Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C*

Phospholipase C (PLC) activity secreted by bacteria as a virulence factor is commonly detected by use of the artificial substrate p-nitrophenylphosphorylcholine (p-NPPC). We examined several commercially available enzymes (phosphodiesterases, phosphomonoesterases, phospholipase A, lipase, protease) for their hydrolytic activity towards p-NPPC and compared these results with those of PLC tests using phospholipid substrates. Our data indicate that, in addition to PLC, several other enzymes which can affect phosphate esters are able to hydrolyze p-NPPC. We therefore suggest to use lipid substrates for correct characterization of bacterial PLCs, especially when whole bacteria or crude enzyme preparations are investigated.     

Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication

Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.     

Zinc metalloproteinase ProA directly activates Legionella pneumophila PlaC glycerophospholipid:cholesterol acyltransferase

Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated.     

The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA

Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum-derived 'Legionella-containing vacuole' (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild-type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt-resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER-derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two-component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS- and LetA-controlled interactions of L. pneumophila with phagocytes.     

A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae. To persist in the environment, the microbe must be proficient at both replication and transmission. In laboratory cultures, as nutrients become scarce a stringent response-like pathway coordinates exit from the exponential growth phase with induction of traits correlated with virulence, including motility. A screen for mutants that express the flagellin gene poorly identified five activators of virulence: LetA/LetS, a two-component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA of Salmonella; the stationary-phase sigma factor RpoS; the flagellar sigma factor FliA; and a new locus, letE. Unlike wild type, post-exponential-phase letA and letS mutants were not motile, cytotoxic, sodium sensitive or proficient at infecting macrophages. L. pneumophila also required fliA to become motile, cytotoxic and to infect macrophages efficiently and letE to express sodium sensitivity and maximal motility and cytotoxicity. When induced to express RelA, all of the strains exited the exponential phase, but only wild type converted to the fully virulent form. In contrast, intracellular replication was independent of letA, letS, letE or fliA. Together, the data indicate that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating L. pneumophila to a transmissible form.     

Acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides, has phospholipase, lysophospholipase, diacylglycerollipase, and acyltransferase activities in vitro.

Human acyloxyacyl hydrolase (AOAH) is a leukocyte enzyme that hydrolyzes acyloxyacyl bonds in the lipid A region of bacterial lipopolysaccharide (LPS), thereby detoxifying the LPS. We report here that the enzyme also acts in vitro on glycerophospholipids, lysophospholipids, and diacylglycerol. While AOAH preferentially removes palmitate or stearate from the sn-1 position of phospholipid and diacylglycerol substrates that have unsaturated acyl chains in the sn-2 position, it is able to cleave both palmitates from sn-1,2-dipalmitoylphosphatidylcholine and sn-1,2-dipalmitoylglycerol. This apparent preference for removing saturated (or shorter) acyl chains from glycerolipids is consistent with its ability to cleave laurate more rapidly than palmitoleate from lipopolysaccharide (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449). AOAH also catalyzes acyl transfer from LPS and phosphatidylethanolamine to acceptor lipids; approximately equal amounts of laurate and myristate are transferred from LPS to monooleoylglyceryl ether, forming acyloleoylglyceryl ether. The demonstration that AOAH has phospholipase, lysophospholipase, diacylglycerol lipase, and acyltransferase activities in vitro suggests that the enzyme may have roles in addition to LPS deacylation (detoxification) in phagocytic cells.     

The phospholipase activities present in preheparin mouse plasma are inhibited by antiserum to hepatic lipase

• 1.1. Preheparin plasma from mice, but not rats or man, contains high levels of phospholipase A and lysophospholipase activities which are distinct from lecithin:cholesterol acyltransferase (LCAT).
• 2.2. Neither the phospholipase A nor the lysophospholipase activities in preheparin plasma are inhibited by incubation in the presence of protamine sulphate or high salt concentrations.
• 3.3. When mouse plasma is incubated in the presence of an antiserum specific for rat hepatic triacylglycerol lipase (HTGL), the phospholipase activities are abolished.
• 4.4. These observations suggest that the phospholipase activities are attributable to the action of HTGL, which, in the mouse appears to be a freely circulating enzyme, whereas for other species this enzyme only appears in the blood following administration of heparin.

     

Occurrence of Lysophosphatides in Bacteriophage T4rII-Infected Escherichia coli S/6

Hydrolysis of phospholipids was observed to start about 15 min after Escherichia coli S/6 cells were infected with T4rII bacteriophage mutants. Hydrolysis continued through the latent period and well past the time when cell lysis occurs. The hydrolytic products that accumulated were free fatty acids, 2-acyl lysophosphatidylethanolamine, and various lysocardiolipins. These products indicated the action of phospholipase A(1). From 15 to 22 min after infection, there were equivalent amounts of fatty acids and lysophosphatides in extracts of cellular lipids. Thereafter, free fatty acids were produced in excess. This suggests that lysophospholipase was active at the later time. We also observed a stoichiometric relation between loss of phosphatidylglycerol and increase of cardiolipin plus lysocardiolipins. This continued well past the normal lysis time (25 min). The appearance of lipase activities during the latent period seems to be specific to infection with rII mutants. Neither the wild-type bacteriophage nor rI mutants produced similar activities by 22 min after infection.     

Cloning and characterization of a lysosomal phospholipase A2, 1-O-acylceramide synthase.

Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A(2) and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A(2) activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized with N-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS.     

Characterization of two Pseudomonas aeruginosa mutants with defective secretion of extracellular proteins and comparison with other mutants

We have isolated 2 new pleiotropic mutants of Pseudomonas aeruginosa strain PAO with defective secretion of extracellular proteins (Xcp mutants). One of these mutants was compared to 2 different, previously isolated secretion mutants. All had similar phenotypes and were unable to release at least 4 exoproteins (lipase, elastase, alkaline phosphatase, and phospholipase C), whilst alkaline protease was still secreted. The exoproteins appeared to be blocked in the periplasmic space. No difference in molecular weight was detected between cell-bound forms of elastase and alkaline phosphatase in the different mutants and the corresponding extracellular forms from the wild-type strain. Genetic mapping showed that the mutations were located in the 55′ region of the chromosome.     

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.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Purification of rabbit myocardial cytosolic acyl-CoA hydrolase, identity with lysophospholipase, and modulation of enzymic activity by endogenous cardiac amphiphiles

Rabbit myocardial cytosolic acyl coenzyme A (acyl-CoA) hydrolase activity was purified to near-homogeneity by ammonium sulfate precipitation and ion-exchange, gel filtration, chromatofocusing, and hydroxylapatite chromatographies. Kinetic analysis of the purified protein demonstrated a maximum velocity of 24 mumol/(mg . min) and an apparent Michaelis constant of 50 microM. Cytosolic acyl-CoA hydrolase and lysophospholipase activities cochromatographed in every fraction of every step. The purified protein was a single band (Mr 23 000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. These results suggest that cytosolic lysophospholipase and palmitoyl-CoA hydrolase activities are catalyzed by a single polypeptide with dual activities. Palmitoyl-CoA competitively inhibited lysophospholipase activity (Ki = 4 microM). Low concentrations (20 microM) of lysophosphatidylcholine or L-palmitoylcarnitine increased palmitoyl-CoA hydrolase activity at low palmitoyl-CoA concentrations but had little effect at high concentrations of palmitoyl-CoA. In contrast, high concentrations (100 microM) of lysophosphatidylcholine or L-palmitoylcarnitine inhibited palmitoyl-CoA hydrolase activity. The results suggest that interactions between endogenous cardiac amphiphiles and palmitoyl-CoA hydrolase contribute to the regulation of intracellular long-chain acyl-CoA concentrations and therefore potentially modulate fluxes of fatty acid through several biochemical pathways.     

A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa

The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.     

Secreted phospholipases of the dimorphic fungus, Candida albicans; separation of three enzymes and some biological properties

Several phospholipases are secreted into the culture medium by growing yeast cells of Candida albicans 3125. DEAE-Sephadex column chromatography of concentrated culture filtrate revealed three separable fractions with phospholipase activities. Analysis of products of hydrolysis showed that the enzyme activities were lysophospholipase, lysophospholipase-transacylase and a phospholipase B.     

Suppression of the humoral immune response of Atlantic salmon, Salmo salar L. by the 64 kDa serine protease of Aeromonas salmonicida

Bacteria-free supernatants of broth cultures of Aeromonas salmonicida inhibited the humoral immune response, but not the cell-mediated immune response, of Atlantic salmon to bacteriophage MS2. The immunosuppressive factor was the 64 kDa serine protease secreted by A. salmonicida. The suppressive activity was not due to degradation of epitopes of MS2, and although serine protease degraded the heavy chain of salmon IgM in vitro there was no evidence for significant degradation in vivo. The principal lethal toxin of A. salmonicida, the glycerophospholipid: cholesterol acyltransferase did not inhibit the immune response of salmon.