Germ Tube Formation In Candida Albicans Research Articles

Inhibitory effect of baicalin on germ tube formation and adhesion of Candida albicans

To investigate the effects of baicalin against Candida albicans germ tube formation, and adherence to buccal epitherial and vaginal epitherial cells. Various concentrations of baicalin (100, 50, 10 mg x L(-1)) were incubated with C. albicans suspension, the mixed suspension of C. albicans and human buccal epitherial cells, the mixed suspension of C. albicans and vaginal epitherial cells, respectively. The effects of baicalin on C. albicans germ tube formation, and adherence to buccal epitherial and vaginal epitherial cells were then assessed microscopically. All concentrations of baicalin could inhibit C. albicans germ tube formation, and adherent to buccal epitherial and vaginal epitherial cells,while there was no significant difference between standard and clinical strains. Baicalin could inhibit C. albicans germ tube formation, and adherence to buccal epitherial and vaginal epitherial cells.

A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition

In addition to producing lethal antibiotics, microorganisms may also use a new form of antagonistic mechanism in which signal molecules are exported to influence the gene expression and hence the ecological competence of their competitors. We report here the isolation and characterization of a novel signaling molecule, cis-2-dodecenoic acid (BDSF), from Burkholderia cenocepacia. BDSF is structurally similar to the diffusible signal factor (DSF) that is produced by the RpfF enzyme of Xanthomonas campestris. Deletion analysis demonstrated that Bcam0581, which encodes an RpfF homologue, was essential for BDSF production. The gene is highly conserved and widespread in the Burkholderia cepacia complex. Exogenous addition of BDSF restored the biofilm and extracellular polysaccharide production phenotypes of Xanthomonas campestris pv. campestris DSF-deficient mutants, highlighting its potential role in inter-species signaling. Further analyses showed that Candida albicans germ tube formation was strongly inhibited by either coculture with B. cenocepacia or by exogenous addition of physiological relevant levels of BDSF, whereas deletion of Bcam0581 abrogated the inhibitory ability of the bacterial pathogen. As B. cenocepacia and C. albicans are frequently encountered human pathogens, identification of the BDSF signal and its activity thus provides a new insight into the molecular grounds of their antagonistic interactions whose importance to microbial ecology and pathogenesis is now becoming evident.

Effect of subinhibitory concentration of some established and experimental antifungal compounds on the germ tube formation inCandida albicans

The influence of subinhibitory concentrations of six established and 19 newly synthesized antifungal compounds on the dimorphic transition of three C. albicans strains was evaluated in the filamentation-inducing medium. Amphotericin B was found to produce almost complete inhibition in the germination at a concentration of 1/10 of the corresponding MIC and partial inhibition at a concentration as low as MIC/50. Flucytosine and four azole derivatives were proven ineffective. From the newly synthesized drugs, the incrustoporin derivative LNO6-22, two phenylguanidine derivatives (PG15, PG45), and four thiosalicylanilide derivatives, in particular, showed results comparable to those of amphotericin B, with a high inhibition of germ tube formation at concentrations of MIC/10. In general, concentrations of MIC/50 had no visible effect.

Identification of the dialysable serum inducer of germ-tube formation in Candida albicans.

Yeast cells of Candida albicans are induced by serum at 37 degrees C to produce germ tubes, the first step in a transition from yeast to hyphal growth. Previously, it has been shown that the active component is not serum albumin but is present in the dialysable fraction of serum. In this study, serum induction of germ-tube formation is shown to occur even in the presence of added exogenous nitrogen sources and is therefore not signalled by nitrogen derepression. The active component in serum was purified by ion-exchange, reverse-phase and size-exclusion chromatography from the dialysable fraction of serum and was identified by NMR to be d-glucose. Enzymic destruction of glucose, using glucose oxidase, demonstrated that d-glucose was the only active component in these fractions. Induction of germ-tube formation by d-glucose required a temperature of 37 degrees C and the pH optimum was between pH 7.0 and 8.0. d-Glucose induced germ-tube formation in a panel of clinical isolates of C. albicans. Although d-glucose is the major inducer in serum, a second non-dialysable, trichloroacetic acid precipitable inducer is also present. However, whereas either 1.4 % (v/v) serum or an equivalent concentration of d-glucose induced 50 % germ-tube formation, the non-dialysable component required a 10-fold higher concentration to induce 50 % germ-tube formation. Serum is, therefore, the most effective induction medium for germ-tube formation because it is buffered at about pH 8.5 and contains two distinct inducers (glucose and a non-dialysable component), both active at this pH.

Antimycotic Activity and Phagocytosis Effects of Econazole in Combination with Ibuprofen Isobuthanolammonium Against Vaginal Strains

Vaginal infections caused by Candida spp., other yeasts and Trichomonas vaginalis are problematic mainly due to the various factors involved in development of infection and to the failure of common treatments. In this study we investigated the presence of synergistic activity of econazole and ibuprofen isobuthanolammonium against 310 different vaginal isolates, by using the microdilution broth assay to test in vitro antimicrobial activity and the effect of the two drugs on phagocytosis and intramacrophagic cellular killing of mouse peritoneal macrophages. The effect of sub-inhibitory concentrations of econazole / ibuprofen isobuthanolammonium combination on Candida albicans germ tube formation was also evaluated. The in vitro antifungal activity of econazole was notably improved by addition of ibuprofen isobuthanolammonium. Macrophage killing of C. albicans was significantly increased by the two drugs and also germ-tube formation was significantly affected. We conclude that the addition of ibuprofen isobuthanolammonium to econazole provides better in vitro antifungal activity. However, further studies are needed to elucidate the in vivo action of this formulation.

The Role of Candida albicans Germ Tube Formation in Adherence to Cultured Enterocytes

The Role of Candida albicans Germ Tube Formation in Adherence to Cultured Enterocytes

Prostaglandin E2 enhances and gamma interferon inhibits germ tube formation in Candida albicans

Prostaglandin E2 (PGE2), an immunosuppressive monokine that increases intracellular cyclic AMP (cAMP) levels, stimulated Candida albicans germ tube formation. Dibutyryl cAMP (dB-cAMP) and isoproterenol, other compounds that increase cAMP levels, also stimulated germination. Gamma interferon (IFN-gamma), a product of cellular immune system activation, inhibited Candida germ tube formation, even in the presence of PGE2, dB-cAMP, and isoproterenol. Thus, PGE2 and IFN-gamma as well as having opposing roles in the suppression or activation of cell-mediated immunity, are also antagonists for the yeast-to-hyphal transition of C. albicans.

Proline-induced germ-tube formation in Candida albicans: role of proline uptake and nitrogen metabolism.

Proline-induced germ-tube formation and cell-cell aggregation in four strains of Candida albicans were completely inhibited when the pH of the medium was 5.0 or lower, whereas morphogenesis induced by N-acetylglucosamine (GlcNAc) was unaffected even at pH 4.5. The pH sensitivity of proline-induced germ-tube formation was not caused by a modulation of proline uptake, which was unchanged over the pH range 4.5-6.5. The proline uptake system was specific, constitutive and subject to ammonium repression, and only one permease was detected, with a Km of 179 microM. Cultures deprived of nitrogen in the presence of glucose were derepressed for proline uptake but the yeast-mycelial transition could not be mediated by either proline or GlcNAc. The inhibition of morphogenesis was reversed when the nitrogen starvation was relieved by the addition of ammonium ions, proline, or certain amino acids. These results indicate that the nitrogen status of the cells is critical for the morphogenesis of C. albicans.

The role of glucose in the pH regulation of germ-tube formation in Candida albicans.

It has been reported that Candida albicans can form germ-tubes only in the narrow pH range of 6-8, and that by changing only the pH one can regulate germ-tube formation. We found that the pH minimum for germ-tube formation could be dramatically lowered by eliminating the glucose present in many induction solutions. Lee's medium lacking glucose, ethanol, N-acetyl-D-glucosamine, and proline induced germ-tubes at pH values as low as 3 under most conditions. The presence of as little as 1 mM-glucose in these induction solutions was sufficient to cause the cells to grow either as yeasts with multiple buds or as pseudohyphae when the pH was 3.7. However, when C. albicans was grown in any of the above induction solutions (with the exception of ethanol), containing 200 mM-glucose buffered at pH 5.8, not only were germ-tubes formed, but their rate of formation and length were also increased. Preincubation of the cells in a solution buffered at pH 3.7 and containing 200 mM-glucose, before exposure to induction solutions lacking glucose at pH 3.7 or at pH 5.8, did not inhibit germ-tube formation. Likewise, addition of glucose after 45 min exposure to an induction solution was without effect. Theophylline and dibutyryl cAMP did not counteract the action of glucose. Other sugars which suppressed germ-tube formation at low pH were fructose, galactose, mannose, xylose, gluconic acid and the nonmetabolizable sugar 3-O-methylglucose. These results indicate that pH does not directly regulate dimorphism in C. albicans, and that glucose or its metabolites may play an important role.

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[14C]proline were taken up by 1×106 cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [3H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[14C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.

Antimorphogenic effects of 2-deoxy-D-glucose inCandida albicans

2-Deoxy-d-glucose (dGlc) is able to inhibit both N-acetyl-hexosamine-induced chlamydosporogenesis and N-acetyl-glucosamine- or proline-induced germ-tube formation in Candida albicans. This inhibition is exerted also at dGlc concentrations which do not affect growth in the yeast form and do not reduce either the uptake or the incorporation of N-acetyl-d-glucosamine (GlcNAc) into yeast or hyphal cell material. Inhibition of germtube formation by dGlc does not occur in serum and is fully reversed by glucose. It is suggested that dGlc acts as a potent antimorphogenic effector in C. albicans.

The secretion of N-acetylglucosaminidase during germ-tube formation in Candida albicans.

N-Acetylglucosaminidase was induced by either N-acetylglucosamine or N-acetylmannosamine in several strains of Candida albicans. Enzyme activity was not induced in a N-acetylglucosamine non-utilizing mutant which is unable to express the first three steps in the N-acetylglucosamine catabolic pathway. The enzyme, purified 500-fold, had a specific activity of 36.8 units (mg protein)-1 and catalysed the hydrolysis of p-nitrophenyl-beta-n-acetylglucosamine, N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. No activity was observed toward colloidal chitin, hyaluronic acid or mucin. The cellular distribution of N-acetylglucosaminidase was determined by measuring in situ enzyme activity before and after acid treatment of intact cells. N-Acetylglucosaminidase (80-88% of the total cellular activity) was rapidly secreted to the periplasm when the enzyme was induced either during yeast growth at 28 degrees C or germ-tube formation at 37 degrees C. Export of the enzyme from the periplasm into the medium was fourfold greater during germ-tube formation, and after 6 h incubation the amount of enzyme released into the medium represented 70% of cell-associated enzyme activity.

Magnesium and the regulation of germ-tube formation in Candida albicans.

Candida albicans requires Mg2+ for germ-tube formation. Mg-deficient media, metal ion chelators and the ionophore A23187 inhibited germ-tube formation. Cell Mg content during exponential yeast-phase growth remained constant but increased throughout germ-tube formation. The onset of germ-tube formation coincided with a sharp peak in Mg concentration within the cells. Yeast-phase cells of strain CA2, which did not form germ-tubes, had a lower Mg content and failed to accumulate Mg when incubated under conditions for germ-tube formation. Mg also increased the uptake and incorporation of N-acetylglucosamine. These findings point to a central regulatory role for Mg in C. albicans morphogenesis.

Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans.

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.

Candida albicans germ-tube formation with immobilized GlcNAc

Candida albicans germ-tube formation with immobilized GlcNAc

Enzymes of N-acetylglucosamine metabolism during germ-tube formation in Candida albicans.

The enzymes of N-acetyl-D-glucosamine (GlcNAc) metabolism, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase were found to be inducible in Candida albicans. The pattern of induction for these enzymes was the same under conditions of germ-tube formation (37 degrees C) and where yeast cells metabolized GlcNAc with no change in morphology (28 degrees C); this indicates that these enzymes are not control points in the dimorphic development of C. albicans. During induction there was a 40-and 25-fold increase in specific activity for the deacetylase and the deaminase, respectively, and the maximum specific activity correspond to the time when all the GlcNAc had been metabolized. The presence of lomofungin (an inhibitor of transcription) or trichodermin (an inhibitor of translation) in cell suspensions of C. albicans containing GlcNAc prevented the increase in specific activity of these enzymes. 2-Deoxyglucose inhibited germ-tube formation, partially inhibited the induction of the deacetylase (43%) and the deaminase (60%), but did not affect the growth of C. albicans on either Glc or GlcNAc. GlcN-6-phosphate was a competitive inhibitor of the deacetylase with a Ki of 1.4 mM while the other product of the reaction, acetate, did not inhibit the enzyme. The Km value for GlcN-6-phosphate on GlcN-6-phosphate deaminase was 0.24 mM. Incubation of starved yeast cells with GlcNAc produced a four-fold increase in the specific activity of UDP-GlcNAc-pyrophosphorylase at either 28 degrees C or 37 degrees C.

Changes in lipid composition during starvation and germ-tube formation in Candida albicans

The changes in the lipid components of Candida albicans have been determined during growth, starvation, and germ-tube formation. 14C-Labeled cells were used to determine the extent of synthesis and degradation of the different lipid fractions. On a dry weight basis the percentage of total lipid increased from 18% for blastospores to 22% after starvation but decreased to 11% after 4 h of germination. The major components of the lipid fraction were sterols (36–52%) and phospholipids (28–42%). The free and esterified sterol fractions both increased approximately 45% during starvation. The free sterol content continued to increase over the initial stage of germ-tube formation, then decreased with time. The sterol ester fraction decreased throughout germination to the concentration found in growing blastospores. The changes in specific activity (cpm/μg sterol) of these fractions indicated that sterol esters were precursors for free sterols during germination. The total phospholipid fraction increased during starvation but there was a decrease in both the cellular concentration (60%) and the specific activity during germination. There were only minor changes in the relative concentrations of phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine which indicated coordinate synthesis and degradation of these components. Free fatty acids and triacylglycerides are only minor components of the total lipid pool.

N-Acetyl- d-glucosamine kinase and germ-tube formation in Candida albicans

N-Acetyl- d-glucosamine kinase (GlcNAc kinase, EC 2.7.1.59), the specific enzyme required for the phosphorylation of N-acetyl- d-glucosamine (GlcNAc) to N-acetylglucosamine 6-phosphate, was found to be an inducible enzyme in Candida albicans. The pattern of GlcNAc kinase induction was examined under conditions where germ-tube formation was elicited and where blastospores metabolized GlcNAc with no change in morphology. There was no change in either the time course or the extent of GlcNAc kinase induction, indicating that this enzyme is not a control point for the dimorphic development in C. albicans. A low basal level of GlcNAc kinase activity was found in noninduced cells of C. albicans but this increased 20- to 30-fold after the addition of GlcNAc to the cell suspension. GlcNAc is both an inducer and a substrate for the enzyme; ATP and Mg 2+ were also required for activity. The K mvalues for GlcNAc and ATP are 1.9 and 1.3 m m, respectively. ADP was found to be a noncompetitive inhibitor with respect to GlcNAc with a K ivalue of 7.1 m m. UDP-GlcNAc and glucosamine 6-phosphate had no effect on GlcNAc kinase activity. The presence of ethidium bromide (an inhibitor of transcription) or trichodermin (an inhibitor of translation) in cell suspensions of C. albicans inhibited the increase in GlcNAc kinase specific activity. The glucose analogs, α-methylglucopyranoside and 3- O-methylglucose, enhanced the formation of germ tubes by C. albicans but the induction of GlcNAc kinase was not affected. 2-Deoxyglucose, an inhibitor of germ-tube formation, had no effect on the induction of GlcNAc kinase.

Regulation of chitin synthesis during germ-tube formation in Candida albicans.

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely L-glutamine-D-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate. UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (Ki = 1.2 microM) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenxyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.

Inducibility of Germ-tube Formation in Candida albicans at Different Phases of Yeast Growth

Summary: Yeast cells of Candida albicans growing at 25 °C in glucose/yeast extract medium cannot germinate by dilution into fresh medium at 37 °C before reaching the stationary phase of growth. However, cells at any stage of growth are able to form germ-tubes giving true hyphae when incubated at 37 °C in the presence of inducers such as serum or N-acetyl-d-glucosamine. Thus, stationary phase cells are not endowed with a specific ‘competence' to germinate.