Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.     

Sub-inhibitory Concentrations of Antifungals Suppress Hemolysin Activity of Oral Candida albicans and Candida tropicalis Isolates from HIV-Infected Individuals

Paracoccidioides brasiliensis, a dimorphic pathogenic fungus, causes the principal form of systemic mycosis in Brazil. The literature furnishes only limited data on the ecology of this fungus in the state of Rio Grande do Sul, the southernmost state of Brazil. The purpose of this study was to evaluate the prevalence of fungal infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20 %), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures.     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

Quantification of thigmotropism (contact sensing) of Candida albicans and Candida tropicalis

To quantify the thigmotropism, we adapted the our previous method using a chemotaxifilter system in combination with a bioluminescent adenosine triphosphate (ATP) assay based on firefly luciferase-luciferin system and analyzed the relationship between the ability of germ tube formation and thigmotropism of C. albicans and C. tropicalis. Both the ability to form germ tube and the amount of hyphae exhibiting thigmotropism varied depending upon both the species and strains of Candida. C. albicans formed more germ tubes than C. tropicalis. A good correlation was observed between the ability to form a germ tube and the capacity for thigmotropism, and the results gave a level of significance (p<0.05). Further, SEM observation revealed that relatively long hyphae of C. tropicalis with penetrated through the pores of filter membrane. This phenomenon may be of importance in the development of pathogenesis of C. tropicalis as well as C. albicans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Factors involved in the adherence of Candida albicans and Candida tropicalis to protein-adsorbed surfaces

The adherence of Candida albicans and C. tropicalis to protein-adsorbed surfaces was investigated with surface-modified glass slides to which serum or salivary proteins were covalently bound. A specific adherence like a ligand-receptor interaction was observed between C. albicans and mucin- or salivary protein-immobilized glass slides. This interaction was eliminated by deglycosylation of the slides, suggesting that the receptor may be an oligosaccharide(s) contained mucin or saliva. A similar specific interaction was also observed between C. tropicalis and fibrinogen-immobilized glass surfaces. When the numbers of adherent cells to deglycosylated protein-immobilized glass glides were plotted against zeta potentials and contact angles of these protein-immobilized glass slides, a significant correaltion was observed between the numbers of adherent cells and zeta potentials in the case of C. albicans (r = –0.87), whereas a significant correlation was observed between cell numbers and contact angles (r = 0.82) in the case of C. tropicalis. These results suggest that the forces governing the adherence of fungi to pellicle in dentures may vary depending upon the surface properties of fungi and substrate.     

Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

Background

Severely burned patients may develop life-threatening nosocomial infections due to Pseudomonas aeruginosa, which can exhibit a high-level of resistance to antimicrobial drugs and has a propensity to cause nosocomial outbreaks. Antiseptic and topical antimicrobial compounds constitute major resources for burns care but in vitro testing of their activity is not performed in practice.

Results

In our burn unit, a P. aeruginosa clone multiresistant to antibiotics colonized or infected 26 patients over a 2-year period. This resident clone was characterized by PCR based on ERIC sequences. We investigated the susceptibility of the resident clone to silver sulphadiazine and to the main topical antimicrobial agents currently used in the burn unit. We proposed an optimized diffusion assay used for comparative analysis of P. aeruginosa strains. The resident clone displayed lower susceptibility to silver sulphadiazine and cerium silver sulphadiazine than strains unrelated to the resident clone in the unit or unrelated to the burn unit.

Conclusions

The diffusion assay developed herein detects differences in behaviour against antimicrobials between tested strains and a reference population. The method could be proposed for use in semi-routine practice of medical microbiology.     

Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

Interspecific complementation analysis by protoplast fusion of Candida tropicalis and Candida albicans adenine auxotrophs.

A protocol employing inositol starvation was used to isolate proline and adenine auxotrophs of Candida tropicalis. Interspecific hybrids between red adenine auxotrophs of C. tropicalis and Candida albicans were formed by protoplast fusion. These C. tropicalis red adenine auxotrophs were shown to fall into two complementation groups by crossing them with a known C. albicans ade1 tester strain. It is suggested that these two groups correspond to the ade1 and ade2 mutants of Saccharomyces cerevisiae and C. albicans and that these defined mutants may be useful in attempts to develop transformation systems for C. tropicalis.     

A subpopulation of Candida albicans and Candida tropicalis biofilm cells are highly tolerant to chelating agents

Many Candida spp. produce surface-adherent biofilm populations that are resistant to antifungal compounds and other environmental stresses. Recently, certain chelating agents have been recognized as having strong antimicrobial activity against biofilms of Candida species. This study investigated and characterized the concentration- and time-dependent killing of Candida biofilms by the chelators tetrasodium EDTA and sodium diethyldithiocarbamate. Here, Candida albicans and Candida tropicalis biofilms were cultivated in the Calgary Biofilm Device and then exposed to gradient arrays of these agents. Population survival was evaluated by viable cell counting and by confocal laser scanning microscopy (CLSM) in conjunction with fluorescent viability staining. At concentrations of > or =2 mM, both EDTA and diethyldithiocarbamate killed c. 90-99.5% of the biofilm cell populations. Notably, a small fraction (c. 0.5-10%) of biofilm cells were able to withstand the highest concentrations of these antifungals that were tested (16 and 32 mM for EDTA and diethyldithiocarbamate, respectively). Interestingly, CLSM revealed that these surviving cells were irregularly distributed throughout the biofilm community. These data suggest that the use of chelating agents against biofilms of Candida spp. may be limited by the refractory nature of a variant cell subpopulation in the surface-adherent community.     

Effect of dietary carbohydrates on the in vitro epithelial adhesion of Candida albicans, Candida tropicalis, and Candida krusei

Adhesion to epithelial surfaces is considered as a critical step in the pathogenesis of oral candidosis. Therefore, the effects of the most commonly consumed dietary carbohydrates on the adhesion of Candida albicans, Candida tropicalis, and Candida krusei to monolayered HeLa cells were investigated. Adherence of C. albicans and C. tropicalis appeared significantly promoted by incubation in defined medium containing a high concentration (500 mM) of fructose, glucose, maltose, and sucrose (p < 0.001). C. albicans organisms grown in sucrose elicited maximal increase in adhesion, whereas adhesion of C. tropicalis and C. krusei was enhanced to the greatest extent when cultured in glucose. Maltose and fructose also promoted adherence of C. albicans and C. tropicalis (p < 0.001), but to a lesser extent than sucrose and glucose. On the other hand, sorbitol-grown yeasts demonstrated a marginal increase in adhesion (p > 0.01). Xylitol only significantly reduced adherence of C. albicans (p < 0.001). These results suggest that the frequent consumption of carbohydrates, such as sucrose, glucose, maltose, or fructose, might represent a risk factor for oral candidosis. The limitation of their consumption by substituting xylitol or sorbitol could be of value in the control of oral Candida colonization and infection.     

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.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.     

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.