Suppression of Anti-Candida Activity of Murine Neutrophils by Progesterone In Vitro: A Possible Mechanism in Pregnant Women's Vulnerability to Vaginal Candidiasis

Sex steroid hormones were examined for their effect on mycelial growth of Candida albicans, and the inhibitory activity of casein-induced murine peritoneal neutrophils against mycelial growth of C. albicans was examined in vitro using a crystal violet staining method or a [³H]glucose incorporation method. Four steroid hormones, danazol, estradiol, estriol and testosterone had no effect on mycelial growth of C. albicans, but progesterone appeared to convert the growth form of C. albicans from hyphal to yeast. Danazol (10^–6 m ) and progesterone (10^–5 m ) suppressed anti-Candida activity of neutrophils of non-treated mice, while testosterone, estradiol, and estriol did not. The anti-Candida activity of neutrophils of estradiol-pretreated mice was clearly suppressed by progesterone even at 10^–6 m which corresponded to its plasma concentration in pregnant women in the third trimester. The physiological significance of this suppressive effect of progesterone was discussed in relation to the vulnerability of pregnant women to vaginal candidiasis.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

Dimorphism-associated changes in plasma membrane H+-ATPase activity of Candida albicans

In situ plasma membrane H⁺-ATPase activity was monitored during pH-regulated dimorphism of Candida albicans using permeabilized cells. ATPase activity was found to increase in both the bud and germ tube forming populations at 135 min which coincides with the time of evagination. Upon reaching the terminal phenotype the mycelial form exhibited higher H⁺-ATPase activity as compared to the yeast form. At the time of evagination H⁺-efflux exhibited an increase. K⁺ depletion resulted in attenuated ATPase activity and glucose induced H⁺-efflux. The results demonstrate that ATPase may play a regulatory role in dimorphism of C. albicans and K⁺ acts as a modulator.Abbreviations PM Plasma membrane - pHi intracellular pH - Pi inorganic phosphorus - TET Toluene: Ethanol: Triton X-100     

An effective medium for the selective growth of yeast or mycelial forms of Candida albicans: Biochemical aspects of the two forms

A new synthetic medium, based on a modification of a commercially available tissue culture medium, allows Candida albicans to be grown in the yeast or mycelial form. Salient features of the system are described and comparisons with previous physiological investigations are discussed. A concise biochemical profile of these two forms of C. albicans is also presented. The results indicate vast metabolic differences between the two forms.     

Cellular and humoral immune responses to Candida albicans in subcutaneously infected mice

Pure mycelial and yeast cultures of Candida albicans were produced in a low sulphate medium. Groups of mice were injected subcutaneously with increasing doses of viable or heat-killed mycelial or yeast cells and the kinetics of delayed-type hypersensitivity (DTH), anti-mycelial and anti-yeast antibodies were studied. Both the dose and the morphological phase of C. albicans showed an influence on the development of the DTH, but the viability is the factor which showed the highest influence on this reaction, since on the one hand mice infected with viable yeast or mycelial cells developed higher DTH levels than mice injected with heat killed cells, and on the other hand this factor seems to play an important role in the kinetics of DTH response. The enzyme-linked immunosorbent assay has been adapted to detect antibodies to yeast and mycelial phase cytoplasmic antigens of C. albicans. In contrast with the DTH reactions, neither dose, morphological phase nor viability played an important role on the antibody titer developed. However, the use of mycelial cytoplasmic antigens seems to be better than the yeasts to detect anti -Candida antibodies over the last days studied.     

Growth ofCandida albicans in a minimal synthetic medium without biotin

Summary Growth ofCandida 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 °C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherdet al. [12], 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 ofC. albicans during the first period of its development.     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

Induction of mycelial type of development in Candida albicans by low glucose concentration

A new simple method for synchronous germ tube production in Candida albicans has been described, based on the further incubation of cells released from stationary grown cultures in aerated mineral medium enriched with vitamins and low glucose concentration (5 mmol/l). At higher initial glucose (e.g. 250 mmol/l) the growth proceeded in yeastlike form. At low glucose concentration germ tubes developed at 28°C which is in contradiction with the results of many authors, considering 37°C besides other factors to be an inevitable requirement. On the other hand the cell population from stationary growth phase was the absolute prerequisite for massive germ tube production. Its importance for other inductive techniques is assumed.The report brings comparative results concerning the physiological and biochemical properties as well as the ultrastructure of the yeastlike and mycelial forms. Neither were found any differences in respiration intensity nor in respiration quotients during the development of both growth forms. Slight dissimilarities resulted from the incorporation experiments (using ¹⁴C labeled adenine, leucine and especially glycine). The mycelial cell walls were found to contain twice as much chitin as the yeastlike form.Some suggestion for further biochemical elucidation of dimorphism in Candida albicans and fungal morphogenesis generally are presented.     

Inhibition of yeast-mycelium transformation by 2-alkylthio-6-amino-and 2-alkylthio-6-formamidobenzothiazoles and theirin vitro antifungal activity

The antifungal effect of 2-alkylthio-6-amino-and 2-alkylthio-6-formamidobenzothiazoles (22 derivatives in all) onAspergillus niger and variousCandida yeasts was tested. No significant effect was observed withA. niger. With the pathogenic yeast speciesC. albicans andC. guilliermondii, the most efficient derivatives (6-formamido-2-propylthio-, 6-formamido-2-butylthio-, 6-formamido-2-pentylthio-and 6-formamido-2-isopentylthiobenzothiazole) exhibited ED₅₀ values in the range from 2.2 to 21 mg/L and were thus 3–35 times more efficient than 2-mercaptobenzothiazole (Dermacid) that is normally used. 6-Amino-2-pentylthiobenzothiazole was found to be an efficient specific inhibitor of the transformation of the yeast form ofC. albicans to its mycelial one, IC_95(M) being 10 mg/L, which was a concentration 25 times lower than MIC_Y+M.     

Differential profiles of soluble proteins during the initiation of morphogenesis in Candida albicans

Candida albicans is a dimorphic fungus that can grow either as yeast or as mycelia. The mycelial form may be required for tissue penetration and therefore may have a role in pathogenesis. The protein profiles of the cell-free S100 fraction from budding yeast cells and germ tube-forming cells (an early stage of the transition between yeast and mycelia) were evaluated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yeast growth or germ tube formation was induced in carbon-starved cells at 37° C by either glucose, galactose or N-acetylglucosamine at pH 4.5 or pH 6.7. More than 400 constitutively synthesised polypeptides were identified on 2-D PAGE by silver staining. A few polypeptides which seem to reflect the release from carbon starvation were detected, but no polypeptides unique to either morphology were observed. Fractionation of S100 preparations by polyethylenimine or heparin-agarose affinity chromatography, which have been used to detect DNA-binding proteins, revealed several proteins that were synthesised on the resumption of cell growth or in response to pH difference. Heparin-agarose also bound novel polypeptides in the size range 130–200 kDa that were preferentially synthesised in germ tube-forming cells. These results suggest that any protein factors that might exert a regulatory role early in germ tube formation are of low abundance, and that a minor group of soluble proteins involved in C. albicans morphogenesis may be differentially synthesised. Received: 11 March 1996 / Accepted: 10 July 1996     

Immune responses to yeast and mycelial forms of Candida albicans in intraperitoneally infected mice

Candida albicans E-139 produced pure mycelial and yeast cultures in a low sulphate medium at different temperatures. The influence of the morphological phase, dose and viability of the fungi on the kinetic of delayed-type hypersensitivity (DTH) and anti-mycelial and anti-yeast antibodies have been studied in mice injected intraperitoneally. The mycelial form elicited higher DTH levels than the yeast phase. This effect seems to be related to its antigenic properties. The effect of dose on the immune response depends on the viability of the fungus. The mycelial cytoplasmic antigens were more effective than the yeast ones in detecting antibodies induced during the experiments, particularly during the later stages of the observation periods, suggesting that such antigens may be useful in the serodiagnosis of Candida infections.     

Comparative activities of glycolytic enzymes in yeast and mycelial forms of Candida albicans

Through use of a synthetic defined medium which allows for the exclusive growth of yeast or mycelial forms of Candida albicans the activity of several major glycolytic enzymes in these forms were examined and compared. The results indicate vast metabolic differences between the forms. These data are discussed in relationship to the phenomenon of morphogenesis in C. albicans which in turn relates to problems in immunology and pathogenics of this important opportunistic organism.     

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores

The effect of germ tube induction on the antigenic variability in C. albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45–47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.Abbreviations GYE glucose-yeast extract agar     

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form

Aims: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. Methods and Results: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 μg ml⁻¹) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast’s transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 μg ml⁻¹. This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. Conclusion: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. Significance and Impact of the Study: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.     

Distribution of the 7S Proteins in Soybean Globulins by Gel Filtration with Sephadex G-200

The level of isocitrate lyase, an enzyme of glyoxylate cycle, in Candida tropicalis was enhanced at the later period of growth when the yeast was cultivated in a semisynthetic glucose medium. On the other hand, such increase in the enzyme activity was not observed in C. lipolytica grown under the same conditions. In the case of C. tropicalis, high concentrations of glucose remaining in the medium permitted the increase in the enzyme activity and the addition of ethanol, one of the major products from glucose, to the glucose medium did not stimulate the enzyme formation, indicating that the enhanced enzyme level in the yeast was not merely attributable to the release from the repression by glucose or to the induction by ethanol. Biotin, one of the growth-stimulating factors for C. tropicalis, affected markedly the level of isocitrate lyase. That is, the supplementation of biotin to the synthetic glucose medium inhibited completely the increase in the enzyme activity, and reversely the absence of biotin stimulated the enzyme formation in the glucose-assimilating cells. Thiamine, another growth-stimulating factor for C. tropicalis, did not show any effect on the level of isocitrate lyase in the yeast. The level of isocitrate lyase in C. lipolytica growing on glucose was not affected by biotin added exogenously.     

The role of galectin‐3 in phagocytosis of Candida albicans and Candida parapsilosis by human neutrophils

Candida albicans causes the majority of invasive candidiasis in immunocompromised adults while Candida parapsilosis is a leading cause of neonatal candidiasis. While much work has focused on how the immune system recognizes and responds to C. albicans, less is known about host interaction with C. parapsilosis. This study investigates the human neutrophil phagocytic response to these species. Neutrophils underwent phagocytosis of C. parapsilosis yeast and C. albicans hyphae much more efficiently than C. albicans yeast. Treatment of neutrophils with a galectin‐3 (gal3) blocking antibody inhibited phagocytosis of C. parapsilosis yeast and C. albicans hyphae, but not C. albicans yeast. The majority of neutrophil gal3 was expressed intracellularly and was secreted from neutrophils after treatment with C. parapsilosis mannan. When neutrophils were treated with exogenous gal3, phagocytosis of both C. albicans and C. parapsilosis yeast increased. Exposure of neutrophils to C. parapsilosis yeast increased phagocytosis of C. albicans yeast and was inhibited by gal3 blocking antibody. Taken together, these data indicate that gal3 secreted from neutrophils may act as a pro‐inflammatory autocrine/paracrine signal in neutrophil phagocytosis and suggest that gal3 has a unique role in neutrophil response to C. parapsilosis yeast and C. albicans hyphae distinct from C. albicans yeast.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

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     

Biofilm formation and adhesive/invasive properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in comparison with <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida dubliniensis and Candida albicans are closely related spp. exhibiting differences in their virulence potency. This study compared clinical isolates of C. dubliniensis with C. albicans from HIV patients with oropharyngeal candidiasis (OPC) and standard strains in power to form biofilm and their adhesive and invasive properties. Members of both spp. were able to form strong biofilms. However, SEM microscopy confirmed that C. albicans undergoes the more effective yeast-to-hyphae transition than C. dubliniensis with prevalent yeast form and limited ability to form filaments. Kinetic patterns indicated that while the first 30 min are critical for sufficient attachment to a polystyrene surface, adhesion to human carcinoma cell lines (Caco-2 and TR 146) needs additional time with maximal saturation observed at 240 min for both spp. The invasion process was tested on 3D RHE (reconstituted human epithelium) with Caco-2 or TR 146 on the collagen surface. C. albicans rapidly produced hyphae that penetrated the tissue layer, demonstrating substantive invasion within 21 h. In contrast, C. dubliniensis attached to the tissue surface and proliferated, suggesting the formation of a biofilm-like structure. After 21 h, C. dubliniensis was able to penetrate the RHE layer and invade unusually, with a cluster of the yeast cells.