Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0023241 (Legionella)
 6,990 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major extracellular enzyme of Legionella pneumophila, a metalloprotease, has been proposed as a pathogenic factor in Legionnaires' disease due to its cytotoxic, tissue-destructive, and phagocyte-inhibitory properties. The relevance of these activities depends on the production of the protease during infection, i.e. by L. pneumophila multiplying intracellularly. In this study, L. pneumophila was demonstrated to produce protease in guinea-pig and human alveolar macrophages infected in vitro. After 24 h infection, approximately 0.1 to 0.2 micrograms of protease per 10(6) bacteria was measured by ELISA in culture supernatants and lysates of the infected cells, whereas no protease could be detected immediately after infection. Immunogold labelling using anti-protease antibody showed the enzyme to be located within phagosomes and distributed throughout the macrophages. Recent observations have shown that this protease could modify host defence mechanisms through inhibition of bacterial killing by neutrophils and monocytes. The intracellular production of the enzyme in infected macrophages demonstrated here further supports a role for the protease in the pathogenesis of Legionnaires' disease.
 ...
PMID:Demonstration of the intracellular production of tissue-destructive protease by Legionella pneumophila multiplying within guinea-pig and human alveolar macrophages. 152 7

The extracellular metalloprotease of Legionella pneumophila, also called tissue-destructive protease or major secretory protein, has been proposed as one of the virulence factors of this organism. Considering the decisive role played by the phagocytic cells in host defense against Legionella infection, we investigated the effect of this protease on the function of human neutrophils and monocytes. L. pneumophila protease inhibited the chemotactic response of neutrophils to F-Met-Leu-Phe and zymosan-activated serum in a concentration-dependent and heat-labile manner. A direct effect of the protease on the chemotactic activity of neutrophils was demonstrated by the continued inhibition of neutrophil chemotaxis when the protease was removed following pre-incubation of the cells. In contrast, the enzyme had no effect on monocyte chemotaxis. The protease inhibited, also in a concentration-dependent and heat-labile manner, the binding of F-Met-Leu-Phe to both cell types. Neutrophil and monocyte oxidative burst response, as measured by superoxide release and chemiluminescence response, was not significantly affected by the enzyme. A slight enhancement of PMA-stimulated superoxide release was induced by the protease in both cell types. Lastly, the protease inhibited the killing of Listeria monocytogenes by neutrophils or monocytes. Inhibition of Listeria killing was concentration-dependent, heat-labile, and did not require the presence of the enzyme in the bactericidal assay. The inhibitory activity of L. pneumophila protease on neutrophil chemotaxis and on the listericidal activity of human neutrophils and monocytes demonstrated in this study provides evidence for a role of this enzyme in the pathogenesis of Legionnaires' disease.
 ...
PMID:Effect of Legionella pneumophila cytotoxic protease on human neutrophil and monocyte function. 158 5

The prt1 gene encoding extracellular protease from Erwinia carotovora subsp. carotovora EC14 in cosmid pCA7 was subcloned to create plasmid pSK1. The partial nucleotide sequence of the insert in pSK1 (1,878 bp) revealed a 1,041-bp open reading frame (ORF1) that correlated with protease activity in deletion mutants. ORF1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 Da. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produces a protease (Prt1) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prt1 activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prt1 promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prt1 transcription start site 205 bp upstream of ORF1. The deduced amino acid sequence of ORF1 showed significant sequence identity to metalloproteases from Bacillus thermoproteolyticus (thermolysin), B. subtilis (neutral protease), Legionella pneumophila (metalloprotease), and Pseudomonas aeruginosa (elastase). It has less sequence similarity to metalloproteases from Serratia marcescens and Erwinia chrysanthemi. Locations for three zinc ligands and the active site for E. carotovora subsp. carotovora protease were predicted from thermolysin.
 ...
PMID:Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene. 191 78

Strains of Legionella spp. produce extracellular proteases than can be detected using synthetic chromogenic peptides. Chromogenic tri- and tetrapeptides show a high degree of sensitivity, specificity and reagent stability when linked to para-nitroaniline (pNA). For example, SucOMe-Arg-Pro-Tyr.pNa (S-2586) is specifically hydrolysed by proteases of Legionella pneumophila and some other Legionella species. A paper disc method to sample protease directly from agar plates has been used to evaluate chromogenic peptides as reagents for diagnostic purposes. Strains of Legionella spp., Pseudomonas spp. and Enterobacteriaceae were examined, together with a recombinant Escherichia coli strain containing the cloned 38 kDa zinc metalloprotease from L. pneumophila, S-2586 was hydrolysed by 282 out of 283 L. pneumophila strains, and by the recombinant E. coli. Two of the six strains representing other Legionella species, and 22 of the 50 strains from the Pseudomonas group were also positive. No reaction was seen with any of the Enterobacteriaceae strains. Although there was functional homology between proteases from several bacterial groups, the high prevalence of S 2586-hydrolysing proteases within L. pneumophila indicates a potential usefulness for phenotypic identification.
 ...
PMID:Rapid identification of Legionella pneumophila zinc metalloprotease using chromogenic detection. 203 13

The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.
 ...
PMID:Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase. 211 Jan 46

The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown. The structural gene for the Msp protease, mspA, was isolated from an L. pneumophila genomic library. In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm. Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E. coli yielded mutants which no longer expressed protease activity and others with increased protease activity. These results suggested that mspA expression might be regulated. Msp was shown to be produced at a much higher level in L. pneumophila grown in rich compared to semidefined media. A Tn9 insertion which abolishes Msp expression was introduced into the L. pneumophila genome. This mspA::Tn9 L. pneumophila strain showed no detectable production of Msp by immunoblot analysis, and it had less than 0.1% of the protease activity found in the wild-type strain. This mutant was fully capable of growing within and killing human macrophages derived from the HL-60 cell line.
 ...
PMID:The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. 216 10

The DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli. This protein, which is 38,000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L. pneumophila protease. Periplasmic extracts of E. coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells. Using transposon mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities. Inactivation of proteolytic activity by transposon insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes. A putative regulatory sequence approximately 200-500 bp upstream of the gene's coding region was identified. A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L. pneumophila Philadelphia 1 as well as clinical isolates of L. pneumophila.
 ...
PMID:Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities. 254 10

Several strains of Legionella pneumophila and other species of Legionella with proteolytic activities were compared by assays, including Southern hybridizations and Western immunoblots, to determine their proteolytic, hemolytic, and cytotoxic activities. Only proteases from strains of L. pneumophila were both hemolytic and cytotoxic, and proteolytic activities extracted from other species of Legionella possessed only hemolytic activity. A 4.0-kilobase DNA sequence encoding the 38-kilodalton metalloprotease from L. pneumophila Philadelphia 1 that we showed previously was responsible for the observed hemolytic and cytotoxic phenotypes (F. D. Quinn and L. S. Tompkins, Mol. Microbiol., 3:797-805, 1989) was used in Southern hybridizations to probe chromosomal DNA from several strains of L. pneumophila and other Legionella species. The probe hybridized to the chromosomal DNA of all serogroups of L. pneumophila but not to any strains of L. dumoffii, L. micdadei, L. feeleii, or L. jordanis that we examined. Additionally, Western immunoblots done with rabbit antisera made to the cloned L. pneumophila protease demonstrated cross-reactions among 38-kilodalton proteins from strains of L. pneumophila, but no reactions were observed with proteins from other species of Legionella. Similarly, the cloned protease from L. pneumophila reacted with convalescent-phase sera from patients infected with L. pneumophila, but not with antisera isolated from patients infected with other Legionella species. Thus, despite some similarities among the proteolytic activities of members of the genus Legionella, including proteolytic and hemolytic phenotypes, metal requirements for zinc or iron, sensitivity to EDTA, and temperature and pH optima, we documented distinct genetic, immunological, and cytotoxicity differences among the proteolytic activities produced by Legionella species.
 ...
PMID:Genetic, immunological, and cytotoxic comparisons of Legionella proteolytic activities. 266 84

To determine the effects, if any, of the Zn-metalloprotease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acanthamoeba cell model, in guinea-pig macrophages, and in a guinea-pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin-resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA- mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea-pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild-type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea-pig model of infection employing the intratracheal inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain. Although deletion of the protease was not sufficient to completely abolish virulence in a guinea-pig model, the mutation caused a delay in the lethal effects of L. pneumophila and attenuated the infection.
 ...
PMID:Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model. 805 22

Legionella pneumophila, the causative agent of Legionnaires' disease and Pontiac fever, replicates within and eventually kills human macrophages. In this study, we show that L. pneumophila is cytotoxic to HL-60 cells, a macrophage-like cell line. We demonstrate that cell death mediated by L. pneumophila occurred at least in part through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented host cell DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether potential virulence factors like the metalloprotease and the macrophage infectivity potentiator of L. pneumophila are involved in the induction of apoptosis. None of these factors are essential for the induction of apoptosis in HL-60 cells but may be involved in other cytotoxic mechanisms that lead to accidental cell death (necrosis). The ability of L. pneumophila to promote cell death may be important for the initiation of infection, bacterial survival, and escape from the host immune response. Alternatively, the triggering of apoptosis in response to bacterial infection may have evolved as a means of the host immune system to reduce or inhibit bacterial replication.
 ...
PMID:Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection. 894 24


1 2 3 Next >>