hemagglutination test, respectively. Serum mannan was tested by enzyme-linked immunosorbent assay (ELISA) using the biotin-streptavidin procedure. The upper limit of negativity of the assay was determined by adding 0.06 to the absorbance of pooled serum from healthy laboratory workers. This value ws about 0.8 ng/ml with ELISA. The D-arabinitol concentration in serum was examined by an enzymatic fluorometric method. Rising antibody titers against C. albicans, mannan antigenemia, and an elevated D-arabinitol creatinine ratio were first observed between the 11th and 12th hospital days. Blood cultures obtained on 8th, 9th, and 11th hospital days grew C. albicans after 3 to 4 days of incubation. Of 11 serum samples, 5 were positive for mannan, whereas D-arabinitol creatinine ratio was positive in 7 of 9 samples. Blood cultures was the earliest evidence of Candida infections in our cases. However, because of saprophytic nature of Candida species, tests for antibodies, antigenemia, and the D-arabinitol creatinine ratio in combination with blood cultures are necessary to confirm systemic candidiasis at an early stage of infection.Abbreviations ELISA enzyme-linked immunosorbent assay     

Complex interaction between different proteinaceous components within the cell-wall structure ofCandida albicans

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface ofCandida 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 theC. 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 immunofluo-rescence (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.     

A new minimal synthetic medium for germ-tube production in Candida albicans

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.