Trehalose hydrolysis is not required for human serum-induced dimorphic transition in Candida albicans: evidence from a tps1/tps1 mutant deficient in trehalose synthesis

Exponential yeast-like cells of a Candida albicans wild-type strain exhibited strong capacity for germ tube formation in a glucose-containing medium (YPD) after induction with human serum at 37 degrees C, whereas the isogenic double disruptant tps1/tps1 mutant, which is deficient in trehalose synthesis, failed to produce germ tubes. In a medium without glucose (YP), the morphological transition fraction was roughly equivalent in both strains. Substitution of glucose by galactose or glycerol increased the number of wild-type proliferating cells able to enter the dimorphic program with no noticeable change in their trehalose content, while stationary cells, which accumulate a large amount of trehalose, did not form germ tubes. When fresh medium was added, a high proportion of these resting cells recovered their ability to carry out dimorphic transition. The tps1/tps1 mutant followed the same pattern of hyphae formation, despite the fact that it was unable to accumulate trehalose either during dimorphism induction or after several stress challenges. Furthermore, trehalose-6-phosphate synthase activity was barely detectable in the mutant. These results strongly suggest that serum-induced dimorphic transition does not require trehalose mobilization; they also support the idea that TPS1 is the only activity involved in trehalose biosynthesis 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 effect of oral bacteria on Candida albicans germ-tube formation

A total of eight bacterial isolates belonging to six species, and a select group of 12 oral Candida albicans isolates, were used to study the effect of bacteria on germ-tube formation. Briefly, each bacterial suspension (10(5-6) cells/ml) was mixed with a C. albicans suspension (10(7) cells/ml) and incubated at 37 degrees C for 90 min with bovine serum, and the percentage germ-tube-positive Candida cells was quantified using a haemocytometer, under light microscopy. In general, out of eight bacteria, Streptococcus sanguis SK21A, Streptococcus salivarius SK56, Escherichia coli ATCC 25922, and S. salivarius OBU3 suppressed germ-tube formation to varying degrees, with different C albicans isolates. Porphyromonas gingivalis Pg 50, Lactobacillus casei ATCC 7469 and Prevotella intermedia OBU4 elicited significant enhancement of germ-tube formation, whereas S. sanguis OBU 2 had no effect. E. coli ATCC 25922 was the only organism to show statistically significant suppression of germ-tube formation (p=0.0312). A significant increase in the germ tube production of C. albicans isolated from HIV-infected compared with HIV-free individuals was also noted. The current results tend to suggest that commensal and transient oral bacterial populations may selectively influence the differential expression of germ-tube-forming ability of C. albicans isolates.     

The Candida albicans PMM1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation

We have constructed an ordered-array genomic DNA library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, PMM1, and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans PMM1 gene, in single copy, to transform temperature-sensitive S. cerevisiae sec53-6 mutant cells, which are defective in PMM activity at 37°C, to growth at 37°C. The C. albicans PMM1 gene is thus the structural and functional equivalent of the SEC53 gene.     

New insights into a mutant of Saccharomyces cerevisiae having impaired sugar uptake and metabolism

Summary A regulatory mutant of Saccharomyces carlsbergensis unable to inactivate fructose-1,6-bisphosphatase was shown to have a normal response to the glucose signal as measured by trehalase and 6-phosphofructose-2-kinase activities. The level of fructose 2,6-bisphosphate, however, was found to be 4- to 5-fold lower than that found in the wild-type strain. A rapid and drastic depletion in ATP was confirmed. A partial revertant for growth on glucose which retained its inability to grow on fructose did not show normal levels of fructose 2,6-bisphosphate; however, ATP levels were restored. Trehalose-6-phosphate synthase activity was found in its phosphorylated, less active form. A high degree of phosphorylation at the level of enzymatic activity and of the sugar phosphorylating systems might be responsible for the impairment of control between hexose transport and metabolism, as well as for the absence of trehalose accumulation.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - PFK1 6-phosphofructose-l-kinase - FBPasel fructose-1,6-bisphosphatase - PFK2 6-phosphofructose-2-kinase - PEP phosphoenolpyruvate     

Adaptive response of microglial cells to in vitro infection by Candida albicans isolates with different genomic backgrounds

It has been recently demonstrated that Candida albicans isolates with distinct genomic backgrounds (namely, b and c genotypes) express different susceptibility to antifungal activity by human monocytes in vitro. We show here that, although comparable in their ability to undergo dimorphic transition and in susceptibility to phagocytosis by microglial cells, the b and c isolates show striking differences in terms of intracellular survival. Only the c genotype resists indeed to intracellular killing and eventually replicates inside microglial cells, that in turn respond to fungal infection, preferentially towards the c genotype, with nuclear factor-kappaB (NF-kappaB) activation and increased Mip1alpha production. These data indicate that C. albicans-microglial cell interaction is strictly dependent upon fungal genotype, strengthening the potential significance of genotyping as prognostic parameter in clinical infections by C. albicans.     

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A pattern of active accumulation of trehalose during growth on glucose medium, TAC(+) phenotype, is controlled by a polymeric series of maltose fermentation (MAL) genes. An essential requirement for expression of the TAC(+) phenotype is that the MAL gene be in the constitutive state, MAL ^c. Mutation of a constitutive MAL allele to a maltose- inducible or nonfermenting (mal) state, alters the pattern of trehalose metabolism so that little or no trehalose accumulation occurs during growth on glucose medium. The TAC(+) phenotype is obtained in MAL ^c strains whether or not -glucosidase formation is sensitive or resistant to carbon catabolite repression. However, trehalose accumulation is sensitive to glucose levels even in MAL ^c strains in which -glucosidase formation is insensitive to catabolite repression. The effects of constitutive MAL genes on trehalose accumulation cannot be accounted for by an increase in trehalose-6 phosphate synthase or a decrease in trehalase as determined in vitro. A mechanism is proposed in which the gene-product of a MAL gene serves as a common positive regulator for expression of four genes coding respectively for maltose permease, maltase, -methylglucosidase and a component of the trehalose accumulation system.Paper I appeared in Cell. and Molec. Biology 25: 345–354, 1979     

In vitro study of contact-mediated killing of Candida albicans hyphae by activated murine peritoneal macrophages in a serum-free medium.

Activated peritoneal macrophages obtained from Listeria-immune mice were demonstrated to kill nonphagocytosable Candida albicans hyphae by contact-mediated mechanisms in a serum-free synthetic medium. The actual killing of hyphae was confirmed by a microculture technique utilizing the dimorphic nature of the fungus. The most efficient candidacidal activity was demonstrated by the macrophages obtained from mice first immunized with live Listeria monocytogenes and then elicited with heat-killed L. monocytogenes cells. Resident macrophages from control mice showed only low candidacidal activity against C. albicans hyphae and yeast cells. Direct physical contact appeared to be required for macrophages to efficiently kill oversized C. albicans hyphae. Efficient in vitro killing of hyphae also required relatively high effector/target cell ratios (50 or higher). The contact-mediated candidacidal activity of activated macrophages was not significantly abrogated by oxygen-radical scavengers, suggesting the involvement of oxygen-independent mechanisms. These results suggest that the enhanced nonspecific immunity to candidiasis seen in Listeria-immune hosts can be attributed, at least in part, to activated fungicidal macrophages. The ability of macrophages to detect and destroy both yeast and hyphal C. albicans cells is clearly an important element of the host defense against candidiasis.     

Synthesis, accumulation and hydrolysis of trehalose during growth of peanut rhizobia in hyperosmotic media

We examined and compared the activities of synthetic and hydrolytic enzymes involved in trehalose metabolism, in three peanut rhizobia strains grown in control, hypersaline, and non-ionic hyperosmotic media. Results indicated that the effects of hyperosmolarity on the synthesis and the degradation of the disaccharide were diverse. In the salt-tolerant slow-growing strain Bradyrhizobium sp. ATCC 10317, we observed increased synthesis and accumulation of trehalose under hyperosmolarity imposed by either NaCl or PEG-8000. In the other two peanut rhizobia strains, the disaccharide level did not change under hypersalinity. In the salt-sensitive slow-growing strain Bradyrhizobium sp. USDA 3187, intracellular trehalose diminished in late stationary phase-cells grown with PEG, this reduction was accompanied by both an increased activity of synthetic enzymes and a decreased activity of trehalase. In the salt-tolerant fast-growing strain Rhizobium sp. TAL 1000, we also observed a reduction of intracellular trehalose under PEG-mediated growth, this decrease was early and transiently accompanied by an enhancement of trehalase activity, afterwards, the activity of synthetic enzymes augmented.     

Influence of parenterally injected trehalose in mammals having trehalase activity at different sites]

When trehalose is injected via parenteral pathway into animals lacking kidney trehalase (rat), more than 75 per cent of this disaccharide is eliminated in urine. When the injected animals possess an active kidney trehalase (guinea-pig, rabbit), there is only a low urinary trehalose excretion. Moreover, in rabbit, a marked hyperglycaemia is observed which is due to the rapid hydrolysis of trehalose by kidney trehalase.     

Formation of colonies of clonogenic stromal precursors in bone marrow and splenic cell cultures in the presence of streptococcal antigens in the culture medium

The presence of streptococcal M protein and A polysaccharide in culture medium is shown to have an inhibitory effect on the growth of clonogenic stromal precursors in cultures of healthy murine bone marrow and of healthy guinea pig bone marrow and spleen. The efficacy of colony formation dropped 1.5- to 2-fold in the presence of antigens in a concentration of 25 μg/ml in the medium. The inhibitory effect was absent if antigens were added to adhesive cell cultures. The addition of antigens to cultures originating from animals immunized with streptococcus resulted in inhibition of the efficacy of colony formation in complete cultures and in cultures of adhesive cells. The presence of streptococcal antigens in guinea pig stromal fibroblast cultures of different strains did not affect their growth or colony formation. These data indicate that the effects of streptococcal antigens appear to be aimed at the stromal cells not directly, but rather via another cellular category in the bone marrow and splenic cell cultures, probably lymphocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 11, pp. 489–492, November, 1994     

白念珠菌的菌体密度与生物被膜形成及tyrosol分泌

目的 观察不同接种浓度条件下白念珠菌生被物膜的形成,研究菌体密度在白念珠菌生物被膜形成及密度感应分子tyrosol产生中的作用.方法 白念珠菌标准株SC5314和临床株通过YPD增菌,细胞计数和梯度稀释将白念珠菌细胞悬液调配至5×10~6个/ml、5×10~5个/ml、5×10~4个/ml、5 ×10~3个/ml不同浓度,随后将不同浓度的白念珠菌接种到培养皿中,形成自念珠菌生物被膜.1.5、3、4.5、24、36 h高效液相色谱检测白念珠菌tyrosol产生情况,在同样的时间点对生物被膜取样进行扫描电镜观察.结果 菌体密度对白念珠菌生物被膜tyrosol产生存在作用;接种浓度低的白念珠菌分泌tyrosol少,接种浓度高的白念珠菌分泌tyrosol较多.生物被膜形成24 h产生tyrosol最多,随后减低.菌体密度影响白念珠菌早期生物被膜的形成;扫描电镜观察到接种浓度低的白念珠菌细胞出芽少,分裂繁殖少;而接种浓度高的白念珠菌出芽较多,分裂繁殖更多;随着时间的延长,接种浓度高的情况下更早形成成熟生物被膜.结论 菌体密度与白念珠菌生物被膜形成及tyrosol的产生之间存在相关性.     

Further evidence for the alternative pathway of trehalose synthesis linked to maltose utilization in Saccharomyces

Summary Yeast strains bearing a deficiency in trehalose-6-phosphate synthase activity are unable to accumulate trehalose on any carbon source unless they contain one of the MAL genes. If the gene is inducible then synthesis of trehalose occurs specifically during growth on maltose when the MAL gene is constitutive then trehalose accumulation can also be seen when cells are grown on glucose. Different systems for trehalose synthesis were suggested: one of them would require the UDPG-linked trehalose synthase whereas the second would utilize an alternative pathway. We proposed a mechanism by which the gene-product of a MAL gene would serve as a common positive regulator for the expression of the genes coding for maltose permease, -glucosidase and some component of the trehalose accumulation system. In order to elucidate this novel pathway a strain lacking UDPG-linked trehalose synthase activity and harboring a defect in maltose uptake was constructed. Excessive maltose uptake resulted in accumulation of intracellular maltose, and twice as much trehalose as in a control strain. Partial inhibition of hexokinase by xylose affected the ratio between internal maltose and trehalose and significantly reduced glycogen synthesis. Sodium fluoride also blocked glycogen synthesis but allowed for trehalose accumulation. Moreover, a mutant which lacks hexokinase I and II was unable to accumulate trehalose when grown on glucose in spite of the presence of a constitutive MAL2 gene. These results suggest that trehalose synthesis would require G-6-P formation derived from maltose. Such a deviation would allow for slowing down the glycolytic flux which, in turn, would favour efficient maltose utilization. Therefore, trehalose synthesis during growth in media containing glucose serves as an additional parameter for assessing constitutivity of MAL genes.     

Partially purified antibodies used in a solid-phase radioimmunoassay for detecting candidal antigenemia.

The development of a solid-phase radioimmunoassay procedure for the detection of Candida albicans antigens in serum of mice is described. Antibodies against C. albicans that were used in the radioimmunoassay procedure were partially purified from immune serum by a C. albicans antigen-coupled affinity column. Elution of anti-C. albicans antibodies from the column was by glucose and mannose; 4 mg of protein was recovered per ml, which contained 50% of the candidal agglutinin activity of immune serum. Also, 81% of the protein (partially purified antibody) recovered was adsorbed by whole C. albicans cells. Anti-C. albicans antibodies were either coupled to Sepharose 4B for use as the solid phase to bind candidal antigen in serum of infected animals, or radioiodinated (125I) for use as a tracer molecule to bind to the candidal antigen solid-phase complex. Although control experiments indicated that at least 100 ng of candidal antigen should be present in a serum specimen for a positive radioimmunoassay test, candidal antigenemia was detected in 70.4% of infected mice even in cases where blood cultures for C. albicans were negative. With further refinement and adaptability to human serum, the radioimmunoassay test may become a helpful tool for use in the diagnosis of systemic candidiasis.     

Lack of correlation between trehalase activation and trehalose-6 phosphate synthase deactivation in cAMP-altered mutants of Saccharomyces cerevisiae

The rise in cAMP level that follows the addition of glucose or 2,4-dinitrophenol (DNP) to stationaryphase cells of Saccharomyces cerevisiae was accompanied by a marked activation of trehalase (3-fold increase) and a concomitant deactivation of trehalose-6 phosphate synthase (50% of the basal levels). In glucose-grown exponential cells, which are deficient in glucose-induced cAMP signalling, the addition of glucose also prompted a decrease in trehalose-6 phosphate synthase, but had no effect on trehalase activity. Mutants defective in the RAS-adenylate cyclase pathway (ras1 ras2 bcy1 strain), as well as mutants containing greatly reduced protein kinase activity either cAMP-dependent (tpk ^w1 BCY1 strains) or cAMP-independent (tpk1 ^w1 bcy1 strains), were unable to show glucose- or DNP-induced trehalase activation but still displayed a clear decrease in trehalose-6 phosphate synthase activity upon addition of these compounds. These data suggest that the activity of trehalose-6 phosphate synthase, as opposed to that of trehalase, is not controlled by the cAMP signalling pathway in vivo. Trehalose-6 phosphate synthase was competitively inhibited by glucose (Ki=15 mM) and resulted unaffected by ATP in assays performed in vitro.     

Identification of Candida glabrata by a 30-second trehalase test

Rapid (30-s) trehalase tests done with material from colonies of 482 yeasts suspended in a drop of trehalose solution on a commercially supplied glucose test strip were positive for 225 (99.1%) of 227 Candida glabrata isolates grown on either of two differential media, Candida ID medium or CandiSelect medium. The test was positive for only 3 (1.2%) and 12 (4.7%) of 255 isolates of other medically important yeast species grown on the same two media, respectively. A rapid maltase test done with a subset of 255 yeast isolates was negative for all but 1 of 64 trehalase-positive C. glabrata isolates, raising the specificity of the rapid testing for C. glabrata to 98.4 to 100%, depending on the isolation medium used. Rapid trehalase and maltase tests done independently in two laboratories with 217 yeast isolates showed sensitivities of 96.0 to 98.0% and specificities of 98.2 to 99.4% for identification of C. glabrata from colonies grown on Candida ID medium. The specificity was much lower because of frequent false-positive trehalose test results when the source of colonies was Sabouraud agar formulated with 4% glucose. We conclude that direct recognition of C. albicans as blue colonies on Candida ID isolation medium coupled with the performance of the 30-s trehalase and maltase tests for C. glabrata among the white colonies on this medium will allow the rapid presumptive identification of the two yeast species most commonly encountered in clinical samples.     

Listeria monocytogenes infection of Caco-2 cells: role of growth temperature

The aim of the present study was to evaluate the role of temperature in the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular food-borne pathogen. The capacity of bacteria grown at 37, 25 and 4°C to develop haemolytic activity, to enter the Caco-2 enterocyte-like cell line and to multiply intracellularly was investigated. We demonstrated that L. monocytogenes penetration was not significantly influenced by the growth temperature of cultures and that bacteria grown at low temperature were capable of synthesizing internalin and, during the infection process, of restoring the haemolytic phenotype which is normally lacking in the extracellular environment at 4 and 25°C.It can be concluded that L. monocytogenes, frequently present in numerous environmental sources and also in refrigerated food products, produces at low temperature, the virulence factors necessary to invade intestinal cells.     

Biochemical examination of sera during systemic Candida infection in mice.

Candida pathogenesis was examined by intravenous challenge of mice with either C. albicans or C. guilliermondii. Animals were moribund 12 h postchallenge with C. albicans and were found to have the greatest number of organisms in the heart and kidney, severe interstitial myocarditis, and elevated serum levels of blood urea nitrogen, creatine phosphokinase, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, and lactic dehydrogenase. In contrast, challenge with C. guilliermondii resulted in a significantly lower renal census, no myocarditis, and no significant change in the concentration of these serum constituents. Challenge with nonviable C. albicans did not produce the effects observed with viable organisms. Moreover, challenge with filamentous C. albicans resulted in biochemical alterations of lower magnitude and in lower mortality rates. These results indicated that altered serum biochemistries were correlated with the histopathology of fatal Candida infection and that there were distinct differences with C. guilliermondii and the dimorphic phases of C. albicans.