The morphology of colony variants of three species of Candida.

The colonies of 12 isolates of 3 Candida spp. with variant colony forms were studied by scanning and transmission electron microscopy. Small colonies were formed by 4 isolates each of C. albicans and C. parapsilosis and by 1 of C. tropicalis. These had an abnormally high proportion of degenerate yeast cells with an associated increase in granular cytoplasmic material intercellularly. The increased matrix in these small colonies formed a thick superficial coat over the organisms. Rough colonies were formed by 1 isolate each of C. albicans, C. tropicalis and C. parapsilosis. The convoluted regions of these colonies contained many pseudohyphal cells but few degenerate cells and little granular or fibrillar material in their intercellular matrices. The shape of colonies of Candida spp. may be altered by variations in the viability or the morphology of the organisms.     

Reduced azole susceptibility of oral isolates of Candida albicans from HIV-positive patients and a derivative exhibiting colony morphology variation.

Approximately 50% (15/28) of a selection of oral isolates of Candida albicans from separate individuals infected with the human immunodeficiency virus (HIV) exhibited low susceptibility to ketoconazole as determined by hyphal elongation assessment. Nine of these isolates exhibited colony morphology variation or switching at 37 degrees C, of which six expressed low ketoconazole susceptibility. To determine whether colony morphology variation could give rise to derivatives with reduced azole susceptibility, several high-frequency switching variants of three HIV-patient isolates were recovered and assessed. All but one of the variants expressed similar azole susceptibility profiles to their respective parental strains. However, the C. albicans derivative 132ACR expressed significantly reduced susceptibility to ketoconazole in comparison to its parental strain 132A. In whole cells, on the basis of total growth the switched derivative 132ACR was markedly less susceptible than its parental isolate 132A to ketoconazole at 10 microM. A much smaller difference was observed with fluconazole at 10 microM, with the switched derivative 132ACR exhibiting a threefold lower susceptibility compared with the parental isolate 132A. The incorporation of [14C]acetate in control and azole-treated cells of both organisms was higher for the parental strain. When cell lysates of strain 132A and its derivative 132ACR were incubated with [14C]mevalonic acid and ketoconazole, the IC50 for 14C-label incorporation into C-4 demethyl sterols was fivefold higher for lysates of the switched derivative 132ACR compared with those of the parental strain 132A. With fluconazole the IC50 value for the derivative 132ACR was 25-fold higher than for strain 132A. The 14-sterol demethylase of the switched derivative 132ACR was possibly less sensitive to azole inhibition than that of the enzyme of strain 132A. These studies indicated that colony morphology variation in vitro can generate derivatives with stable, reduced azole susceptibility without prior exposure to azoles.     

The effect of nystatin on the morphology of Candida albicans.

The effect of nystatin on Candida albicans was observed by light and scanning electron microscopy; it caused abnormal growth. Some cells had deep depressions in the cell walls; budding and germ tube formation were reduced.     

Farnesol concentrations required to block germ tube formation in Candida albicans in the presence and absence of serum

Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candida albicans strains A72 and SC5314 by using six different conditions known to trigger germination. For defined media, 1 to 2 microM farnesol was sufficient. However, with serum at 2 to 20%, up to 250 microM farnesol was required. Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.     

Freeze-fracture studies of the plasmalemma of Candida albicans after treatment with econazole-nitrate.

In electron microscopic studies the interior of the plasmalemma of Candida albicans was revealed by means of the freeze-fracture technique. The superficial structures of the extracellular (E) and protoplasmic (P) fracture faces differed negligibly from structures on the corresponding fracture faces of Saccharomyces cerevisiae. Following treatment with 2.2 x 10(-5) M econazole nitrate a layer, present on the P face in the form of a tight matrix of globular proteins, dissolved into isolated groups of particles whose globular elements sometimes formed hexagonal patterns. As the damage progressed, fissure-shaped membrane invaginations on the P face disappeared. Parts of the outer lipid layer of the plasmalemma were torn off the cell wall and adhered in fragments to the P face. The ultrastructural changes in the plasmalemma induced by econazole nitrate temporally correlate with an increase in the permeability of the cell envelope found in physiological studies performed by other authors.     

The effects on immune response of levamisole treatment following infection of U-937 macrophages with Candida albicans

The aim of this study was to investigate the effects on the immune response of levamisole alone and in conjunction with Candida albicans stimulation in human macrophage cell culture by determining the alterations in the levels of cytokine release. Levamisole treatment was performed before, during and after infecting U-937 human macrophage cells with C. albicans. In cell supernatants, interleukin (IL)-1b, IL-12, IL-18, tumour necrosis factor alpha (TNF-α) levels were measured by ELISA. In vitro levamisole treatment accompanied by C. albicans stimulation significantly increased IL-12, IL-1β and IL-18 production in macrophage cells (p < 0.05). It was observed that when administered before C. albicans infection, levamisole significantly increased IL-12 and IL-1β production in macrophage cells (p < 0.05). Another finding was that when applied to macrophage cells simultaneously with C. albicans infection, or before infection with C. albicans, levamisole suppressed the TNF-β production stimulating effect of C. albicans (p < 0.05). These results indicated that levamisole could be useful in treating patients infected with C. albicans or in protecting individuals under the risk of being infected with this pathogen. There is a need for further experimental and clinical studies on this hypothesis.     

Morphogenesis of Candida albicans and cytoplasmic proteins associated with differences in morphology, strain, or temperature.

The extent of change in cytoplasmic proteins which accompanies yeast-to-mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium. The two strains selected for this study were strain 4918, which produces pure mycelial cultures in low-sulfate medium at 37 degrees C and yeast cells at 24 degrees C, and strain 2252, which produces yeast cells exclusively at both 24 and 37 degrees C in low-sulfate medium. The proteins of both strains were labeled at both temperatures with [35S]sulfate, cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation, and the labeled proteins were analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. Ten protein spots were identified on the two-dimensional gels of the 4918 mycelial-phase extract which were not present in the 4918 yeast-phase extract. These proteins appeared to be modifications of preexisting yeast-phase proteins rather than proteins synthesized de novo in the mycelial cells because 5 were absorbed by rabbit anti-yeast-phase immunoglobulin and each of the 10 was also present in extracts of strain 2252 grown at 24 and 37 degrees C, indicating that they were neither unique to filamentous cells nor sufficient for induction or maintenance of the mycelial morphology. Thirty-three proteins were identified in the 4918 yeast-phase extract which were not present in the 4918 mycelial-phase extract. Pulse-chase experiments revealed the synthesis of new proteins during yeast-to-mycelial conversion, but none of these was unique to mycelial cells. No differences in the major cytoplasmic proteins of any of the yeast- or mycelial-phase extracts were identified. This finding suggests that the major structural proteins of the cytoplasm are not extensively modified and argues instead that proteins unique to either phase may serve a regulatory function.     

Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors.

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N-terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N- and C-termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10-10-10-9 M. The HIV-1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.     

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.     

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.     

Fungitoxicity of 1,4-naphthoquinones to Candida albicans and Trichophyton mentagrophytes.

Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2.     

Resistance of the Candida albicans filamentous cycle to environmental change.

The filamentous growth cycle in C. albicans was resistant to changes in environment brought about either by the serial transfer of growing cells to fresh nutrients or by sudden changes of temperature after the first h of growth. In further experiments older culture filtrates, exhausted of their ability to induce mycelial growth, were reactivated by addition of fresh nutrients or water. The data provided evidence against the existence of both a mycelial stimulatory and inhibitory compound in the growth medium. It is concluded that although the environment initially dictates what proportion of blastospores are committed to filamentation it has no further effect on the process.     

Basement membrane protein and matrix metalloproteinase deregulation in engineered human oral mucosa following infection with Candida albicans.

A variety of morphological changes in the basement membrane (BM) are known to occur in inflammatory diseases. Modifications of the BM can be associated with significant changes in protein content. Candida albicans (C. albicans) is normally a commensal organism and is a member of the natural flora of a large number of healthy individuals. However, under certain conditions, C. albicans can invade host tissues, causing inflammation and tissue damage. The aim of this study was to investigate the effect of C. albicans on the expression and production of structural (laminin-5 and type IV collagen) and inflammatory [matrix metalloproteinases (MMPs) and their inhibitors] proteins by human oral epithelial cells. Using engineered normal human oral mucosa infected with 10(5) C. albicans/cm2 for different periods of time, we were able to demonstrate that this yeast promotes significant laminin-5 and type IV collagen gene activation and protein secretion. These effects were accompanied by MMP-2 and MMP-9 gene activation. Interestingly, only the levels of active MMP-9 rose. The increase in MMP levels was paralleled by a decrease in the secretion of type 2 matrix metalloproteinase tissue inhibitors (TIMP-2). Our results demonstrated that C. albicans has a significant effect on tissue structure through BM protein and MMP modulation. This might help C. albicans overcome the mechanical and biological defenses of the tissue and allow it to disseminate, causing severe infections. If C. albicans uses MMPs (mainly MMP-9) to disseminate, inhibition of this protease could be of interest in treating a variety of inflammatory disorders, including oral candidiasis.