Candida albicans: genetics, dimorphism and pathogenicity.

Candida albicans is a dimorphic fungus that causes severe opportunistic infections in humans. Recent advances in molecular biology techniques applied to this organism (transformation systems, gene disruption strategies, new reporter systems, regulatable promoters) allow a better knowledge of both the molecular basis of dimorphism and the role of specific genes in Candida morphogenesis. These same molecular approaches together with the development of appropriate experimental animal models to analyze the virulence of particular mutants, may help to understand the molecular basis of Candida virulence.     

Effect of sodium nitroprusside on H+-ATPase activity and ATP concentration in Candida albicans

ATP hydrolysis by plasma membrane H+-ATPase from Candida albicans has been investigated in presence of nitric oxide and various nutrients (sugars and amino acids). Sodium nitroprusside (SNP) was used as nitric oxide donor. It was found that ATP concentration decreased in SNP treated cells which was more in presence of sugars like glucose, xylose and 2-deoxy-D-glucose and amino acids as compared to their respective controls. The activity of H+-ATPase from plasma membrane decreased by 70 % in SNP treated cells. Both in vivo and in vitro treatments of SNP showed almost similar effects of decrease in ATPase activity. Effect of SNP was more pronounced in presence of nutrients. Interestingly, it was observed that vanadate did not show any independent effect in presence of nitric oxide. Several workers have reported similar type of results with other P-type ATPases. For the first time, it was observed in the present study that in presence of nitric oxide, H+-ATPase activity decreased like other P-type ATPases. Our study indicated that NO had a significant effect on ATP synthesis and activity of H+- ATPase. In the presence of NO, the ATP concentration was decreased indicating it affected mitochondrial electron transport chain. It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with H+- ATPase of plasma membrane by changing its conformation resulting in decreased activity.     

A characterization of pH-regulated dimorphism in Candida albicans

When cells of the dimorphic yeast Candida albicans are grown to stationary phase in defined liquid medium at 25°C, they accumulate as singlets in G_l of the cell cycle. When these pluripotent, stationary phase singlets are released into fresh medium at 37°C, they synchronously evaginate after an average period of 135 to 140 minutes and form either buds or mycelia, depending upon the pH of the medium into which they are released. This method of dimorphic regulation offers the distinct advantage of comparability and serves as a very precise method for temporal comparisons of molecular and cytological events related to the establishment of the alternate growth phenotypes. In the present report, we have carefully examined the effects of individually varying pH or temperature on the length of the pre-evagination period, the population synchrony for evagination, and the phenotype of daughter cells. Exact phenotypic transition points, optima, and upper limits are defined for both temperature and pH. In addition, a method of pH-regulated dimorphism is developed in which the original temperature shift is removed from the inductive process. Finally, a common transition phenotype is described for cells reverting from the initial mycelial to budding phenotype when either pH or temperature traverse their respective transition points. The advantages as well as limitations of pH-regulated dimorphism are discussed in detail.     

Inhibition of H+ Extrusion by Phosphocreatine in Candida albicans

Vanadate, a potent inhibitor of P-type ATPases, reduces the electrochemical gradient considerably. H⁺-extrusion in cells of Candida albicans, a pathogenic yeast, was strongly inhibited in the presence of 25mM phosphocreatine (PCr) by about 83%. H⁺-extrusion was further inhibited by 25 mM PCr in the presence of vanadate; 89% with 1 mM, 92% with 2 mM and 99% with 5 mM vanadate. 2 mM vanadate caused 90%, 92% and 96% inhibition in the presence of 20 mM, 30 mM and 40 mM PCr, respectively. Creatine (Cr) had a negligible effect on H⁺ - extrusion. The inhibition caused by 1 mM, 2 mM and 5 mM vanadate alone was 66%, 77% and 88%, respectively. PCr and vanadate inhibit proton extrusion with almost equal magnitude. It can be concluded that phosphate moiety of PCr interacts with the ATPase and is similar to vanadate interaction. Since PCr is having such a drastic inhibitory effect on ATPase activity we can say that it is playing a significant role in holding a check on this pathogenic fungus in healthy human hosts.     

Effect of nitric oxide on H+ -efflux in presence of various nutrients in Candida albicans

In the present study tentative link has been established between H+ -efflux and effect of NO in presence of various nutrients (glucose, 2-deoxy-D-glucose, xylose, proline, glutamic acid and lysine) in C. albicans using sodium nitroprusside (SNP) as a potent source of NO. It was observed that there was a decreasing trend in pH with time, in control, while SNP treated cells showed an initial decline in pH for 10-15 min, followed by an increase in pH up to 30 min. In presence of glucose there was an enhancement in H+ -efflux by 9-fold whereas proline, glutamic acid and lysine showed enhancement by 3, 6 and 1.5-fold respectively. Similar trends in increase in pH after 15 min in SNP treated cells of Candida was observed in presence of all nutrients used. It was demonstrated for the first time that H+ -ATPase of C. albicans was affected by NO.     

Candida albicans dimorphism and virulence: Role of copper

Previously reported observations that Candida albicans grows in the yeast phase at 30° C and the mycelial phase at 37° C and that the former phase is more virulent than the latter were confirmed. A novel factor, copper, was discovered to suppress filamentation. Injection of copper into mice permitted the filamentous phase to be as virulent as the yeast phase. In subsequent studies on candidosis, copper assays should be performed on relevant body fluids to determine if there might be a correlation between elevated copper and heightened susceptibility to the fungus.     

Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] 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.     

Control of dimorphism in a biochemical variant of Candida albicans

The cellular morphology of a biochemical variant of Candida albicans could be controlled by the ratio of carbon dioxide to oxygen in the culture system or by individual amino acids. Predominantly pseudohyphal morphology was observed (i) at a CO(2) to O(2) ratio of 2:1 and (ii) without the addition of carbon dioxide, when either glycine, d- or l-ornithine, l-serine, l-methionine, l-phenylalanine, or l-tyrosine was the sole nitrogen source in the culture medium. When ammonium chloride, ammonium sulfate, l-glutamic acid, l-glutamine, or l-proline was the nitrogen source, yeastlike growth was observed in the presence or absence of CO(2). More adenosylmethionine was present in pseudohyphal than in yeastlike cells, and pseudohyphal cell wall preparations contained less methionine than cell walls from the yeastlike form. These results suggest a correlation between sulfur amino acid metabolism and dimorphism.     

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     

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.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

In Vitro Effect of Amphotericin B on Candida albicans,Candida glabrata and Candida parapsilosis Biofilm Formation

Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) ≤1 μg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC ≤1 μg/mL. Based on these results, amphotericin B displays species- and strain-depending activity against Candida spp. biofilms.     

Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1-3 mutant and Tpk2p is able to phosphorylate an established PKA-acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.     

Role of nutritional status of the cell in pH regulated dimorphism of Candida albicans

Stationary phase cells of Candida albicans are under the control of glucose repression, as indicated by the inhibition of germ tube formation by glucose. This 'glucose effect' was absent in starved cells which were derived from similar stationary phase cells. Moreover, starved cells required glucose for germ tube formation, suggesting that it was depletion of energy reserves which was the main factor overriding the 'glucose repression machinery' during starvation. High concentration of phosphate in Lee's medium was the reason for the reduced ability of the starved cells to form germ tubes at pH 4.5 (20% of cells compared to 88% at pH 6.8). However, when phosphate was replaced or its concentration reduced, germ tube formation occurred as frequently at pH 4.5 as at pH 6.8. This 'phosphate effect' was not observed in stationary phase cells, as they were already repressed by glucose.     

The electrochemical gradient of H+ in Candida albicans and its relevance to the uptake of nutrients

The electrochemical gradient of protons, delta microH+, in Candida albicans was estimated between pH 3.5 and 8.5. The electrical potential difference (delta psi) and the chemical proton gradient (delta pH) were measured by steady-state distribution of tetraphenylphosphonium ion and of propionic acid across the plasma-membrane, respectively. In the pH range tested, the intracellular pH was maintained fairly constant at values between 7.3 and 8.1. On the other hand, there was an up to three fold enhancement of delta psi under similar conditions. The uptake of a neutral (glycine), an acidic (L-glutamate) and a basic (L-arginine) amino-acids and of the aldopentose (D-xylose) was determined under different values of delta microH+, which was manipulated by varying the pH of the cell suspension. The rate of uptake of D-xylose and glycine appeared to follow delta microH+ while the uptake velocity of L-arginine could be correlated to changes in delta psi. The rate of uptake of L-glutamate, although at highest among the rates of tested nutrients, was, however, largely independent of delta microH+. This and other reasons (discussed below) indicate that delta microH+ may not be the sole driving force of nutrients uptake in C. albicans.     

Evaluation of morphogenic regulatory activity of farnesoic acid and its derivatives against Candida albicans dimorphism

A series of farnesoic acid derivatives was prepared and their morphogenic regulatory activities were evaluated. Their inhibitory activities against yeast cell growth and yeast-to-hypha transition examined in Candida albicans cells are dependent upon the chain length as well as the substitution patterns on the isoprenoid template. The preliminary structure-activity relationship of these compounds is described to elucidate the essential structural requirements.