Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-gamma. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.     

Changes in external trehalase activity during human serum-induced dimorphic transition in Candida albicans

Yeast-like cells (blastoconidia) of Candida albicans growing exponentially on a glucose-containing medium (YPD) exhibited low external trehalase activity and stored a negligible amount of intracellular trehalose. The addition of human serum at 37 degrees C to exponential cultures promoted a high degree of germ-tube formation with no significant changes in trehalase activity or trehalose content. In contrast, stationary cells accumulated a large amount of trehalose, while external trehalase remained at a low and practically constant level. However, resting cultures were unable to enter the dimorphic program, except when they were supplemented with fresh YPD and serum together. Only under these conditions was trehalase activated and trehalose hydrolyzed. Specific inhibition of external trehalase by validoxylamine A caused a certain delay in, and a lower level of, germ-tube formation, but did not totally block the dimorphic conversion. These results suggest that external trehalase is not involved in the serum-induced morphological transition in C. albicans.     

Inductions of germ tube and hyphal formations are controlled by mRNA synthesis inhibitor in Candida albicans.

Candida albicans is a pathogenic dimorphic fungus. When yeast cells were pre-incubated in YPD medium at 25degreesC and released into HFM7 medium containing 4% serum at 37degreesC, germ tubes emerged within 0.5 h. To determine whether mRNA or protein synthesis was necessary for germ tube formation, we examined the effects of mRNA and protein syntheses inhibitors on this formation. In the presence of cycloheximide, cells were unbudded and no germ tube was observed. However, in the presence of actinomycin D, germ tube formation was observed while budding growth and true hyphae elongation were blocked. Next, we measured mRNA or protein accumulation during induction of germ tube formation in the presence of the inhibitors. In the presence of cycloheximide, protein was not synthesized, while in the presence of actinomycin D, mRNA synthesis decreased to 6.3% and protein synthesis to 37.7%. The condition we found which allows only germination but not budding or filamentation might be convenient to use in screening genes involved in the initial stage of morphological change in C. albicans.     

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

Combined inactivation of the Candida albicans GPR1 and TPS2 genes results in avirulence in a mouse model for systemic infection

Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.     

Effect of pH, carbon source and K+ on the Na+-inhibited germ tube formation of Candida albicans.

The effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of the pathogenic fungus Candida albicans was examined in the arginine-phosphate modified (APM) medium. All C. albicans cells formed germ tubes in APM medium at pH 5.0-9.0. Na+ inhibited germ tube formation in a concentration dependent manner ranging from 0.2 to 1.0 M, and was further influenced by the pH of the medium. The inhibitory effect of Na+ was lowest at pH 8.0, and germ tube formation ceased at 1.0 M Na+ for any pH (4.0-9.0). At pH > or = 6.0, non-germ tube-forming cells did not show yeast growth; whereas at pH < or = 5.0, Na+ inhibited only germ tube formation but did not inhibit yeast growth. The inhibitory effect of Na+ was stronger in glucose medium than in galactose medium as carbon source. K+, at 0-0.8 M, had almost no effect on germ tube formation. However, in the presence of Na+, a very low concentration of K+ (0.5 mM) was able to release the cells from Na+ arrest and produced an increase in the rate as well as the percentage of germ tube formation. Intracellular Na+/K+ ratios increased with the increase in extracellular Na+ concentration, whereas the ratios decreased and remained within nontoxic levels when the extracellular K+ concentration was increased.     

Germination of Candida albicans induced by proline.

Blastospores of Candida albicans germinated in proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glatamate, asparate, and pyruvate families also fostered germinaton but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of L-proline inhibited proline-stimualted germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts.     

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G₁ arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the K_m values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.     

Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.     

Disruption of the Candida albicans TPS2 gene encoding trehalose-6-phosphate phosphatase decreases infectivity without affecting hypha formation

Deletion of trehalose-6-phosphate phosphatase, encoded by TPS2, in Saccharomyces cerevisiae results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated whether Tre6P phosphatase could be a useful target for antifungals in Candida albicans. We have cloned the C. albicans TPS2 (CaTPS2) gene and constructed heterozygous and homozygous deletion strains. As in S. cerevisiae, complete inactivation of Tre6P phosphatase in C. albicans results in 50-fold hyperaccumulation of Tre6P, thermosensitivity, and rapid death of the cells after a few hours at 44 degrees C. As opposed to inactivation of Tre6P synthase by deletion of CaTPS1, deletion of CaTPS2 does not affect hypha formation on a solid glucose-containing medium. In spite of this, virulence of the homozygous deletion mutant is strongly reduced in a mouse model of systemic infection. The pathogenicity of the heterozygous deletion mutant is similar to that of the wild-type strain. CaTPS2 is a new example of a gene not required for growth under standard conditions but required for pathogenicity in a host. Our results suggest that Tre6P phosphatase may serve as a potential target for antifungal drugs. Neither Tre6P phosphatase nor its substrate is present in mammals, and assay of the enzymes is simple and easily automated for high-throughput screening.     

Biofilm formation by Candida species on the surface of catheter materials in vitro.

A model system for studying Candida biofilms growing on the surface of small discs of catheter material is described. Biofilm formation was determined quantitatively by a colorimetric assay involving reduction of a tetrazolium salt or by [3H]leucine incorporation; both methods gave excellent correlation with biofilm dry weight (r = 0.997 and 0.945, respectively). Growth of Candida albicans biofilms in medium containing 500 mM galactose or 50 mM glucose reached a maximum after 48 h and then declined; however, the cell yield was lower in low-glucose medium. Comparison of biofilm formation by 15 different isolates of C. albicans failed to reveal any correlation with pathogenicity within this group, but there was some correlation with pathogenicity when different Candida species were tested. Isolates of C. parapsilosis (Glasgow), C. pseudotropicalis, and C. glabrata all gave significantly less biofilm growth (P < 0.001) than the more pathogenic C. albicans. Evaluation of various catheter materials showed that biofilm formation by C. albicans was slightly increased on latex or silicone elastomer (P < 0.05), compared with polyvinyl chloride, but substantially decreased on polyurethane or 100% silicone (P < 0.001). Scanning electron microscopy demonstrated that after 48 h, C. albicans biofilms consisted of a dense network of yeasts, germ tubes, pseudohyphae, and hyphae; extracellular polymeric material was visible on the surfaces of some of these morphological forms. Our model system is a simple and convenient method for studying Candida biofilms and could be used for testing the efficacy of antifungal agents against biofilm cells.     

抗白念珠菌芽管单克隆抗体的制备及特性鉴定

目的制备抗白念珠菌芽管的单克隆抗体mAb03.2C1-C2,并对其特性进行分析,以探讨其应用于实验室检测的可行性。方法利用杂交瘤技术制备分泌抗白念珠菌芽管胞壁外膜抗原mAb的杂交瘤细胞株,对mAb的Ig亚类进行鉴定。用间接免疫荧光(IIF)法对mAb的抗体活性及特异性进行测定。在白念珠菌芽管形成条件下,加入mAb,观察其对白念珠菌芽管诱导的抑制及白念珠菌对上皮细胞、内皮细胞黏附的抑制。复苏冻存的杂交瘤细胞株,分析其持续分泌mAb的能力。结果获得1株mAb03.2C1-C2,其Ig亚类为IgG1。该mAb仅与白念珠菌芽管特异性结合,并能显著降低白念珠菌芽管的生成率以及在芽管形成条件下对上皮细胞、内皮细胞的黏附,其抑制作用与该mAb的浓度呈正比。结论制备的mAb03.2C1-C2抗体效价高,在体外能抑制白念珠菌芽管的形成,降低白念珠菌的侵袭力。而且其对白念珠菌芽管具有高度的特异性,可用于白念珠菌和都柏林念珠菌的快速鉴别。     

Deletion of the CaBIG1 gene reduces beta-1,6-glucan synthesis, filamentation, adhesion, and virulence in Candida albicans

The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in beta-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of beta-1,6-glucan in the cell wall was decreased in the Cabig1Delta strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Delta mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Delta mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Delta mutant.     

Germ-tube formation by oral strains of Candida tropicalis.

Candida species isolated from the mouths of healthy children and of patients with denture stomatitis included strains of Candida tropicalis that formed germ tubes when incubated in serum. Twenty-six germ-tube-forming strains of C. albicans and of C. tropicalis were subcultured weekly for 9 wk on blood agar and on Sabouraud's agar and the ability of each subculture to form germ tubes was measured. All the strains of C. albicans formed almost as many germ tubes after nine weekly subcultures as they did when first isolated. By contrast, although all 26 strains of C. tropicalis formed germ tubes when first isolated, all had lost the ability to do so after six serial weekly subcultures. Germ-tube formation should not be the sole criterion for the identification of oral C. albicans strains.     

In vitro effect of fibrinogen on Candida albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.     

Binding of resting platelets to Candida albicans germ tubes.

The binding of resting platelets to Candida albicans germ tubes was studied by means of an affinity column in which germ tubes were physically immobilized. Adhesion of platelets to the column was dependent on both the germ tube concentration and the number of platelets applied. It was found that the interaction of C. albicans germ tubes with platelets is specific and should be mediated by a fungal protein receptor. The results obtained by scanning electron microscopy confirmed that resting platelets can fix directly onto germ tubes. In addition, this study showed that attachment of platelets onto C. albicans is associated with morphological changes. Platelets lost their discoid shape, became globular, generated spikes or pseudopods, and then flattened on the yeast cells.     

Role of yeast cell growth temperature on Candida albicans virulence in mice.

Previous studies have suggested that yeast cell growth temperature may influence the relative virulence of the opportunistic dimorphic fungus Candida albicans. To test this possibility, mice were challenged with C. albicans yeast cells which were grown at either room temperature or 37 degrees C, and their survival was monitored daily. Mice which received room temperature-grown cells died faster. The interaction of glycogen-elicited polymorphonucleated neutrophils (PMNs) with C. albicans yeast cells grown at the two temperatures was examined, because PMNs have been shown to have a critical role in preventing development of candidiasis in normal individuals. In the absence of serum (i.e., nonopsonic conditions), more PMNs conjugated and engulfed C. albicans cells grown at room temperature than those grown at 37 degrees C. However, PMNs were less able to kill cells grown at room temperature than cells grown at 37 degrees C. Cells grown at room temperature also produced abundant germ tubes after engulfment and were thus more likely to escape killing by phagocytes. These results suggest that cells grown at room temperature are more virulent because they are less likely to be killed by phagocytes and are more likely to disseminate. The possibility that expression of cell surface hydrophobicity is involved in these events is discussed.     

Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans.

The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined. Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration [MIC]) to 0.004% (1/128 MIC). GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls. At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01). A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5%. Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum. Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01). GTF by C. albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO. This may have therapeutic implications.     

Inhibition of hyphal growth of Candida albicans by activated lansoprazole, a novel benzimidazole proton pump inhibitor.

The effect of activated lansoprazole (AG 2000), a novel benzimidazole proton pump inhibitor, against hypha formation of Candida albicans was examined in hypha-forming medium pH 7 (HFM7) after 20 h. AG 2000, at 50-800 microM, did not inhibit germ tube formation. However, it inhibited elongation of germ tubes to form hyphae and favored conversion of germ tubes to resume yeast growth at concentrations of > or =200 microM. Pre-treatment of AG 2000 with a sulfhydryl reagent (1:1), such as 2-mercaptoethanol. blocked the inhibitory property of AG 2000 on hypha formation.