Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Comparison of phospholipase production in shape Cryptococcus neoformans isolates from AIDS patients and bird droppings

Secreted phospholipase has been recently proposed as a virulence determinant in Cryptococcus neoformans as well as Candida albicans. This issue of cryptococcal phospholipase requires screening of phospholipase production in a larger number of isolates from clinical and environmental sources. In this study we examined phospholipase production in a total of 67 C. neoformans isolates from AIDS patients and bird droppings by using the egg-yolk plate method. Phenoloxidase activity, capsule size and growth at 37 °C were also measured in these strains in order to observe a possible relationship between phospholipase production of different C. neoformans strains and its virulence. Four of the 21 AIDS strains at 28 °C and 1 at 37 °C did not produce phospholipase, respectively. In contrast, 38 and 34 of the 46 bird dropping strains were negative for phospholipase production at 28, and 37 °C, respectively. Statistical analysis revealed a significant difference in phospholipase production, capsule size and growth ability at 37 °C, but not phenoloxidase activity, between the AIDS and the bird dropping strains. The highly prevalent distribution of phospholipase activity in the AIDS strains suggests a role of the enzyme in invading the host. This revised version was published online in June 2006 with corrections to the Cover Date.     

A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts

Phospholipase A and lysophospholipase activities were measured in the culture fluid and in the blastospores of Candida albicans. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of Candida parapsilosis and Saccharomyces cerevisiae) a correlation was found between this activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Non-pathogenic yeasts, including C. albicans isolates which did not adhere and did not kill mice, had lower phospholipase activities.     

Factors affecting the phospholipase activity of Candida species in vitro

The phospholipase activity of 41 isolates of oral Candida species was determined by a plate assay. Seventy nine per cent of the C. albicans isolates were phospholipase producers whereas none of the C. tropicalis, C. glabrata or C. parapsilosis isolates produced the enzyme. The degree of phospholipase activity (Pz value) of individual isolates was remarkably constant despite the large variation in activity among different isolates. Experiments with 10 phospholipase positive C. albicans isolates indicate that phospholipase production in vitro is limited to a narrow pH range (c. 3.6-4.7) and is suppressed by increasing concentrations of sucrose and galactose in the media (r = 0.9). Hence, candidal phospholipases seem to play a complex role in the aetiopathology of human candidoses.     

Purification and characterization of secretory proteinase of Candida albicans.

Proteolytic activity of medically important yeasts was tested in both YCB-BSA agar and medium. All of 134 strains of Candida albicans, 13 of 18 strains of Candida tropicalis and 11 of 18 strains of Candida parapsilosis had this activity, while none of 52 Candida glabrata strains or of 11 Cryptococcus neoformans strains tested had proteolytic activity. Strains of C. albicans fell into five groups based on the level and time-course of in vitro proteinase productivity. Five strains randomly selected from each group were tested for pathogenicity in mice. The strain possessing the strongest pathogenicity was used to purify proteinase. The molecular weight of the proteinase was approximately 44,000 daltons and its isoelectric point was pH 4.2. Optimal pH of the proteinase was 3.2 and the enzyme was stable below pH 7.0 and lost its activity above pH 8.0 at 37 C in a 60-min incubation. The 23 amino acid sequence of the proteinase N-terminus was determined.     

Phospholipases as possible virulence factor in Vibrio parahaemolyticus

Phospholipase C activity was elevated in pathogenic Vibrio parahaemolyticus isolated from patients. Phospholipase A activity was more pronounced in the nonpathogenic V. parahaemolyticus strains isolated from water. Extracts of the strains containing phospholipase C and A activity but no thermostable direct haemolysin (TDH) were capable of producing lesions in guinea pig skin indicating the presence of a toxic factor other than TDH. It is suggested that the toxic factor may be phospholipase C since the purified enzyme from Clostridium perfringens produced a similar reaction in guinea pig skin.     

Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.     

Phospholipid-deacylating enzymes of rat small intestinal mucosa.

1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.     

Effect of different K+ concentrations on Cryptococcus neoformans phenoloxidase activity

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.     

Extracellular enzymatic activity and serotype of Cryptococcus neoformans strains isolated from AIDS patients in Brazil

One hundred and fifty-one Cryptococcus neoformans strains isolated from AIDS patients in Brazil and maintained in the Adolfo Lutz Institute (S?o Paulo, Brazil) were tested for phospholipase, protease and other extracellular enzymatic activities and their serotypes determined. Production of extracellular phospholipase and protease was tested by the agar plate methods. Determination of extracellular enzyme profile of the strains was performed by using the API-ZYM kit system, which can test 19 different enzymes. The serotypes were determined by cell agglutination using the Crypto-check method. Among the 151 strains, 147 were identified as serotype A and four strains were serotype AD. Production of extracellular phospholipase and protease was extensive and observable at early stages of incubation. All of the tested strains were positive for the production of both enzymes. In the API-ZYM tests, more than 90 % of the 151 tested strains were positive for esterase C4 (No. 3), esterase lipase C8 (No. 4), leucine arylamidase (No. 6), phosphatase acid (No. 11), naphtol-AS-BI-phosphohydrolase (No. 12), alpha-glucosidase (No. 16) and beta-glucosidase (No. 17). Differences in enzymatic activities between the Brazilian strains and strains isolated in other countries were observed. The phospholipase, protease and other enzyme activities may play a role in host tissue invasion by C. neoformans.     

Purification and characterization of phospholipase C of Salmonella gallinarum

Phospholipase C was isolated from an outbreak strain of Salmonella gallinarum with ciprofloxacin extraction, dialysis, gel filtration, ion exchange chromatography and chromatofocussing. Purified phospholipase C (mol wt. 65 KDa; isoelectric point, pI 3.5) was resistant to pasteurization, stomach enzyme (pepsin), bacterial protease and lipase but lost its activity on trypsin and chymotrypsin treatment. It was sensitive to pH > or = 8.0. It was haemolytic, embryotoxic, enterohaemorrhagic, lethal to birds, cytotoxic to Vero and MDBK cells, dermonecrotoxic in rabbit and antigenically active protein. Antisera raised against purified phospholipase C neutralized its all biological activities and agglutinated the producer Salmonella strains. Serologically it was proved similar to phospholipase C of Klebsiella pneumoniae and S. weltevreden. Fluorescent antibody technique (FAT) was standardized to detect phospholipase producer strains.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.     

Factors affecting intra- and extracellular phospholipase A1 production bySalmonella newport

The effect of various physico-chemical factors on production of intra- and extracellular phospholipase A₁ bySalmonella newport was investigated. Maximum intracellular enzyme levels were observed when cells were grown in brain heart infusion broth, after 12 h of incubation at 37°C. Highest level of extracellular phospholipase A₁, however, was seen in synthetic medium (pH 7.0) after 24 h of incubation at 37°C. Agitation during incubation had no effect on the intracellular enzyme synthesis but enhanced extracellular enzyme levels. Addition of surfactants to the growth media significantly decreased both intra- and extracellular phospholipase A₁ production.     

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.     

Rapid detection of lytic antimicrobial activity against yeast and filamentous fungi.

A rapid method for assessing the lytic activity of antimicrobial agents against yeast and fungi has been developed. The assay is based on the release of the intracellular enzyme, maltase (alpha-glucosidase). The released maltase activity was measured colorimetrically by the production of p-nitrophenol from p-nitrophenyl-alpha-D-glucopyranoside (PNPG). The lytic activity of different antimicrobial compounds was measured against yeast cells or germinating spores of filamentous fungi. Lytic anti-yeast activity could be detected within 20 min incubation at 30 degrees C against Saccharomyces cerevisiae, Candida albicans, and Cryptococcus neoformans. Lytic anti-fungal activity appeared after 2 h of incubation at 30 degrees C against germinating spores of Aspergillus niger and Botrytis cinerea. Whole cells of either yeast or fungi did not hydrolyze sufficient PNPG within 3 h at 30 degrees C to yield a detectable color change. Lytic activity of enzymes (e.g., Lyticase), antibiotics (e.g., Amphotericin B), and an antibiotic-producing strain of bacteria were detected using the assay. The anti-yeast assay has been adapted to a 96-well microtiter format. Both assays provided a rapid, sensitive, and reproducible detection of lytic anti-yeast and anti-fungal activity.     

Technological properties of Enterococcus faecium isolated from ewe's milk and cheese with importance for flavour development

Eight Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina were screened for biotechnological properties relevant to flavour development. The API ZYM test showed absence of proteases, presence of high amounts of peptidases, and high esterase-lipase activities. Low extracellular proteolytic activity was observed. Most strains produced diacetyl in milk, with E. faecium OvL 214 and OvL 254 being the best producers. Biomass and growth rate increased when citrate was added to the medium, suggesting that these strains could use citrate as a main energy source. After 24 h of incubation, citrate was completely consumed in complex medium supplemented with glucose and citrate. An average of 17% residual citrate was detected in complex media supplemented with citrate. For all strains, esterase activity was detected up to alpha-naphthyl-caproate. They hydrolyzed alpha-naphthyl derivatives of fatty acids in this order: C3 > C6 > C4 > C8 > C2. Post-electrophoretic detection of esterase activities revealed the presence of multiple esterases. Hydrolysis of tributyrin, tricaprylin, and milk fat was observed in cell-free extracts. Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina present the metabolic potential to contribute to cheese flavour development.     

Steroidogenic effect of exogenous phospholipase C on bovine adrenal fasciculata cells

Phospholipase C (Bacillus cereus) added to the incubation medium stimulated the steroidogenic activity of bovine adrenal zona fasciculata cell suspensions to a level similar to that induced by optimal concentration of ACTH. This effect was not related to an increase of cyclic AMP; it was calcium-dependent and was also induced by an other bacterial phospholipase C (from Clostridium perfringens) whereas phospholipases A2 and D were ineffective. Phospholipid metabolism was examined in these cells after radiolabeling with [14C]-glycerol or [32P]orthophosphate. Phospholipase C induced a very fast (5 seconds) increase in cellular [14C]-1,2-diacylglycerol followed by [32P] labeling of phosphatidic acid and phosphatidylinositol. These events preceded the stimulation of steroidogenesis which was detectable after 2 minutes of incubation. These observations suggest that activation of an endogenous phospholipase C activity may be considered as an early event in the response of bovine adrenocortical cells to steroidogenic effectors such as angiotensin II and acetylcholine.     

Isolation and fundamental characteristics of a phospholipase B inhibitor from autolyzed Torulaspora delbrueckii.

Phospholipase B inhibitor was found in the autolyzate of yeast cells, Torulaspora delbrueckii. The inhibitor was purified to homogeneity by ethanol precipitation, gel filtration with Sephadex G-10, ion-exchange chromatography with DEAE-Sephacel, and gel filtration with Asahipak GS-320. On thin-layer chromatography the purified inhibitor was detected with the Hanes-Isherwood reagent, which is used to detect phosphorus. The activity of the inhibitor was not affected by heat treatment at 100 degrees C for 1 h. Heating at 100 degrees C for 1 h in 1 M HCl and 1 M NaOH lowered activity to 76 and 80% of the original values, respectively, but heating at 110 degrees C for 24 h in 6 M HCl completely abolished activity. The inhibitor was highly soluble in water, but practically insoluble in alcohol, acetone, ether, and chloroform. The degree of inhibition of enzyme activity was not proportional to the concentration of inhibitor. The inhibitor inhibited both membrane-bound and water-soluble phospholipase B activity from T. delbrueckii at the same level; however, the inhibitor did not inhibit the activity of phospholipase A2 from snake venom (Naja naja).     

Lipid rafts in Cryptococcus neoformans concentrate the virulence determinants phospholipase B1 and Cu/Zn superoxide dismutase

Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae, and the pathogenic fungus Candida albicans. Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans. These characteristics include insolubility in Triton X-100 (TX100) at 4 degrees C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-beta-cyclodextrin (MbetaCD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37 degrees C. Treatment of viable cryptococcal cells in suspension with MbetaCD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six- to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment.     

Phospholipase C activity in cultured human skin fibroblasts

In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion.