Dimorphism-associated changes in amino acid transport of Candida albicans

Abstract Amino acid uptake was followed during pH-regulated dimorphism of Candida albicans . It was observed that transport activities of various amino acids differed with the morphological phenotype. The uptake rates of l-alanine , l -phenylalanine and of l -lysine were lower and those of l -methionine were higher in elongated hypha (germ tube), while the rates of glycine, l -glutamic acid and l -proline were similar in bud and hyphal phenotypes. Minimum threshold of amino acids transport activity is required at the time of phenotypic commitment in a diverging population of Candida albicans .     

Mechanism of the effect of levorin on transport of amino acids inCandida albicans

By use of incubation media of various ionic compositions and pH's, it was possible to reveal that inhibition of the transport of neutral amino acids having long hydrophobic chains (leucine, phenylalanine) inCandida albicans by the polyene antibiotic levorin was mainly due to redistribution—caused by the antibiotic—of monovalent cations in the cells and protoplasts of the yeast. In an acidic medium, levorin was also found to induce a decrease of proton release from the cells, accompanied by some inhibition of leucine transport. Transport of neutral amino acids having short hydrophobic chains (alanine, glycine, proline) was inhibited by this polyene antibiotic, irrespective of the ionic composition or pH of the medium.     

Dependence of inhibition by levorin of amino acid incorporation into protoplasts and subcellular proteins of Candida albicans on the change of endocellular potassium concentration]

Levorin is found to decrease more efficiently potassium concentration in C. albicans protoplasts under their incubation in the presence of sodium than in the medium containing the equivalent amount of potassium. Minimal inhibitory concentration of levorin for resistant C. albicans cells incubated on potassium-depeleted medium was in 4 times lower than for cells incubated in potassium-enriched medium. The decrease of membrane permeability for 14C-amino acids and their incorporation into membrane, ribosomal and soluble proteins under the effect of levorin was more pronounced when protoplasts were cultivated in sodium-containing medium than in potassium-containing one. In both media the inhibition of 14C-amino acid incorporation by levorin into ribosomal and cytosol proteins was more efficient than into membrane proteins, but these differences were less pronounced in case of potassium-containing medium.     

Effect of amigluracil and levorin on membrane permeability and protein synthesis in Candida albicans protoplasts]

It was shown that amigluracyl, a water soluble derivative of methacyl which decreased the nephrotoxic effect of polyens activated the membrane permeability in Candida albicans for a mixture of 14C-amino acids but had no significant effect on protein synthesis in this microorganism. The level of inhibition of the membrane permeability in C. albicans for the amino acids and protein synthesis in the fungus by levorin did not practically depend on the presence of amigluracyl in the incubation medium. The minimum levorin concentration inhibiting the growth of Candida albicans in the presence or absence of levorin was 0.039 gamma/ml. Therefore, amigluracyl may be used in combination with polyenic antibiotics for the treatment of mycoses.     

Action of polyene antibiotics on ribosome binding with Candida albicans membranes]

The effect of treatment of the protoplasts and cell membranes of C. albicans with polyenic entibiotics on formation of the ribosomal-membrane complex was studied in vitro. It was shown that amphotericin B and nystatin had no effect on this process. Significant suppression of the ribosome binding with the membranes was observed only in vivo in the presence of levorin. The role of the structural changes occurring in the membranes on formation of the polyen-sterol complex, as well as the role of the lipid components of the membrane in binding of the ribosomes is discussed.     

Peptide transport in Candida albicans.

The tripeptide L-methionyl-L-methionyl-L-[METHYL-14C]methionine was taken up into Candida albicans by a saturable system with a pH optimum of 3.5, a temperature optimum of 37 degrees C and an apparent Km of 3.3 x 10(-5) M. Metabolic inhibitors such as sodium azide and dinitrophenol completely prevented uptake. Neither methionine nor dimethionine effectively competed with trimethionine uptake. (Leu)3, Gly-Met-Gly, acetyl-(Met)3, D-Met-L-Met-L-Met and Met-Met-Ile effectively competed with (Met)3 uptake, whereas (Lys)3, L-Met-L-Met-D-Met, D-Met-D-Met-D-Met, (Met)3 methyl ester and (Ala)3 did not. Trimethionine was rapidly hydrolysed by a peptidase after entry into the cell.     

Transport of basic amino acids in Candida albicans

In Candida albicans, ATCC 46977, transport of basic amino acids is mediated by two systems (S1 and S2). Kinetic data and competitive inhibition studies of the different systems showed that transport of L-lysine, L-arginine and L-histidine have distinct specificities. System S1 of L-lysine and L-arginine was highly specific for the respective single basic amino acid. However, S2 of L-lysine and S1 of L-histidine were shown to be specific systems for most of basic amino acids. S2 of L-arginine was different from S2 of L-lysine and S1 of L-histidine. The effect of a thiol reagent, N-ethylmalemide, revealed that S2 of L-lysine and S1 of L-histidine were sensitive to this reagent, while all other systems were insensitive. The transport activity of different systems of L-lysine, L-arginine and L-histidine was followed during the growth of C. albicans. It was observed that different basic amino-acid systems have maximum activity during different stages of C. albicans growth.     

The effect of altered ergosterol content on the transport of various amino acids in Candida albicans.

Candida albicans cells have low levels of ergosterol when grown in ascorbic acid-supplemented media. When cells are grown in hydroquinone-supplemented media, the ergosterol levels became higher as compared to normal cells. The uptake of lysine, glycine, glutamic acid, proline, methionine and serine is reduced in hydroquinone-supplemented cells. In contrast to hydroquinone-supplemented cells, the rate and level of accumulation of these amino acids are higher in ascorbic acid-supplemented cells. Nystatin-resistant isolates of C. albicans with low ergosterol contents also exhibit an increased rate and level of accumulation of these amino acids. The uptake of phenylalanine and leucine remained unaffected by such a change in ergosterol levels brought about by different supplementation of the media. The results demonstrate a correlation between ergosterol levels and amino acids uptake. Contrary to various reports, the rate of K+ efflux does not seem to correlate with the amino acid uptake in C. albicans cells.     

The effect of altered lipid composition on the transport of various amino acids in Candida albicans.

Candida albicans cells grown on alkanes of different chain lengths (C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, and C₁₈) exhibited a low growth rate and gradual increase in the total lipid content with the increase in the length of alkanes. There was a significant change in the phospholipids and sterols content of various alkane-grown cells compared to glucose-grown cells. In glucose-grown cells, the transport of various amino acids, e.g., proline, glutamic acid, lysine, glycine, phenylalanine, serine, methionine, and leucine was found to be energy dependent and against a concentration gradient. In alkane-grown cells, the transport of lysine, proline, serine, and methionine was reduced, however, there was no effect on the uptake of glycine, glutamic acid, phenylalanine, and leucine. The results were interpreted as different carrier(s) responsible for amino acid uptake responsed differently to the change of lipid environment.     

氨基酸对白念珠菌形态学影响的研究

目的初步探讨单个氨基酸对白念珠菌形态学的影响。方法用0．67％的酵母氮源基础培养基和2％葡萄糖配制成SD合成培养基,37％恒温摇床培养,研究单个天然氨基酸对白念珠菌形态学的影响,并分别通过不添加碳源和厌氧条件下培养观察对精氨酸诱导的菌丝的影响。结果在含10mmol／L的L－精氨酸的SD液体培养基中,可见大量的菌丝。在含10mmol／L的L一半胱氨酸、L．苏氨酸、L－缬氨酸和L－色氨酸的sD液体培养基中,可见典型的酵母细胞,未见菌丝。在含10mmol／L的其他单个氨基酸的SD液体培养基中可见混合的酵母和菌丝结构。在不含氨基酸或含各种天然氨基酸的SD固体培养基上,白念珠菌的菌落均光滑。但在含10mmol／L的L-精氨酸固体培养基上,光滑的菌落周围可见小的突起,镜下可见菌丝。无氧条件下,无论有无碳源,含精氨酸的SD培养液中白念珠菌只能形成酵母细胞,生长部分受到抑制。结论精氨酸可以诱导白念珠菌菌丝形成,厌氧条件下精氨酸不能诱导白念珠菌菌丝形成。     

Protein synthesis and amino acid pool during yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans

Protein synthesis at different stages of yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans was evaluated by following incorporation of radioactive amino acids into the acid-insoluble cellular material. In passing from the early germ-tube formation (60-90 min) to the mature hyphal cell (240-270 min) there was a marked decrease in the capacity for protein synthesis. Apparently, this decrease was not due to a decreased amino acid uptake into the soluble cellular pool or to exhaustion of carbon/energy source in the inducing medium with consequent arrest of growth. Protein synthesis, however, did not decay when amino acids at high concentration were added to the medium fostering the yeast-mycelial transition and this effect was potentiated by glucose. Analysis of the intracellular amino acid pool showed that both germ-tubes and hyphal cells were relatively depleted of several amino acids as compared to the yeast-form cells, whereas in the hyphae there was a higher concentration of glutamic acid/glutamine, the latter being the predominant component. These modulations in amino acid pool composition were not seen when yeasts were converted to hyphae in an amino acid-rich induction medium. This study emphasizes that yeast-form cells of C. albicans may efficiently convert to the mycelial form even under a progressively lowered rate of protein synthesis, and suggests that initiation of hyphal morphogenesis in the presence of N-acetyl-D-glucosamine is somehow separated from cellular growth.     

Photoinhibition of chlamydosporulation of Candida albicans]

Candida albicans produced chlamydospores after 24 h in the dark at 27 degrees C, but the process was inhibited under adequate irradiation of light. The in vivo absorption spectra showed a main peak at 414 nm, and less important peaks at 430, 446, 477, 519, 549 and 560 nm. No bands were detected beyond 600 nm. A total inhibition of chlamydosporulation occurred at 414 nm (monochromatic light) for an initial energy of 2,000 ergs cm-2 s-1. A 4,000 ergs cm-2 s-1 irradiation energy was necessary to observe a marked inhibition at 460, 500 and 530 nm (les chlamydospores and/or immaturity); this energy must be raised to 300,000 ergs cm-2 s-1 to observe a similar effect at 575 and 630 nm. Biological activity spectra were in full concordance with absorption spectra at 414 nm; no interpretation of absorption band at 460 nm is given, but total or partial inhibition could be explained by modulation of protoporphyrin activity.     

An inducible proline transport system in Candida albicans.

1. When Candida albicans cells were preincubated with proline or grown in the presence of proline as the sole nitrogen source they exhibited a rapid increase in the influx of proline (the inducible transport system). 2. The induction appeared to be specific for proline and also demonstrated in other Candida species. 3. Both the inducible and constitutive proline uptake systems exhibited similar characteristic features. 4. The nature of the inducer for proline uptake in C. albicans appeared to be free proline. 5. The development of the inducible proline transport system was dependent on concomitant synthesis of RNA and protein and the induction was not affected by glucose or any other carbon sources used.