Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A phospholipase-deficient mutant, termed JL762, was obtained from a virulent strain of Listeria monocytogenes by screening a bank of 5,000 Tn1545 transposon-induced mutants on 2.5% egg yolk brain heart infusion agar. As previously shown (J. Mengaud, C. Geoffroy, and P. Cossart, Infect. Immun. 59:1043-1049, 1991), the transposon insertion took place inside the gene mpl, which encodes a zinc metalloprotease. By Western blot (immunoblot) analysis, we showed that loss of phospholipase activity was associated with loss of a 29-kDa zinc-dependent phosphatidylcholine-phospholipase C (PC-PLC) in culture supernatant of JL762 and of EGD-SmR incubated with ion chelator. As the parental strain, JL762 still produced in supernatants approximately 33-kDa proteins antigenically closely related to the 29-kDa PC-PLC. These results strongly suggest that the zinc metalloprotease of L. monocytogenes might play a role in the maturation of the 29-kDa PC-PLC. Although the uptake and the intracellular growth of bacteria were not affected in vitro, we found that the virulence of mutant JL762 was strongly impaired in the mouse.
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PMID:Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene. 131 8

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.
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PMID:A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. 158 25

The prfA gene of Listeria monocytogenes encodes a protein that activates transcription of the listeriolysin gene (lisA). In order to explore the role of the prfA gene product in the pathogenesis of listerial infection, we constructed a site-directed insertion mutation in prfA by the chromosomal integration of a novel suicide vector containing a portion of the prfA coding region. This mutation not only transcriptionally silenced the listeriolysin (lisA) gene but also abrogated production of specific RNA transcripts corresponding to the phosphatidylinositol-specific phospholipase C (pic) and metalloprotease (mpl) genes, two further virulence gene products expressed only by pathogenic Listeria strains. The strain was also found to be avirulent when tested in a mouse model of listerial infection. The concomitant loss of multiple characteristics such as production of LisA, Pic, Mpl, and loss of virulence in a mouse infection model is the result of a mutation in a single gene and demonstrates that the prfA gene product is a positive regulator of multiple virulence determinants in L. monocytogenes.
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PMID:Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. 172 45

The nucleotide sequence of a 3.5-kb BamHI fragment from Listeria monocytogenes 12067, a human clinical isolate of serotype 4b, has been determined. The DNA fragment harbors the gene for listeriolysin, part of the gene for a phosphatidylinositol-specific phospholipase C, and part of the gene for a metalloprotease. Comparison of the sequence with corresponding sequences from two other L. monocytogenes isolates revealed a significant number of nucleotide differences. Several of the differences give rise to amino acid substitutions. The most variable region was the examined part of the mpl gene, whereas the lisA gene showed a relatively high degree of conservation, particularly at the amino acid level. To analyze the pattern of sequence variability in the lisA gene, a 160-bp region covering nine nucleotide differences was sequenced from 36 isolates of different origins. This work showed that the strains can be grouped into two major types according to the nucleotide sequences. Oligonucleotide probing of a larger number of L. monocytogenes isolates showed that the observed differences can be used to subdivide the species. The data suggest a correspondence between the sequence type of the lisA gene and flagellar antigens. Assays based on hybridization or the polymerase chain reaction with type-specific oligonucleotides may provide fast and easy alternative methods for strain typing.
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PMID:Listeria monocytogenes isolates can be classified into two major types according to the sequence of the listeriolysin gene. 193 53

The synthesis of at least 14 extracellular toxins and enzymes in Staphylococcus aureus is regulated by a set of trans-acting elements from the agr (accessory gene regulator) locus. We have shown that the delta-lysin gene (hld) that is transcribed from a promoter immediately upstream of the agr locus, and which is positively controlled by agr, is part of this regulatory system. Deletion replacement mutagenesis of the chromosomal hld gene had the same pleiotropic effect on the synthesis of several virulence factors as agrA mutations. Characteristically, these mutants had an almost complete block in the synthesis of alpha-toxin, serin- and metalloprotease, whereas synthesis of protein A was greater than 10-fold higher than in the parental strain. Corresponding changes in the levels of alpha-toxin and protein A mRNAs were demonstrated by northern blotting experiments. The effects of the hld deletion mutation could be fully complemented by the hld gene on a plasmid. A plasmid insertion mutation in the 3' non-coding region of hld had a similar effect on exoprotein synthesis, indicating a role of the hld transcript in the regulation of exoprotein synthesis. This was confirmed by the finding that the effects of alpha-toxin and protein A synthesis by the hld deletion replacement mutation could be fully complemented by a hld allele in which we had introduced an early stop codon in the delta-lysin structural gene. However, the mutant hld allele could not complement the defect in production of extracellular proteases, indicating that delta-lysin may act in conjunction with its mRNA to regulate the expression of some exoprotein genes.
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PMID:The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. 232 18

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.
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PMID:Apparent phosphate retrieval system in Bacillus cereus. 250 29

Intracellular growth of Listeria monocytogenes begins after lysis of the primary vacuole formed upon bacterial entry into a host cell. Listeriolysin O (LLO), a pore-forming hemolysin encoded by hly, is essential for vacuolar lysis in most cell types. However, in human epithelial cells, LLO- mutants are capable of growth, suggesting that gene products other than LLO are capable of mediating escape from a vacuole. In this study, we investigated the role of other bacterial gene products in lysis of the primary vacuole in the human epithelial cell line Henle 407. Double internal in-frame deletion mutants were constructed by introducing a mutated hly allele into strains harboring deletions in either of the phospholipase C (PLC)-encoding genes or a metalloprotease-encoding gene. Bacterial escape from the primary vacuole, intracellular growth, and cell-to-cell spread were evaluated in Henle 407 cells. The results indicated that, in the absence of LLO, the broad-range PLC and the metalloprotease were both required for lysis of the primary vacuole in Henle 407 cells. Although phosphatidylinositol-specific PLC was not required, the efficiency of escape was reduced in an LLO phosphatidylinositol-specific PLC double mutant. These observations suggest that the relative importance of LLO, the phospholipases, and the metalloprotease may vary in different cell types or in cells from different species. In addition, these studies provide insight into the mechanisms of action of virulence determinants involved in the lysis of vacuolar membranes.
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PMID:The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. 759 Oct 98

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.
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PMID:GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways. 808 Dec 27

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
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PMID:A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways. 813 15

Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the global activator gene gacA. Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus, the gacA gene appears to be a general stationary-phase regulator.
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PMID:Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0. 815 Feb 59


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