Phospholipase Production Research Articles

Phospholipase D production using immobilized cells of Streptoverticillium cinnamoneum

Production of phospholipase D (PLD) by Streptoverticillium cinnamoneum immobilized within porous particles was investigated in repeated batch fermentation. The enzyme productivity in repeated batch fermentation was 2.2-fold that obtained in batch fermentation without immobilization, since many of the immobilized cells could be utilized as seed cells for each subsequent batch cycle.

Phospholipase D-derived Products in the Regulation of 12-O-Tetradecanoylphorbol-13-acetate-stimulated Prostaglandin Synthesis in Madin-Darby Canine Kidney Cells

Madin-Darby canine kidney (MDCK) cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of ethanol synthesize phosphatidylethanol (PEt) instead of phosphatidic acid (PA) and diglyceride (DG). We have used ethanol to block the production of phospholipase D (PLD)-derived PA and DG (from PA hydrolysis) to study their role in signal transduction. In MDCK cells, TPA-stimulated prostaglandin E2 (PGE2) synthesis was inhibited by ethanol at concentrations which inhibit PA and DG formation. In addition, TPA elicited a prolonged increase in PGE2 synthesis that is dependent upon continuous activation of PLD. The TPA-stimulated translocation of protein kinase Calpha (PKCalpha) from cytosol to membrane was unaffected by ethanol. This suggests that PLD-derived products act downstream of PKC in TPA-stimulated prostaglandin synthesis. The calcium ionophore, A23187, did not activate PLD, and PGE2 synthesis in response to A23187 was unaffected by ethanol. TPA increased prostaglandin endoperoxide H synthase (PGHS) activity and increased the amount of immunodetectable prostaglandin endoperoxide H synthase 2 (PGHS-2). A23187 did not induce PGHS-2 and A23187-stimulated PGE2 synthesis appears to be due to the constitutively expressed PGHS-1. Blocking the formation of PLD-derived products, PA and DG, inhibited the induction of PGHS-2 by TPA. These results indicate that prolonged PGE2 synthesis in response to TPA is due to the continuous induction of PGHS-2, which is dependent upon PLD activation. In contrast, induction of PGHS-2 by epidermal growth factor was not affected by ethanol. Epidermal growth factor did not induce PKCalpha translocation nor activate PLD. Taken together, these data suggest that PLD-derived PA or DG act as second messengers in the induction of PGHS-2 by PKC-dependent pathways. The demonstration that inhibition of TPA-induced PA formation inhibits Raf-1 translocation in MDCK cells (Ghosh, S., Strum, J. C., Sciorra, V. A., Daniel, L. W. , and Bell, R. M. (1996) J. Biol. Chem. 271, 8472-8480) suggests that PA is the active PLD metabolite in TPA-stimulated signaling.

Phospholipase C Production of Listeria monocytogenes SLCC 5764 Cultured with Various Salt Concentrations, pHs, and Temperatures

Listeria monocytogenes SLCC 5764 was propagated at 4, 25, or 37°C in tryptic soy broth (TSB) containing 428, 787, or 1,112mM NaCl; or adjusted with either acetic acid to pH 5.2, 5.7, or 7.0 or hydrochloric acid to pH 5.0, 5.7, or 7.0; or made alkaline with sodium hydroxide to pH 7.5, 8.5, or 9.5, to determine the effects on the production of phospholipase C (PLC). Altering the growth environment caused alterations in PLC production. The PLC activity was highest after cells were propagated in TSB containing 428mM NaCl at each growth temperature. Little PLC activity was detected at 4 or 25°C in media with the lowest and highest salt concentrations. Similarly, little PLC activity was detected at 4 or 37°C at each pH from cells grown in media adjusted with either acid. The PLC activity was greater after the cells were grown in media adjusted with either acid at pH 7.0 at 25°C. For growth in alkaline media, the PLC activity was highest after the cells were propagated in media at pH 7.5 at each growth temperature.

Continuous production of phospholipase D by Streptomyces lydicus D-121 immobilized with cross-linked chitosan beads

Continuous production of phospholipase D (PLD) in an air-lift reactor system was investigated using growing cells of Streptomyces lydicus D-121 immobilized within cross-linked chitosan beads as porous supports. In a continuous culture with the immobilized growing cells, both the activity and productivity were highest at a dilution rate of 0.330 h −1 which was higher than the maximum specific growth rate of the free cells (0.247 h −1). The highest PLD activity was obtained at pH 6.0–6.5. The average activity was 12.7 U/ml during 120–384 h in a continuous culture with immobilized cells, while the productivity of the continuous culture (4.19 × 10 3 U/ l·h) was about 3.3–3.6-fold higher than that of the batch culture (1.16 × 10 3 U/ l·h) and the repeated-batch culture in the reactor (1.27 × 10 3 U/ l·h), and about 3-fold higher than that in the case of continuous culture with free cells (1.36 × 10 3 U/ l·h).

Phospholipase A2-activating protein induces the synthesis of IL-1 and TNF in human monocytes.

Phospholipase A2-activating protein (PLAP) is an important mediator of eicosanoid generation. PLAP can also be found in high concentrations in synovial fluid from patients with rheumatoid arthritis, and injection of PLAP into animal joints results in an inflammatory, rheumatoid-like lesion. We have demonstrated previously that TNF-alpha and IL-1 beta stimulate formation of PLAP before phospholipase A2 (PLA2) enzyme activation and production of eicosanoids. To further explore the mechanisms found in the inflammatory response, we examined the ability of PLAP to stimulate release of TNF and IL-1 from human peripheral blood monocytes. TNF and IL-1 protein levels were measured by ELISA, and IL-1 and TNF mRNA were determined by Northern blotting. PLAP, PLAP peptide, and melittin, a bee venom PLA2 activator with homology with PLAP, all increased IL-1 and TNF production in a time- and dose-dependent manner. Heat-denatured PLAP and actin (an irrelevant protein) failed to exert this effect. PLAP stimulation of TNF and IL-1 could be enhanced with co-treatment of cells with free fatty acids, such as arachidonic or linoleic acid, but it was not blocked completely by PLA2 inhibitors. These results demonstrate not only that synthesis of PLAP can be stimulated by cytokines, but also that PLAP may regulate cytokine synthesis and thus perpetuate an immune or inflammatory response.

International Malignant Hyperthermia Workshop and Symposium

International Malignant Hyperthermia Workshop and Symposium

Candida krusei: biology, epidemiology, pathogenicity and clinical manifestations of an emerging pathogen.

Early reports of Candida krusei in man describe the organism as a transient, infrequent isolate of minor clinical significance inhabiting the mucosal surfaces. More recently it has emerged as a notable pathogen with a spectrum of clinical manifestations such as fungaemia, endophthalmitis, arthritis and endocarditis, most of which usually occur in compromised patient groups in a nosocomial setting. The advent of human immunodeficiency virus infection and the widespread use of the newer triazole fluconazole to suppress fungal infections in these patients have contributed to a significant increase in C. krusei infection, particularly because of the high incidence of resistance of the yeast to this drug. Experimental studies have generally shown C. krusei to be less virulent than C. albicans in terms of its adherence to both epithelial and prosthetic surfaces, proteolytic potential and production of phospholipases. Furthermore, it would seem that C. krusei is significantly different from other medically important Candida spp. in its structural and metabolic features, and exhibits different behaviour patterns towards host defences, adding credence to the belief that it should be re-assigned taxonomically. An increased awareness of the pathogenic potential of this yeast coupled with the newer molecular biological approaches to its study may facilitate the continued exploration of the epidemiology and pathogenesis of C. krusei infections.

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.

Growth in acidic media increases production of phosphatidylinositol-specific phospholipase C byStaphylococcus aureus

Production of phosphatidylinositol-specific phospholipase C (PIPLC), an exoenzyme of some strains ofStaphylococcus aureus, has been epidemiologically associated with virulence. To investigate the elaboration ofS. aureus PIPLC, we have evaluated in vitro conditions that maximize production of enzymatically active PIPLC. PIPLC activity was assessed by measuring the release of3H-inositol-phosphate from the substrate3H-phosphatidylinositol. Lowering the pH ofS. aureus cultures from 7.0 to 5.4 progressively increased the yield of PIPLC. The final yield of PIPLC was at least five-fold greater when the initial culture pH was 5.4 compared with 7.0. Low pH enhanced PIPLC activity recovered from twoS. aureus strains capable of high PIPLC production, but not from a strain producing little PIPLC. At both pH 5.0 and 7.4, PIPLC production peaked during mid- to late-logarithmic phase. We conclude that an acidic starting pH of culture media increases the yield of PIPLC activity elaborated during active growth ofS. aureus.

Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of Clostridium perfringens

The phospholipase C (alpha-toxin) gene (plc) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the plc gene were examined in Escherichia coli JM109. When the 0.7-kb region just upstream of the putative -35 site of the gene was deleted, production of phospholipase C increased approximately 10-fold. Northern blot hybridization analysis of the plc transcript showed that the upstream region inhibited transcription from the plc promoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5-6 tracts between positions -66 and -40 of the plc gene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plc promoter is a locus of sequence directed DNA-bending. Nested deletions of the upstream region were created from its 5' end by exonuclease III and the effects of deletions on the expression of the plc gene were examined. When the 77-bp fragment containing the two (dA)5-6 tracts were deleted, phospholipase C production increased markedly. These results indicate that the intrinsic DNA curvature upstream of the plc promoter is involved in the negative regulation of the plc gene transcription.

Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.

Mechanical Factors in the Etiology of Premature Rupture of the Membranes

The physiologic cascade that results in PROM is cyclic and probably can be entered at many points--through the production of collagenases, peroxidases, phospholipases, or prostaglandins. It can be initiated or exacerbated by bacteria. In addition, PROM is the result of direct bacterial insults or host-mediated autodestruction in response to bacterial presence or challenge. It may be affected by physical properties and stresses that are mechanical. This review of the mechanical factors that support normal chorioamnion membranes may provide an understanding of where the support can be eroded, thus leading to PROM. With this basic overview of the pathophysiology contributing to PROM, the clinician can justify clinical decisions better, depending on the patient's presentation and gestational age. The judicious use of various tocolytic agents, antimicrobial agents, and/or corticosteroids singly or in combination is predicated on the effect each of these iatrogenically administered agents will have on the mother and fetus. As more investigations are done, we will gain greater insight into the mechanical factors involved in causing PROM.

Structural and functional changes in rabbit ileum by purified extracellular phospholipase a ofSalmonella newport

As phospholipases of Salmonella species may play a role in the pathogenesis of gastrointestinal tract diseases. Salmonella newport, the causative agent of infantile diarrhoea was examined for the production of phospholipase. The enzyme was purified by gel filtration chromatography and was found to be a protein of molar mass ranging from 43 to 67 kDa. The purified enzyme alone or in combination with organisms, produced both structural and functional changes in rabbit ileum, contributing towards pathogenesis of diarrhoea due to this organism.

Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans.

A large number of diverse cell surface proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. One proposed function for the GPI anchor is that it facilitates the release of the protein from the cell by acting as a target for anchor-specific phospholipases. We and others have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD) (Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem, Biophys. Res. Commun. 150, 476-482; Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984). Because the GPI-PLD recognizes a conserved portion of the anchor, all GPI-anchored proteins are potential substrates for the enzyme. We demonstrate in this communication the production of the plasma GPI-PLD by the islets of Langerhans. GPI-PLD enzymatic activity was found in dog pancreatic microsomes, but not pancreatic juice. Both the pancreatic and plasma enzymes were divalent cation-dependent and had identical substrate specificities. Purified murine islets of Langerhans, as well as alpha and beta cells, contained and released GPI-PLD activity. A GPI-PLD DNA fragment was amplified by polymerase chain reaction from a normal human islet cDNA library; the amplified fragment hybridized with the GPI-PLD cDNA clone. These findings represent the first demonstration of the production of the plasma GPI-PLD by a specific tissue site as well as cell type.

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A 2 by Saccharomyces cerevisiae

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.

Phospholipase A2 and mediation of the activation of short-circuit current in the rat colonic mucosa

Melittin (0.5-2 micrograms ml-1) increased the short-circuit current (ISC) in mucosa-submucosa and mucosa preparations of the rat colon descendens in a dose-dependent manner. In the preparation with the submucosal plexus, quinacrine and indomethacin completely blocked the effect of melittin, indicating activation of phospholipase A2 and production of prostaglandins induced by the drug. The melittin response was also partially sensitive to the lipoxygenase inhibitor, nordihydroguaiaretic acid. Complete inhibition by tetrodotoxin and atropine gives evidence for the involvement of cholinergic neurons in the mediation of the response induced by melittin. In contrast, in the preparation without the submucosal plexus the effect of melittin was only partially inhibited by quinacrine, indomethacin, or by neuronal blockers, suggesting direct interactions of melittin with the epithelium in addition. The effect of melittin resembles to the action of bradykinin, which is neuronally mediated and quinacrine-sensitive in the mucosa-submucosa preparation, and quinacrine-resistant and not neuronally mediated in the mucosa preparation. In the mucosa-submucosa preparation, the melittin response is even partially sensitive to the bradykinin receptor antagonist [D-Phe7]-bradykinin. The results provide evidence for the presence of a quinacrine-sensitive phospholipase A2 in the preparation with and that without the submucosa.

Phospholipase C activity in microorganisms associated with reproductive tract infection

Phospholipase C (lecithinase or phosphatidylcholine phosphorylase) catalyzes the hydrolysis of lecithin into phosphorylcholine and 1,2-diglyceride. Bacterial production of phospholipase C may damage reproductive tract tissues by both direct and indirect mechanisms. Use of the synthetic substrate p-nitrophenylphosphorylcholine phospholipase C activity was determined in 204 isolates representative of those found in female genital tract. Multiple aerobic (28%) and anaerobic (28%) reproductive tract microorganisms showed phospholipase C activity. Phospholipase C-producing isolates included strains of Bacteroides fragilis, B. bivius, B. thetaiotaomicron, Gardnerella vaginalis, and group B streptococcus. Phospholipase C activity was heterogenous; not all isolates that belong to a particular species showed activity. Phospholipase C production may be a possible virulence factor produced by a number of microflora commonly implicated in various reproductive tract infections or conditions, as well as in some instances of preterm birth.

Factors affecting production and activity of phospholipase C by Yersinia enterocotttica

The production of phospholipase C by Yersinia enterocolitica strain SG was optimum at 37 degrees C at pH 6.5. No enzyme activity could be detected when the organism was grown at extreme pH values (pH greater than 8.5 or less than 5.0). The enzyme production was maximum when the organism was grown under static conditions in TSB medium. All solvents and salts inhibited the enzyme activity, whereas loss of activity was 95% in presence of methanol (20%) and 99% in presence of sodium azide (0.2 mol/l). The enzyme activity was increased twofold in the presence of cysteine and decreased by 98% in the presence of sodium perchlorate (0.2 mol/l).

Plasma levels of the lyso-derivative of platelet-activating factor in acute severe systemic illness.

1. Evidence suggests that activation of phospholipase A2 and production of eicosanoids and platelet-activating factor (PAF) are involved in various responses associated with severe tissue damage and shock. It was postulated that the plasma level of the precursor and degradation product of PAF, lyso-platelet-activating factor (lyso-PAF), might be increased in acute severe illness. 2. After plasma extraction, lyso-PAF was acetylated in vitro to PAF, which was measured by bioassay using 5-[14C]hydroxytryptamine-labelled rabbit platelets. Measurements were made in 18 severely ill patients (five with cardiogenic shock; five with severe infection, five after repair of abdominal aortic aneurysm, two with acute pancreatitis; 13 males, five females). Plasma lyso-PAF in these patients was 33 +/- 15 (SD)ng/ml (range 5-111 ng/ml), whereas values in normal males (40-65 years) ranged from 102 to 253 ng/ml (n = 15) and in females from 74 to 174 ng/ml (n = 10). Depression of plasma lyso-PAF did not relate closely to the patient group nor to specific therapy, but repeated measurements in each of 10 patients showed an increase in plasma lyso-PAF (P less than 0.002), associated with clinical improvement. 3. Evidence was obtained indicating that neither the presence of an inhibitor in the assay system nor reconversion of PAF to lyso-PAF in vitro produced the unexpected depression of plasma lyso-PAF. 4. The mechanisms responsible, which may have therapeutic implications, remain to be elucidated.

Apparent phosphate retrieval system in Bacillus cereus

Bacillus cereus secretes three different phospholipases C. We studied the effect of Pi levels in the growth medium on the production of these exoenzymes. Production of both phosphatidylcholine-preferring phospholipase C and sphingomyelinase C was repressed by Pi in the growth medium, whereas production of phosphatidylinositol phospholipase C was unaffected. We also found that B. cereus secretes a phosphate-repressed alkaline phosphatase activity. Together with a previously reported highly efficient, active uptake system for Pi, these three phosphate-repressed exoenzyme activities seem to be part of a phosphate retrieval mechanism that operates under growth-limiting concentrations of Pi. In natural soil systems, which are the natural habitats of B. cereus, the scarcity of Pi is the major growth-limiting factor. A phosphate-repressed metalloprotease activity was also detected in culture supernatants of B. cereus. It is unclear whether this exoenzyme activity also participates in the proposed phosphate-scavenging system.