Molecular sexing in the Mediterranean fruit fly, Ceratitis capitata.

Molecular methods have been devised for sexing Mediterranean fruit fly (medfly) individuals using minimal amounts of material from any stage of the life cycle. Molecular sexing methods are particularly valuable when material is obtained from pre-adult stages and sex identification based on morphological characters is not possible. These methods may also be useful for adult stage material in situations where only limited amounts or poorly preserved specimens are available. The sexing methods described here use the polymerase chain reaction (PCR) to amplify sequences known to originate from the sex chromosomes of this species. One method co-amplifies homologous regions of the ITS1 ribosomal DNA from both the X and Y chromosomes. Males and females are distinguished based on the restriction fragment pattern produced after digestion of the PCR products with the restriction enzyme ApoI. A second method identifies males based on the positive amplification of a repetitive DNA sequence originating from the Y chromosome. Both methods are shown to be capable of establishing the sex identity of individuals using only minimal amounts of material from any stage of the life cycle.

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.

Effect of iron concentration on siderophore synthesis and pigment production by Candida albicans.

Twelve strains of Candida albicans were grown in defined medium which had been deferrated by ion-exchange chromatography and then supplemented with FeCl3 to give iron concentrations ranging from 0.026 microM (growth-limiting) to 0.8 microM (excess). All of the strains secreted hydroxamate-type siderophores; phenolate siderophores were not detected. Isolates of C. lusitaniae, C. glabrata and C. parapsilosis also secreted hydroxamate but not phenolate-type iron chelators. Siderophore synthesis by C. albicans was maximal during growth in 0.026-0.2 microM iron. These low concentrations of iron also induced the synthesis of a green pigment, with maximal production at 0.026 microM. The pigment could be partially separated from hydroxamate siderophore activity on a column of Sephadex G-10 indicating that it probably does not function as an iron chelator.

Production of extracellular matrix by Candida albicans biofilms.

Growth of Candida albicans biofilms and production of extracellular matrix were monitored by dry weight, colorimetric and radioisotope assays, and by scanning electron microscopy. Under static incubation conditions synthesis of matrix material was minimal, but increased dramatically when developing biofilms were subjected to a liquid flow with the result that the cells were enveloped in extracellular polymer. These findings suggest that production of matrix material could contribute to the resistance of biofilm cells to antifungal agents in vivo.

Fucose-specific adhesins on germ tubes of Candida albicans.

Lectin-like adhesins of hyphal-form Candida albicans were investigated by conventional fluorescence microscopy, fluorescence microscopy with image analysis, spectrofluorimetry and flow cytometry. Labelling was done with neoglycoprotein probes consisting of sugars (fucose, mannose, glucose, galactose, lactose) covalently linked to bovine serum albumin (BSA), which itself was labelled with fluorescein. The fucose probe bound to both the yeast and germ-tube portions of hyphal-form cells, not especially at the tip, but in the adjacent region of the germ-tube portion. Probes with the other sugars did not label the hyphal-form cells. Fucose-probe binding to the cells was optimal at pH 5.0 in citrate buffer, and was a time-dependent reaction requiring 30-60 min and reaching saturation concentration at 100 microg ml(-1). Each hyphal-form cell of C. albicans grown in 199 medium was calculated to have about 2 x 10(7) fucose probe-binding sites. There appeared to be no requirement for Ca2+ or Mg2+ in binding. Binding of the fucose probe to the hyphal-form cells was higher at 37 degrees C than at 22 degrees C or 4 degrees C. Fluorescence intensity of the fucose-labelled yeast forms was not increased over the hyphal-form cells. A germ-tube-deficient mutant when exposed to hyphal-form growth conditions for 2 h showed much less binding of the fucose probe than the wild-type which produced germ tubes. Confirmation of specificity and the need for a carrier molecule was obtained by showing that Fuc-BSA (without fluorescein) effectively inhibited the binding of the fucose probe, although L-fucose itself was inactive, as was Gal-BSA.

Antifungal resistance of candidal biofilms formed on denture acrylic in vitro.

Denture biofilms represent a protective reservoir for oral microbes. The study of the biology of Candida in these biofilms requires a reliable model. A reproducible model of C. albicans denture biofilm was developed and used to determine the susceptibility of two clinically relevant C. albicans isolates against 4 antifungals. C. albicans, growing as a biofilm, exhibited resistance to amphotericin B, nystatin, chlorhexidine, and fluconazole, with 50% reduction in metabolic activity (50% RMA) at concentrations of 8, 16, 128, and > 64 microg/mL, respectively. In contrast, planktonically cultured C. albicans were susceptible (50% RMA for the same antifungals was obtained at 0.25, 1.0, 4.0, and 0.5 microg/mL, respectively). In conclusion, results obtained by means of our biofilm model show that biofilm-associated C. albicans cells, compared with cells grown in planktonic form, are resistant to antifungals used to treat denture stomatitis.

Adhesion of pathogenic Candida species to host surfaces.

Candida infections are becoming increasingly important in compromised hosts. The pathogenic Candida species vary in their ability to adhere to mucosal and other surfaces and there is a clear correlation between adhesion and virulence. Adhesion mechanisms of the most pathogenic species, Candida albicans, are being studied in detail.

Candida proteinases and candidosis.

Infections caused by the opportunistic yeast pathogen, Candida albicans, are becoming increasingly important. Superficial Candida infections, particularly those of the mouth and vagina, are very common; for example, candidal vaginitis plaques millions of women worldwide, often proving refractory to treatment. Systemic candidosis is much rarer, but it is an important hazard of modern medical procedures such as transplant surgery, i.v. hyperalimentation, and immunosuppressive therapy. One significant virulence factor of C. albicans is its ability to secrete extracellular acid proteinase. This attribute is shared by C. tropicalis and C. parapsilosis, but not by other less pathogenic Candida species. The enzymes produced by these yeasts are all carboxyl proteinases capable of degrading secretory IgA, the major immunoglobulin of mucous membranes. Some have keratino- or collagenolytic activity. Two secretory proteinases of C. albicans have been purified and characterized; their properties are reviewed. Possible applications of this work to the treatment and diagnosis of candidosis are discussed.

Adhesion of Candida species to epithelial surfaces.

Successful colonization and infection of host tissues by the pathogenic Candida species depend upon the ability of these organisms to adhere to mucosal surfaces. The different species vary in their ability to adhere, and there is a clear correlation between adhesion and virulence. The mechanism of adhesion of the most pathogenic species, Candida albicans, to epithelial cells has been studied in detail and is thought to involve lectin-like interactions between specific binding molecules (adhesins) on the yeast surface and complementary receptor molecules on the epithelial cell surface. Current information suggests that the protein portion of mannoprotein located in fibrils on the yeast surface serves as the adhesin and interacts with glycoside receptors on epithelial cells. The evidence for this proposed mechanism is critically discussed.

Interaction of Candida albicans with neutrophils: effect of phenotypic changes in yeast cell-surface composition.

The susceptibility of four strains of Candida albicans to phagocytosis and intracellular killing by rabbit peritoneal neutrophils was investigated. Two of the strains, isolated from active infections, were known to synthesize a surface layer of mannoprotein fibrils in response to growth on 500 mm-galactose; the other strains, from asymptomatic carriers, lacked this capability. The presence of serum opsonins greatly enhanced phagocytosis of all four strains and, following opsonization, phagocytosis of an infective strain was equally rapid after growth on either 500 mm-galactose or 50 mm-glucose. In the absence of opsonins, galactose-grown infective strains were phagocytosed faster than either glucose-grown infective strains or galactose-grown carrier strains. These differences in phagocytic uptake were paralleled by differences in neutrophil chemiluminescence response. Intracellular killing of galactose-grown infective strains was only half that of glucose-grown infective strains or galactose-grown carrier strains after incubation for 60 min. Pretreatment of neutrophils with extracellular polymeric material, which contains the surface fibrils, completely inhibited intracellular killing. These results indicate that production of the fibrillar layer promotes yeast virulence by increasing resistance to intracellular killing, although it may enhance phagocytosis in locations where opsonic activity is poor.

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.

Extracellular polymer of Candida albicans: isolation, analysis and role in adhesion.

Extracellular polymeric material (EP) was isolated from culture supernatants of Candida albicans grown on carbon sources (50 mM-glucose, 500 mM-sucrose or 500 mM-galactose) known to promote yeast adhesion to different extents. Galactose-grown yeasts, which are the most adherent, produced more EP than sucrose-grown organisms, particularly after incubation for 5 d, while glucose-grown yeasts (the least adherent) gave the lowest yield. EP produced on all three carbon sources was of similar composition and contained carbohydrate (65 to 82%; mannose with some glucose), protein (7%), phosphorus (0.5%) and glucosamine (1.5%). Serological studies indicated that these EP preparations were immunologically identical but that galactose-grown yeasts had more antigenic determinants than sucrose-grown organisms while glucose-grown yeasts had the fewest determinants. Antigenic differences were apparent between EP preparations of some strains of C. albicans. Pretreatment of acrylic strips with EP to form a polymeric coating promoted yeast adhesion to the acrylic surface, but similar pretreatment of buccal epithelial cells with EP inhibited subsequent yeast adhesion. These results indicate that EP originates from the cell surface of C. albicans and that it contains the surface component(s), probably mannoprotein in nature, responsible for yeast adhesion.

Characterization of a fucoside-binding adhesin of Candida albicans.

Candida albicans GDH 2346 produces extracellular polymeric material (EP) which contains a mannoprotein adhesin with a lectin-like affinity for fucose-containing glycosides. EP isolated from culture supernatants of this strain was used as starting material for purification of the adhesin. The purification protocol involved a stepwise treatment of EP with N-glycanase, papain, and dilute alkali to cleave the protein and carbohydrate portions of the mannoprotein molecule. Fucoside-binding protein fragments were then recovered by affinity adsorption with the trisaccharide determinant of the H (type 2) blood group antigen which terminates in a residue of L-fucose. The purified adhesin was devoid of carbohydrate and inhibited yeast adhesion to buccal epithelial cells 221 times more efficiently, on a protein weight basis, than did EP. Adhesion inhibition reached a maximum of 78 to 80% at an adhesin concentration of 10 micrograms ml-1. Our results indicate that this protein is the major adhesin of yeast-phase cells of C. albicans GDH 2346 but that one or more secondary adhesion mechanisms may operate.

Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents.

Extracellular polymeric material (EP), comprising the matrix of Candida albicans biofilms, was isolated and its composition was compared with that of EP obtained from culture supernatants of planktonically grown (suspended) organisms. Both preparations consisted of carbohydrate, protein, phosphorus and hexosamine, but biofilm EP contained significantly less total carbohydrate (41%) and protein (5%) than planktonic EP. It also had a higher proportion of glucose (16%) and contained galactose, suggesting that it might possess components unique to biofilms. To investigate whether the EP matrix plays a role in the resistance of biofilms to antifungal agents, susceptibility profiles of biofilms incubated statically (which have relatively little matrix) were compared with those for biofilms incubated with gentle shaking (which produce much more matrix material). Biofilms grown with or without shaking did not exhibit significant differences in susceptibility to any of the drugs tested, indicating that drug resistance is unrelated to the extent of matrix formation. However, biofilms formed on two different types of polyvinyl chloride catheter, obtained from different manufacturers, showed differences in susceptibility to amphotericin B, suggesting that drug resistance may arise as a result of highly specific, surface-induced gene expression.

Effect of iron deprivation on surface composition and virulence determinants of Candida albicans.

Six strains of Candida albicans were grown in defined medium which had been deferrated by ion-exchange chromatography and then supplemented with FeCl3 to give iron concentrations ranging from 0.026 microM to 0.8 microM. Growth in 0.026 microM-iron (measured as increase in biomass) was reduced by 26-59% as compared with that in excess (0.8 microM) iron. With five of the strains, adhesion to buccal epithelial cells was maximal after growth in 0.2-0.4 microM-iron, but strain GDH 2023 adhered best when grown in 0.026 microM-iron. Differences in yeast cell-wall composition were revealed by Zymolyase treatment of whole cells and by 125I-labelling of surface proteins. SDS-PAGE of iodinated proteins, followed by autoradiography, showed quantitative but no qualitative differences in protein profiles of iron-deficient and iron-replete organisms. The ability of all strains to form germ tubes in serum was near-maximal after growth in 0.2-0.4 microM-iron but was inhibited by up to 93% following growth in lower concentrations. These results indicate that expression of important virulence attributes by C. albicans is highly dependent on available iron and that expression in vivo may therefore be significantly different from that observed under conventional laboratory conditions.

Resistance of Candida albicans biofilms to antifungal agents in vitro.

Biofilms formed by Candida albicans on small discs of catheter material were resistant to the action of five clinically important antifungal agents as determined by [3H]leucine incorporation and tetrazolium reduction assays. Fluconazole showed the greatest activity, and amphotericin B showed the least activity against biofilm cells. These findings were confirmed by scanning electron microscopy of the biofilms.

Biofilm formation by Candida species on the surface of catheter materials in vitro.

A model system for studying Candida biofilms growing on the surface of small discs of catheter material is described. Biofilm formation was determined quantitatively by a colorimetric assay involving reduction of a tetrazolium salt or by [3H]leucine incorporation; both methods gave excellent correlation with biofilm dry weight (r = 0.997 and 0.945, respectively). Growth of Candida albicans biofilms in medium containing 500 mM galactose or 50 mM glucose reached a maximum after 48 h and then declined; however, the cell yield was lower in low-glucose medium. Comparison of biofilm formation by 15 different isolates of C. albicans failed to reveal any correlation with pathogenicity within this group, but there was some correlation with pathogenicity when different Candida species were tested. Isolates of C. parapsilosis (Glasgow), C. pseudotropicalis, and C. glabrata all gave significantly less biofilm growth (P < 0.001) than the more pathogenic C. albicans. Evaluation of various catheter materials showed that biofilm formation by C. albicans was slightly increased on latex or silicone elastomer (P < 0.05), compared with polyvinyl chloride, but substantially decreased on polyurethane or 100% silicone (P < 0.001). Scanning electron microscopy demonstrated that after 48 h, C. albicans biofilms consisted of a dense network of yeasts, germ tubes, pseudohyphae, and hyphae; extracellular polymeric material was visible on the surfaces of some of these morphological forms. Our model system is a simple and convenient method for studying Candida biofilms and could be used for testing the efficacy of antifungal agents against biofilm cells.

Effects of phosphate limitation of growth on the cell-wall and lipid composition of Saccharomyces cerevisiae.

The phosphorus content of phosphate-limited Saccharomyces cerevisiae was only 71% of that of non-limited yeast. Walls prepared from phosphate-limited cells contained slightly less phosphorus than control walls. No evidence was obtained for the presence in these walls of uronic acid or succinyl residues. The carbohydrate content of walls of phosphate-limited yeast was less than that of non-limited walls, and this was reflected in a decreased glucan content. There was only a slight decrease in glucosamine content while the protein content increased. The major change in the lipid composition of phosphate-limited yeast was a decrease in both sterol esters and triacylglycerols. There was a decrease in total lipid content, but increased production of phosphatidylethanolamine and phosphatidylcholine. The phosphatidylserine content was decreased. These results suggest that there are fewer intracellular low-density vesicles in phosphate-limited yeast.

Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents.

A perfused biofilm fermentor, which allows growth-rate control of adherent microbial populations, was used to assess whether the susceptibility of Candida albicans biofilms to antifungal agents is dependent on growth rate. Biofilms were generated under conditions of glucose limitation and were perfused with drugs at a high concentration (20 times the MIC). Amphotericin B produced a greater reduction in the number of daughter cells in biofilm eluates than ketoconazole, fluconazole, or flucytosine. Similar decreases in daughter cell counts were observed when biofilms growing at three different rates were perfused with amphotericin B. In a separate series of experiments, intact biofilms, resuspended biofilm cells, and newly formed daughter cells were removed from the fermentor and were exposed to a lower concentration of amphotericin B for 1 h. The susceptibility profiles over a range of growth rates were then compared with those obtained for planktonic cells grown at the same rates under glucose limitation in a chemostat. Intact biofilms were resistant to amphotericin B at all growth rates tested, whereas planktonic cells were resistant only at low growth rates (