Phosphatidylinositol-specific Phospholipase C Enzyme Research Articles

Expression, purification, and subcellular localization of phospholipase C in Dunaliella salina

Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina plays important roles in growth and stress responses. However, the molecular basis of PLC action in D. salina remains little understood. To gain insight into the potential biological functions of this enzyme, we cloned a phospholipase C gene from D. salina in a previous study, named DsPLC (GenBank No. KF573428). Here, we present the prokaryotic expression, purification, and characterization of the DsPLC gene. The entire coding region of DsPLC was inserted into an expression vector pET32a, and the DsPLC gene was successfully expressed in Escherichia coli. The DsPLC protein was purified and identified using a polyclonal antibody and western blotting. Expressing DsPLC fused with a green fluorescent protein (GFP) in onion showed that DsPLC-GFP was localized to the intracellular membrane. Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h. Our results support the importance of PLC enzymes in plant defense signaling. This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.

Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity

Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca2+ ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general “pattern”-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors.This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

Staphylococcus aureus is an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains of S. aureus possess a plc gene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity of S. aureus to cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context of S. aureus biology. Among a collection of genetically diverse clinical isolates of S. aureus, community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants of S. aureus, we identified both the agr quorum-sensing system and the SrrAB TCS to be positive regulators of plc gene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator of plc. Furthermore, plc regulation was linked to oxidative stress both in vitro and in vivo in a SrrAB-dependent manner. A Δplc mutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis of S. aureus infections.

FCS of Mutated Phosphatidylinositol-specific Phospholipase C Enzymes Monitors the Interplay of Substrate and Activator Lipid Binding

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are activated on the surface of vesicles by nonsubstrate phospholipids with the extent of activation tuned by the lipid composition. Bacillus thuringiensis PI-PLC can be activated by a small amount of phosphatidylcholine (PC) towards its substrate PI. Fluorescence correlation spectroscopy (FCS) has been used to study fluorescently labeled PI-PLC and a series of mutants (P42G, K44A, K44E, Y88A, and Y246/247/248S) binding to small vesicles of a substrate analogue (phosphatidylglycerol) and PC as a function of the vesicle composition. For PI-PLC and most of the mutant proteins there is a synergistic effect of the two types of phospholipids in anchoring the enzyme to a vesicle. If a mutation alters the affinity of substrate at the active site, binding should vary with XPC and be enhanced in PC-rich region; on the other hand, if a mutation alters the affinity of activator, Kd will increase with increased XPC. In this way, careful determination of the apparent Kd helps to sort out effects of enzyme mutations on activator and substrate sites. FCS is particularly useful in exploring the contribution of Kd to PI cleavage by mutant PI-PLC enzymes. For a fixed XPC, if the bulk concentration of vesicles is above Kd, enzymatic activities should be similar, as long as vesicle binding is the critical step. However, it should drop dramatically when the bulk concentration is below Kd measured by FCS (a long as the substrate analogue is a good mimic for substrate). The results provide a direct analysis of vesicle binding and catalytic activity and shed light into how occupation of the activator site enhances activity. Currently, we are extending the analysis of this system with tethered small unilamellar vesicles and single-molecule methods.

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8A resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp --> Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

Dissection of the Steps of Phospholipase Cβ2 Activity That Are Enhanced by Gβγ Subunits

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate in a two step reaction that involves a cyclic intermediate. The PLCbetafamily are activated by both the alpha and betagamma subunits of heterotrimeric G proteins. To determine which catalytic step is affected by Gbetagamma subunits, we compared the change in PLCbeta(2) activity catalysis toward monomeric short-chain phosphatidylinositol (PI) substrates and monomeric water-soluble cyclic inositol phosphates as well as long-chain PI in bilayer and micellar interfaces in the absence and presence of Gbetagammasubunits. Unlike other PLC enzymes, no cyclic products were detected for either wild-type PLCbeta(2) or a chimeric protein composed of the PH domain of PLCbeta(2) and the catalytic domain of PLCdelta(1). Using cIP as a substrate to examine the second step of the reaction, we found that the presence of Gbetagamma subunits stimulated this step by a higher level than that for the overall reaction (k(cat) 1.5-fold (cIP) as opposed to 1.20-fold for soluble diC(4)PI). Detergents above their CMC can generate the same kinetic activation of PLCbeta(2) as Gbetagamma, suggesting that hydrophobic compounds stabilize the activated state of the enzyme. The most pronounced effect of Gbetagamma is that it relieves competitive product inhibition. Taken together, our results show that activation of PLCbeta(2) occurs through enhancement in the catalytic rate of hydrolysis of the cyclic intermediate and increased product release, and that hydrophobic interactions play a key role.

Platelet Cellular Prion Protein (PRPC) Is Associated with alpha Granules.

The cellular prion protein (PrPc) is a membrane glycoprotein expressed on many human cells including blood platelets. We have previously shown that human platelets rapidly up-regulate PrPc on their plasma membranes after activation (Holada et al., Br J Haematol. 1998; 103(1):276–82.). Our preliminary results also showed that platelet PrPc is resistant to cleavage by phosphatidylinositol specific phospholipase C (PIPLC). In this study we investigated intracellular localization of platelet PrPc and its resistance to PIPLC of different origin and under different experimental conditions. To determine the intracellular localization of platelet PrPc, we used flow cytometry and PrPc directed monoclonal antibodies (MAb) 1562 and 6H4, which recognize residues 109–112, and 144–152, in human PrPc sequence, respectively. The specificity of MAb binding was confirmed by its inhibition with competing peptides. We compared the increase in PrPc expression after platelet activation with increase in expression of P-selectin (CD62) - an α-granular protein, and LIMP (CD63) - a lysosomal and δ-granular protein. Gel filtered platelets were activated by ADP (1–100 μM) or TRAP (0.5–50 μM). Increasing concentrations of agonists induced coexpression of P-selectin and PrPc on the platelet surface at lower concentrations than were required for expression of LIMP (e.g. 5 μM ADP vs. 50 μM ADP to reach 40% of maximal expression), suggesting that PrPc did not associate with lysosomes. To further address the question of the origin of intraplatelet PrPc, we evaluated the expression of platelet PrPc in two patients with Hermansky-Pudlak (H/P) syndrome and two patients with Grey platelet syndrome (GPS). H/P platelets have a low number of δ-granules, but normal numbers of α-granules and lysosomes. When compared to controls, the H/P platelets had decreased mepacrine staining, demonstrating a defect of δ-granules. The expression of LIMP was equivalent on resting control (15.2 geometric mean of FL1 (GMF)) and H/P platelets (14.4 GMF), but was substantially decreased on H/P platelets after full platelet activation (38.2 vs. 168.8 GMF). In comparison, similar levels of PrPc and α-granular P-selectin were expressed on normal (35.7 and 726 GMF) and H/P patient (33.9 and 689 GMF) activated platelets. Platelets of GPS patients are deficient in α-granules. Resting GPS platelets demonstrated higher expression of P-selectin (9.9 GMF) and PrPc (25.8 GMF) than normal platelets (4.9 and 14.3 GMF, respectively). In contrast to normal platelets, GPS platelets failed to up-regulate P-selectin (76 vs. 653 GMF) and PrPc (30.1 vs. 59.9 GMF) after activation. In order to confirm a resistance of PrPc to PIPLC in normal platelets, the presence of glycosyl-phosphatidylinositol (GPI) anchor was verified by the treatment of platelet PrPc with hydrofluoric acid, which resulted in a 2–4 kDa decrease of its molecular weight corresponding to the removal of GPI anchor. Interestingly, PIPLC enzymes of three different origins (B. thuringiensis, B. cereus, B. subtilis) did not cleave GPI anchor even after the solubilization of platelet membranes by Triton X-100, which should make anchor more accessible to the enzyme. In conclusion, our results suggest that platelet intracellular PrPc is associated with α-granules, but not with lysosomes, and δ-granules. Platelet membrane PrPc is resistant to PIPLC, likely due to GPI-modification rather than GPI accessibility.

Cross-linking Phosphatidylinositol-specific Phospholipase C Traps Two Activating Phosphatidylcholine Molecules on the Enzyme

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein. The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.

1α,25(OH) 2D 3 causes a rapid increase in phosphatidylinositol-specific PLC-β activity via phospholipase A 2-dependent production of lysophospholipid

1α,25(OH) 2D 3 activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A 2 (PLA 2). The purpose of this study was to determine if 1α,25(OH) 2D 3 activates PI-PLC directly or through a PLA 2-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1α,25(OH) 2D 3. Inhibitors and activators of PLA 2 were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1α,25(OH) 2D 3, was increased within 30 s with peak activity at 1–3 min. PI-PLC activity in resting zone cells was unaffected by 1α,25(OH) 2D 3. 1β,25(OH) 2D 3, 24 R,25(OH) 2D 3, actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1α,25(OH) 2D 3 regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA 2-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1α,25(OH) 2D 3; PLA 2-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF 3) reduced the effect of 1α,25(OH) 2D 3. Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1α,25(OH) 2D 3 stimulated PI-PLC and PKC activities via Gq; GDPβS inhibited activity, but pertussis toxin did not. RT–PCR showed that the cells express PLC-β1a, PLC-β1b, PLC-β3 and PLC-γ1 mRNA. Antibodies to PLC-β1 and PLC-β3 blocked the 1α,25(OH) 2D 3 effect; antibodies to PLC-δ and PLC-γ did not. Thus, 1α,25(OH) 2D 3 regulates PLC-β through PLA 2-dependent production of lysophospholipid.

Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.

Heat Stability of Virulence-Associated Enzymes from Listeria monocytogenes SLCC 5764

The enzyme activities of listeriolysin O (LLO), phosphatidylinositol-specific phospholipase C (PI-PLC), catalase (CA), and superoxide dismutase (SOD) from Listeria monocytogenes SLCC 5764 were examined after heat treatment, growth at elevated temperatures, and anaerobic growth. Growth at elevated temperatures may influence virulence as expressed by virulence enzyme activity. The enzymes were heated for 0 and 10 min at temperatures ranging from 40 to 100°C. The production of LLO was examined when the bacterium was grown at elevated temperatures, and the production of LLO, CA, and SOD were examined after anaerobic growth. LLO was the most heat-labile factor, losing almost all activity when heated above 50°C. CA was more heat stable, showing no decrease in activity until it was heated at 55°C. The PI-PLC enzyme was most heat resistant: the activity decreased between 40 and 50°C and continued to decrease when heated between 65 and 100°C. When L. monocytogenes was grown at elevated temperatures, it produced less LLO than when grown at the optimum growth temperature of 37°C. When the organism was grown under anaerobic conditions, the levels of CA and LLO decreased, while the SOD level remained unchanged.

Phosphatidylcholine activation of bacterial phosphatidylinositol-specific phospholipase C toward PI vesicles.

The effect of different phospholipids on the kinetic behavior of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis toward PI vesicles has been investigated. Cosonicated PC/PI vesicles displayed enhanced hydrolysis of PI when less than 0. 20 mole fraction PC was incorporated into the vesicle; higher mole fractions of PC led to a decrease from the maximum activity mimicking surface dilution of substrate. Since the PC could affect PI-PLC binding to vesicles, the effect of separate PC vesicles on enzymatic hydrolysis of PI vesicles was examined. Separate phosphatidylcholine vesicles were found to activate PI-PLC-catalyzed cleavage of PI vesicles up to 7-fold. The activation was completely abolished when the PC vesicle was composed of cross-linked molecules. In the absence of enzyme, fluorescence resonance energy transfer studies did not detect any fusion between PI and PC vesicles if the total lipid concentration was below 2 mM. Higher total lipid concentrations (>20 mM) increased PC transfer between PC and PI vesicles, producing a PI vesicle population with small amounts of PC in the outer monolayer. This suggested that the activation of PI-PLC toward PI vesicles reflects the time scale of transfer of PC from PC vesicles to PI vesicles. Cosonicated PC/PI vesicles provide a measure of enzyme activity versus mole fraction of PC that can be used to estimate the extent of vesicle exchange or fusion between separate vesicle pools. The effects of other phospholipid vesicles on PI-PLC hydrolysis of PI were also examined; zwitterionic lipids were activators while anionic phospholipids inhibited activity. The results indicated that PC molecules in the PI interface allosterically bind to PI-PLC and help anchor enzyme in a more active conformation to the PI interface.

Allosteric activation of phosphatidylinositol-specific phospholipase C: specific phospholipid binding anchors the enzyme to the interface.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.

Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling.

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned from Drosophila melanogaster and one is from Artemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.

Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor.

Staphylococcus aureus secretes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) which is able to hydrolyze the membrane lipid PI and membrane protein anchors containing glycosyl-PI. The gene for PI-PLC (plc) was cloned from S. aureus into Escherichia coli. Oligonucleotide probes based on partial protein sequence and polyclonal antibodies raised against the purified protein were used to identify positive clones. E. coli transformed with a plasmid containing the plc gene expressed PI-PLC enzyme activity which was abolished by mutagenesis with a tetracycline resistance gene. The plc gene was present in all 15 S. aureus strains examined but not in any of 6 coagulase-negative staphylococcal species. The plc gene contained 984 bp and coded for a mature protein with a calculated molecular mass of 34,107 Da. Amino acid sequence comparisons indicated that the staphylococcal plc gene was similar (51 to 56%) to the PI-PLCs from Bacillus cereus, Bacillus thuringiensis, and Listeria monocytogenes. The recombinant PI-PLC expressed in E. coli was purified and exhibited biochemical properties identical to those of the native PI-PLC from S. aureus. PI-PLC production was decreased in agr mutant strains of S. aureus. However, PI-PLC production by both agr+ and agr mutant strains exhibited a similar dependence on the type of medium used. These data suggested that PI-PLC production was regulated by both agr-dependent and agr-independent mechanisms.

Phosphatidylinositol-specific phospholipase C, a possible virulence factor of Staphylococcus aureus.

Phosphatidylinositol-specific phospholipase C (PIPLC), an enzyme that can specifically release phosphatidylinositol-linked proteins from host cells, is one of the extracellular enzymes produced by Staphylococcus aureus. To investigate whether PIPLC might be a virulence factor, we assessed PIPLC production by S. aureus strains that had been isolated from healthy carriers and from infected patients with or without toxic shock syndrome. Although none of five vaginal isolates from healthy women was a PIPLC producer, only 10 of 32 selected pathogenic strains that caused significant infections or toxic shock syndrome elaborated PIPLC enzyme activity. Seven of 24 toxic-shock-associated strains, compared with 3 of 8 non-toxic-shock-associated strains, were positive for PIPLC. The majority of strains that produced PIPLC were negative for toxic shock syndrome toxin 1 (P less than 0.05); this association between PIPLC production and strains negative for toxic shock syndrome toxin 1 was even stronger among strains isolated only from patients with toxic shock syndrome (P less than 0.01). Among all 32 pathogenic isolates, PIPLC-producing S. aureus strains were isolated from four of four patients developing adult respiratory distress syndrome and four of five patients with disseminated intravascular coagulation, suggesting a significant association between PIPLC production and adult respiratory distress syndrome and/or disseminated intravascular coagulation (P less than 0.002). On the basis of these results, we propose that PIPLC is a virulence factor of S. aureus and is implicated in the development of adult respiratory distress syndrome and disseminated intravascular coagulation.