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Sixty isolates of Candida albicans, 30 obtained from the oral cavity of denture wearers presenting signs of candidosis and 30 obtained from the oral cavity of denture wearers with normal palatal mucosa were assayed for phospholipase and proteinase production, as well as for adherence to buccal epithelial cells. Likewise, susceptibility of the isolates to antifungals was determined by the NCCLS reference method and the E-test method. Proteinase activity was increased among the strains obtained from oral candidosis patients. In contrast, no significant differences between the two groups of isolates were observed in their adherence ability in vitro, in phospholipase production, and susceptibility to antifungal drugs.

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.

We previously reported that a respiration-competent parent strain (K) of Candida albicans was more susceptible to the intracellular superoxide radical (O2-) generator paraquat (PQ) than was a respiration-deficient mutant (KRD-19), although both showed a similar sensitivity to extracellularly generated O2-. To clarify the cause of the differential PQ lethality, we developed a chemiluminescence method for measuring O2- generated by C. albicans cells by using the probe methyl-Cypridina-luciferin analogue (MCLA), and examined the effects of PQ on O2- generation in both parent and mutant strains. Endogenous O2- generation without stimulation by PQ was unexpectedly low in both strains. PQ-induced O2- generation in the parent strain was maximal in logarithmic phase cells and lowered in stationary phase cells. In contrast, O2- generation in the mutant remained low throughout the growth phase, even when stimulated by PQ. The extent of PQ-induced O2- generation in the parent strain depended on the carbon source added to the assay mixture; in decreasing order, glucose, glycerol, no carbon source. The inhibitor of the cytochrome respiratory chain, antimycin A, suppressed almost completely the PQ-induced O2- generation in the parent strain. It has been established that PQ is converted to its radical form (PQ+) by receiving a single electron in cells. PQ+ then reduces molecular oxygen to O2- by redox cycling. Thus, the high tolerance to PQ of the respiration-deficient mutant can be explained by minimal PQ+/O2- production due to the limited supply of electrons from the impaired respiratory system.

Candida albicans virulence is in part mediated by fibronectin (FN) interaction. We compared the adherence level to FN (using Becton Dickinson FN-coated plates) of several strains of yeast isolated from HIV-1 infected or uninfected subjects affected by candidiasis (30 strains from HIV+ subjects and 18 from HIV- subjects). More adhesive strains were found in HIV+ patients than in HIV- subjects. In particular a mean increase of 120 per cent as regards the total number of adhesive cells and 230 per cent as regards the adhesive cells producing germ tubes was detected in the former group of strains as compared to the latter ( p < 0.001 in both cases). The enhancement of FN expression induced by HIV-1 infection, as we have previously demonstrated, can increase interest in the adherence to FN of C. albicans strains isolated from AIDS-affected patients. Moreover, we also underline the important role played by HIV Nef protein in increasing the C. albicans aggressiveness. In fact a significant inhibitory effect of Nef on the phagocytosis of this yeast by macrophages has been demonstrated and the oxidative processes of these cells seem to be down-regulated by this protein., (Copyright 2000 John Wiley & Sons, Ltd.)

It is important to know responses of the pathogenic fungi to reactive oxygen species by which hosts protect themselves against fungal infection. In the present study, sensitivities to the superoxide radical (O2-) and superoxide dismutase (SOD) were compared between a wild-type parent strain and a respiration-deficient mutant of Candida albicans. When their survival was examined on an agar medium containing an intracellular O2- generator, paraquat (PQ), the parent strain was selectively killed by increasing the PQ concentration. In contrast, when cells of both strains were illuminated in a riboflavin solution, they exhibited similar sensitivity to O2- generated extracellularly by photo-reduced riboflavin. There were no large differences in sensitivity to hydrogen peroxide in the two strains. Thus, the high tolerance of the mutant to PQ was suggested to result from low intracellular O2- generation by PQ due to the respiratory deficiency. It is generally accepted that fungal cells contain manganese (Mn)-SOD in the mitochondria and copper and zinc (CuZn)-SOD in the cytoplasm. Cyanide-insensitive SOD activity (attributable to Mn-SOD) was dominant in the parent strain throughout growth phases, whereas cyanide-sensitive activity (attributable to CuZn-SOD) occurred in the mutant. The activity bands of Mn- and CuZn-SODs were clearly separated by electrophoresis of the cell extracts of both strains on non-denaturing polyacrylamide gels. The electrophoretic profiles obtained were consistent with the results of the activity assay. These results showed that the respiratory deficiency affected oxidative stress sensitivity and SOD in C. albicans.

We have examined the influence of subinhibitory concentrations of several antifungals, the different glucose and ammonium sulphate concentrations in the culture medium as well as the strain variability on the expression in vitro of stress mannoproteins reactive with salivary sIgA in C. albicans and other Candida spp isolates. Irrespective of the conditions used, no reactivity with salivary sIgA was observed in yeast cells grown at 25 degrees C. However, when grown at 37 degrees C, all of the 10 C. albicans strains, but only 9 out of 28 non-C. albicans isolates studied showed reactivity with salivary sIgA. Cells grown at 37 degrees C in medium containing maximum concentrations of glucose and ammonium sulphate expressed the antigens reactive with sIgA during longer periods of time than the cells grown in medium with minimal concentrations of the same compounds. The regulatory role showed by the concentration of glucose and ammonium sulphate on the antigenic expression was subordinated, nevertheless, to the most important factor, the temperature of incubation. Only isolates showing low susceptibility expressed the antigens reactive with sIgA under the influence of subinhibitory concentration of antifungals. However, induced resistance to one of the antifungals tested (5 fluorocytosine) allowed the antigenic expression at elevated subinhibitory concentrations even in previous susceptible strains. In conclusion, in addition to the temperature, factors such as characteristics of the strain, the concentration of glucose and ammonium sulphate in the culture medium and the resistance to antifungals played a role on the expression of C. albicans antigens reactive with sIgA, which could be of clinical relevance in the course of infection.

Citreoviridin, a mycotoxin produced by some molds of the genera Penicillium and Aspergillus, inhibits the growth of bacteria of the Bacillus genus. Since significant information was not available on the effects of citreoviridin on eukaryotic cells and viruses, this molecule was tested on CD4+ T-lymphoid cell lines, on HIV-1 and on Candida albicans, which sometimes complicates HIV-infection. Antiviral activity was detected in H9 HTLV IIIB cells, a clone chronically infected by HIV-1. Citreoviridin reduced p24 in the supernatant of H9 HTLV IIIB in a dose-dependent manner with a pharmacological selectivity index of 2.6. In C. albicans, the effects of the mold-derivative were evaluated on some parameters associated with pathogenicity and virulence: cellular proliferation, germ tube production, expression of heat shock mannoproteins, release of proteases and phospholipases. At a 12.5 microM dose, citreoviridin showed a marked inhibitory effect on all parameters analyzed. As regards the mechanism of action, it is possible to hypothesize that the effects of citreoviridin may be due to a reduction of protein synthesis, since it inhibited the replication of HIV-1 at post-integrational stages and reduced the intracellular RNA and protein content in C. albicans.

The presence of red (RNA) and green (DNA) fluorescent material identifying nucleic acids in the chlamydospores of Candida albicans has been well documented. Red fluorescence in chlamydospores is relatively diffused and ranges from small spots, observed in hyphal cells, to the entire protoplasmic content. Green fluorescence is rarely visible in these structures and, when present it can be observed next to the plasmalemma. The initial percentage values of the two curves related to the cell counts of red fluorescence of the suspensor cells and chlamydospores showed remarkable differences between the two structures. About 54% of the chlamydospores showed red fluorescence compared to about 28% of the suspensor cells. It seems from the results obtained in this study that much RNA was produced and/or accumulated in the chlamydospores and suspensor cells, rather than in mycelium where red fluorescence was not observed. The results obtained sustain the hypothesis that a chlamydospore should he considered a fully functional cell that is morphologically and physiologically active and independent from mycelium.

The influence of different fructose concentrations (5, 3, 1 and 0 g/l) was tested on Germ Tube (GT) production by Candida albicans strain AS3P, using a Minimal Synthetic Medium (MSM) without (NH4)2SO4. The results obtained showed good GT production in the presence of all the different fructose concentrations and in the absence of any nitrogen source. The greatest GT production was obtained with 3 g/l of fructose vs 1 g/l of glucose, after 4 hr of incubation. On the other hand fructose consumption was lower than that of glucose at all concentrations over the 4 hour period. The data obtained may suggest that fructose is metabolized in a different way from glucose for GT production by C. albicans.

The influence of different glucose concentrations was tested in minimal synthetic medium on Candida albicans strain. After 18 hours of starvation, germ-tube (GT) production, amount of consumed glucose, oxygen and the pH of the medium were checked every hour from the beginning through the end of the experiment. Optimal GT production was obtained with 1 g/l of glucose. At this concentration the greatest glucose and oxygen consumption were also noted. No pH variations in the medium were observed in all of the glucose concentrations used. At 3 and 5 g/l glucose concentrations a lower GT production were obtained. The Crab-tree effect might interfere with GT production when glucose concentration is higher than 1 g/l. This data may support the hypothesis that GT production is strictly glucose dependent.

Candida albicans starved cells were incubated in minimal synthetic liquid media containing different concentrations of ammonium sulphate (0.00, 0.02, 0.05, 0.10, 0.03, 0.50 g/L). Culture growth was monitored by measuring daily the optical density and by evaluating RNA and protein cellular content after 48 and 96 hours from the inoculum. The environmental availability of ammonium ion influenced the biomass production, that was maximum when its concentration was 0.10 and 0.30 g/L. In addition, an effect on cell duplication time was observed, this was particularly evident when the (NH4)2SO4 concentration was 0.10 g/L. The protein content increased in relation to the increase of ammonium ion availability, with a peak in correspondence to 0.30 g/L and a drop when the greatest concentrations were employed. RNA production was inversely proportional in respect to protein production. The optimal range of ammonium sulphate concentration for C. albicans growth was 0.10-0.30 g/L; over these concentrations there was an inhibitory effect. The rate of the protein and RNA syntheses seems to indicate the growth phase and the nitrogen nutritional conditions of the cultures, respectively.

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.

A prospective 19-month study of 26 human immunodeficiency virus type 1-infected patients with episodes of erythematous or pseudomembranous oral candidiasis was done to evaluate the significance of Candida albicans biotypes in patients treated with antifungal therapy. Changes in the biotype of C. albicans were frequently noted in recurrent oral candidiasis. However, no correlation was found between the various biotypes and the clinical features of oral candidiasis, the clinical stage of human immunodeficiency virus type 1 infection, or the number of CD4+ lymphocytes. On the contrary, a significant correlation appeared among clinical lesion features, CD4+ cell numbers, and time of clinical disappearance of the oral lesions. Changes in the biotype of C. albicans were observed at the end of the antifungal therapy in 17 of 26 patients who had a second appearance of oral candidiasis as well as in 10 of 14 subjects who experienced a third reappearance of oral candidiasis.

Candida albicans strain B 311-10 with and without starvation was cultivated in the minimal synthetic medium of Shepherd et al., modified without biotin, amino acids, low glucose concentration and with decreasing amounts of (NH4)2SO4, to determine the optimal growth requirement for this strain. All the experiments were carried out under sterile conditions at 25 degrees C in a thermostat with initial O.D.s (675 nm) of 0.500 and 0.100. Cell growth was generally monitored everyday for six days with a spectrophotometer by determining the absorbance of the cultures at 675 nm. All the experiments were repeated three times and a statistical analysis of the data with a probability of 99% and 1% of error was performed to confirm the validity of the results. Best growth was obtained with starved cells at an initial O.D. of 0.100 and with a 0.1 g/L concentration of (NH4)2SO4. At this concentration, the growth of C. albicans B 311-10 was best between the first and the fourth day with the maximum at the third day. With (NH4)2SO4 concentrations of 0.05 and 0.5 g/L, cell growth was the same.

Growth of Candida albicans strain B 311-10 was observed in a minimal synthetic biotin-free medium, using different glucose concentrations, during the first 30 hours of its development at 28 degrees C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherd et al., with glucose (15 g/L) and biotin was modified: this vitamin was eliminated and the concentration of glucose was gradually lowered to 0.5 g/L. At 5 g/L of glucose and without biotin very good growth was obtained. Based on our results during the first 30 hours of growth, biotin has no influence on the yeast's growth. This medium would be useful for the study of the physiology of C. albicans during the first period of its development.

Different physical and chemical methods were used to detach the chlamydoconidia of Candida albicans from its mycelium. The action of concentrated H2SO4 acid for a 4-min period on cultures lysed both the mycelium and the outer but not the inner wall layer of the chlamydoconidia. The sulfuric acid procedure is recommended as the best method to obtain mycelium free chlamydoconidia because of its simplicity, rapidity and low cost.

Several isolates of Candida albicans were tested for production of chlamydoconidia and metabolic changes when grown on several different solid and liquid media. A liquid medium, consisting solely of sterilized skimmed milk and a solid medium containing processed cheese stimulated more rapid and greater production of chlamydoconidia than the corn meal agar and the other media tested.

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal synthetic medium of Shepherd et al. modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.