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.     

Isolation, purification and kinetic characterization of plasma membrane H(+)-ATPase of Candida albicans.

The plasma membrane ATPase of Candida albicans was solubilized by Tween 40 and purified to homogeneity on glycerol step gradient. The purified protein appeared as a single band of 100 +/- 4 KDa, represented greater than 98% of the total pure protein on densitometer scan. The purified PM-ATPase which was very specific to MgATP, had Km of about 0.77 mM and a sharp pH optimum at 6.6. Orthovanadate was able to inhibit the enzyme in a non-competitive manner, however, at higher concentrations the nature of inhibition changed to uncompetitive type. Based on molecular size, immuno cross-reactivity and sensitivity to different inhibitors, PM-ATPase of C. albicans appears to be similar to other ion pumps.     

Levels of plasma membrane H+-ATPase do not change during growth and morphogenesis of Candida albicans

Abstract A transient rise in the PM-ATPase activity was observed at the time of commitment of Candida albicans cells to either bud or hyphal formation. However, the changes in PM-ATPase activity did not correlate with the level of enzyme protein detected by ELISA. It was found to be fairly constant during differentiation, implying that there was no de novo synthesis of the protein. Post-translational modification(s) of enzyme protein is suggested to account for variation in PM-ATPase activity during morphogenesis.     

Identification of salivary components that induce transition of hyphae to yeast in Candida albicans

Candida albicans , the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphae and hyphae filaments. The hyphae form is considered the most invasive form of the fungus. The purpose of this study is to investigate the effect of saliva on hyphae growth of C. albicans. Candida albicans hyphae were inoculated in Roswell Park Memorial Institute medium with whole saliva, parotid saliva or buffer mimicking the saliva ion composition, and cultured for 18 h at 37 °C under aerobic conditions with 5% CO₂. Whole saliva and parotid saliva induced transition to yeast growth, whereas the culture with buffer remained in the hyphae form. Parotid saliva was fractionated on a reverse-phase C8 column and each fraction was tested for inducing transition to yeast growth. By immunoblotting, the salivary component in the active fraction was identified as statherin, a phosphoprotein of 43 amino acids that has been implicated in remineralization of the teeth. Synthetically made statherin induced transition of hyphae to yeast. By deletion of five amino acids at the negatively charged N-terminal site (DpSpSEE), yeast-inducing activity and binding to C. albicans were increased. In conclusion, statherin induces transition to yeast of C. albicans hyphae and may thus contribute to the oral defense against candidiasis.     

B7/CD28-dependent CD4+CD25+ regulatory T cells are essential components of the memory-protective immunity to Candida albicans

Protective immunity to the fungus Candida albicans is mediated by Ag-specific Th1 cells. Paradoxically, some Th2 cytokines are required for the maintenance of Th1-mediated immune resistance to the fungus. Therefore, in addition to the Th1/Th2 balance, other mechanisms seem to be involved in the regulation of Th1 immunity to the fungus. Here we show that CD4(+)CD25(+) T cells, negatively regulating antifungal Th1 reactivity, are generated in mice with candidiasis. CD4(+)CD25(+) T cells were not generated in B7-2- or CD28-deficient mice or in condition of IL-10 signaling deficiency. Accordingly, although capable of efficiently restricting the fungal growth, these mice experienced inflammatory pathology and were incapable of resistance to reinfection. CD4(+)CD25(+) T cells poorly proliferated in vitro; were highly enriched for cells producing IL-4, IL-10, and TGF-beta; and required IL-10-producing, Candida hypha-activated dendritic cells for generation. Adoptive transfer of CD4(+)CD25(+) T cells or IL-10-producing dendritic cells restored resistance to reinfection and decreased inflammation in B7-2-deficient mice. These results show that oral tolerance induced by Candida hyphae is required for the occurrence of long-lasting protective immunity after yeast priming. The implication is that preventing reactivation rather than favoring sterilizing immunity to ubiquitous fungal pathogens may represent the ultimate expectation of vaccine-based strategies.     

三颗针对白假丝酵母的抑制作用

目的研究三颗针对白假丝酵母的抑制作用。方法采用溴化噻唑蓝四唑法(MTT)和琼脂平板法测定三颗针对白假丝酵母的最低抑菌浓度(MIC)和最低杀菌浓度(MFC);利用倒置荧光显微镜观察三颗针对白假丝酵母菌丝形成的影响;采用MTT法测定三颗针对白假丝酵母菌丝萌发不同时期的抑制率。结果三颗针对白假丝酵母具有较强的抑制作用。其中,采用琼脂平板法测得三颗针抑制该菌的MIC为2mg/mL,MFC为4mg/mL。三颗针能减缓该菌的生长速度或使其停止生长,且浓度越高作用效果越明显。其中4mg/mL的三颗针作用白假丝酵母6h后,能够完全抑制菌丝形成。对于24h后已经萌发为菌丝态的白假丝酵母,三颗针可抑制菌丝的继续生长,与对照组相比,4mg/mL的三颗针对于萌发2h后的白假丝酵母的菌丝抑制率为76.7%(P0.01)。结论三颗针能抑制白假丝酵母菌丝的生长和萌发。     

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.     

pH及氧气对白念珠菌菌丝形成的影响

目的探讨pH值和氧气对白念珠菌菌丝形成的影响。方法通过调节Muller—Hinton液体培养基的pH值和去除培养基中的氧气来观察白念珠菌的生长曲线、倍增时间和菌丝形成率的变化。结果在无氧气的液体培养基中,白念珠菌生长缓慢,不能产生菌丝结构,只有酵母细胞形成。生长曲线的延缓期内各组没有明显差异,而在生长的对数期pH3和pH4的条件下念珠菌生长速度明显慢于pH5、pH6、pH7、pH8和pH9。菌丝形成率在pH3、pH4和pH5条件下〈20％,而在pH6、pH7、pH8和pH9条件下可高达70％。结论厌氧条件抑制白念珠菌的菌丝形成,只形成酵母细胞。白念珠菌在pH3—9的范围内均能生长,偏酸性环境有利于白念珠菌酵母形成,偏碱性的环境有助于菌丝的形成。     

大蒜素对白念珠菌形态转换的影响研究

目的探讨大蒜素对白念珠菌形态转换的影响及其作用机制。方法倒置显微镜观察白念珠菌菌丝形成的体外动力学过程;采用CLSI-M27-A3微量液基稀释法检测大蒜素对白念珠菌的最小抑菌浓度(minimum inhibitory concentration,MIC);倒置显微镜观察不同浓度大蒜素对白念珠菌在Spider液体培养基中菌丝形成的影响;qRT-PCR法检测在不同浓度大蒜素作用下白念珠菌菌丝相关基因HWP1、ALS1、EFG1、PDE2表达水平的变化。结果白念珠菌在Spider液体培养基中6 h时出现较长菌丝,24 h后镜下可见大量念珠菌菌丝包裹酵母细胞,紧密交错;大蒜素对白念珠菌的MIC值为25μg/mL;倒置显微镜观察(25~100)μg/mL浓度的大蒜素能明显抑制Spider液体培养基中白念珠菌菌丝的生长;qRT-PCR结果显示,在(25~100)μg/mL浓度的大蒜素作用下,白念珠菌菌丝相关基因表达下调。结论大蒜素能有效抑制白念珠菌的形态转换,其作用机制可能与调节菌丝形成相关基因的表达水平有关。     

Regulation of MI transport in retinal pigment epithelium by sugars, amiloride, and pH gradients: potential impairment of pump-leak balance in diabetic maculopathy

Impairment of transport and metabolism of retinal pigment epithelium (RPE) has been recognized to play a role in the development of diabetic macular edema. To understand the mechanism(s) of action of high glucose levels in alteration of RPE metabolism, primary cultures of RPE cells were used as an in vitro model of diabetic retinopathy/maculopathy. RPE cells were grown with 5 mM (control) or 40 mM glucose (a monosaccharide that enters the cells), or 40 mM sucrose (a disaccharide that does not enter the cells), and the extent of Na(+)-dependent active transport of an osmolyte ([3H]-myo-inositol, MI, 10 microM) into cells was determined. While 40 mM glucose down-regulated 3H-MI transport, 40 mM sucrose stimulated it, compared to 5 mM glucose feeding. Addition of 1 mM amiloride, an inhibitor of Na+/H+ exchanger, in the incubation media, significantly inhibited MI transport. Cells treated with high sucrose or high glucose were more sensitive toward amiloride inhibition, compared to controls. Inhibition of either pump or leak pathway alone was not sufficient to completely inhibit MI transport, but simultaneous inhibition of both pathways, by amiloride and ouabain (1 mM each), strongly inhibited osmolyte accumulation. The strongest inhibition of uptake occurred when 150 mM NaCl in the incubation media was replaced by 150 mM choline-Cl, and the percent inhibition of uptake, with choline-Cl, was highest with sucrose-fed cells, compared to normal or high glucose-fed cells. Imposition of a pH gradient [pHi (6.1) less than pH0 (8.0)] across the cell membrane, a condition that stimulates Na+/H+ exchange activity, also reduced MI accumulation. Cellular water content, measured by the extent of [3H]-3-O-methyl glucose uptake, in the presence of balanced salt solution (BSS), BSS containing half the ionic strength (hypotonic solution), or BSS containing 20 mM K+, for induction of cell swelling, varied when cells were fed with various sugars. Cells fed with high glucose were less sensitive toward media tonicity compared to normal. These results suggested that in cultured RPE cells, changes in Na+/H+ exchanger activity (intracellularly or extracellularly), through its inhibition by amiloride, its activation via intracellular acidification, or perhaps by chronic feeding with high sucrose or high glucose, affected the Na(+)-dependent active accumulation of MI. A metabolic factor involved in the development of diabetic macular edema is perhaps associated with glucose-induced alterations in Na+ fluxes (e.g., changes in Na+/H+ exchanger activity), which can secondarily influence osmolyte accumulation, impairment of pump-leak balance, and/or intracellular pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-gamma-producing CD4(+) donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.     

Some Specific Features of the Respiration of Hydrocarbon-utilizable Candida Yeasts

The respiration of both glucose-grown and hydrocarbon-grown cells of Candida tropicalis pK 233 harvested in the stationary phases was not inhibited by cyanide when glucose was used as oxidation substrate, but the former was rather stimulated in the presence of cyanide. When n-alkanes were used as oxidation substrate, cyanide lowered the respiratory activities of both cells to about 50%. With respect to the susceptibility to cyanide, the younger cells growing on n-alkanes were less sensitive in hydrocarbon oxidizing ability than the older cells, whereas the older cells growing on glucose or n-alkanes were more resistant in glucose oxidizing ability than the younger cells. Acetate was oxidized by both glucose-grown and hydrocarbon-grown cells of the yeast. Laurate was oxidized by hydrocarbon-grown cells, but not by glucose-grown cells. The respiration on laurate was inhibited completely by 3.3 mM of cyanide. In general, hydrocarbon-grown cells of Candida tropicalis pK 233 were more sensitive to various respiratory inhibitors than glucose-grown cells, although the oxidation substrates had a significant effect.

The respiration of both glucose-grown and hydrocarbon-grown cells of C. albicans, C. guilliermondii and C. lipolytica harvested in the stationary phases was also resistant to cyanide when glucose was used as oxidation substrate. But the respiration on n-alkanes of these cells was inhibited significantly by 3.3 mM of cyanide except for C. albicans.     

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.     

Synergistic action of nikkomycin X/Z with azole antifungals on Candida albicans.

Fluconazole, ketoconazole and tioconazole were shown to act synergistically in vitro with the antibiotic nikkomycin X/Z on the pathogenic fungus Candida albicans. The phenomenon was demonstrated using a checkerboard technique and growth inhibition experiments. The azole antifungal agents, even at concentrations not affecting growth, decreased the incorporation of the 14C-label from [14C]glucose into chitin of the candidal cell wall. After 3 h incubation with tioconazole, 1 microgram ml-1, the incorporation of the radiolabelled glucose into chitin of intact cells and regenerating spheroplasts of C. albicans was inhibited by 43% and 30%, respectively. Moreover, the relative chitin content was approximately 45% lower than that of control cells. The chitin content increased after prolonged incubation with azoles, thus confirming the known phenomenon of azole-induced uncoordinated chitin synthesis and deposition. On the other hand, azole derivatives had very little effect on the rate of nikkomycin transport into C. albicans cells. A sequential blockade mechanism of synergism is proposed.     

Pre-steady state kinetic studies on H(+)-ATPase from Candida albicans.

The mechanism of ATP hydrolysis by plasma membrane H(+)-ATPase from Candida albicans has been investigated by following the kinetics of H(+) liberation/absorption and the UV difference spectrum in a stopped flow spectrophotometer. A distinct pre-steady state phase of ATP hydrolysis could be defined. While the rapid mixing of P(i) and ATPase produced no transient pH changes, the mixing of ADP leads to the release of 1 H(+) per molecule of ATPase. Rapid mixing of ATP with ATPase releases about 2 H(+) per molecule of ATPase, of which around 1.3 H(+) are reabsorbed. The magnitudes of both H(+) release and absorption were found to be independent of ATP concentration. The rate of H(+) release (k(f)) shows ATP dependence while the rate of H(+) absorption is independent of ATP concentration. The rate of H(+) liberation with ADP, on a concentration basis, was far less as compared with ATP, indicating a low affinity of the ATPase for ADP. No change in the difference spectrum was observed with ADP. The stoichiometry of ATP binding to PM-ATPase was found to be unity from UV-difference spectrum studies. The k(f) values for H(+) release and for the appearance of a difference spectrum following the addition of ATP were found to be similar beyond a 1:1 ratio of ATP:ATPase. The results obtained lead us to propose a 4-step kinetic scheme for the mechanism of ATP hydrolysis.     

Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence [corrected

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process. At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics. The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA. At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI. However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of Candida albicans NADH dehydrogenase complex I in filamentation

The gene encoding the 51-kDa subunit of nicotinamide adenine dinucleotide (NADH) dehydrogenase complex I, a principal component of the mitochondrial electron transport chain, was cloned in Candida tropicalis. The homolog in C. albicans, CaNDH51, was identified, and each allele was successively disrupted by PCR-mediated gene disruption. Wild type, heterozygote, reintegrant, and homozygous null mutants grew as blastoconidia in rich medium containing 3% glucose, but the homozygous null mutant failed to grow in ethanol or acetate. When glucose concentration was varied from 1 mM (0.018%) to 200 mM (3.6%) in a basal salts medium, all strains grew equally well at all glucose concentrations; the wild-type strain, the heterozygote, and the reintegrant exhibited abundant germ tubes, pseudohyphae, and hyphae. In contrast, the ndh51/ndh51 strain failed to display any type of filamentous growth, even in glucose concentrations as low as 1 mM. These results suggest a previously unexplored relationship between mitochondrial electron transport and morphogenesis.     

N-cadherin mediates endocytosis of Candida albicans by endothelial cells

Candida albicans is the most common cause of fungal bloodstream infections. To invade the deep tissues, blood-borne organisms must cross the endothelial cell lining of the vasculature. We have found previously that C. albicans hyphae, but not blastospores, invade endothelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the endothelial cell receptor that mediates the endocytosis of C. albicans. We determined that endocytosis of C. albicans was not mediated by bridging molecules in the serum and that it was partially dependent on the presence of extracellular calcium. Using an affinity purification procedure, we discovered that endothelial cell N-cadherin bound to C. albicans hyphae but not blastospores. N-cadherin also co-localized with C. albicans hyphae that were being endocytosed by endothelial cells. Chinese hamster ovary (CHO) cells expressing human N-cadherin endocytosed significantly more C. albicans hyphae than did CHO cells expressing either human VE-cadherin or no human cadherins. The expression of N-cadherin by the CHO cells resulted in enhanced endocytosis of hyphae, but not blastospores, indicating the selectivity of the N-cadherin-mediated endocytosis. Down-regulation of endothelial cell N-cadherin expression with small interfering RNA significantly inhibited the endocytosis of C. albicans hyphae. Therefore, a novel function of N-cadherin is that it serves as an endothelial cell receptor, which mediates the endocytosis of C. albicans.