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Query: UMLS:C0023241 (Legionella)
 6,990 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
Mol Microbiol 1994 Jun
PMID:Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model. 805 22

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.
Mol Biol Evol 1994 Jul
PMID:Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications. 807 7

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Tn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5' truncated 'lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cis-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig-) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposon-linked mutation into a wild-type background results in a Pig- phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and beta-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.
Mol Microbiol 1994 Feb
PMID:Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene. 819 41

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome-lysosome fusion and association of host cell organelles with bacteria-containing phagosomes ('recruitment'). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome-lysosome fusion to mutants belonging to both phenotypic classes.
Mol Microbiol 1993 Jan
PMID:Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. 838 32

A new method to identify clonal strains of pathogenic bacteria has been developed recently in this laboratory. The method utilizes degenerate random amplified polymorphic DNA primers (D-RAPD) to amplify random fragments in crude bacterial lysates, generating reproducible DNA banding profiles or fingerprints. We use this method to type outbreak and non-outbreak isolates of Legionella pneumophila serogroup 1 from four hospitals near to, and affiliated with the University of Pittsburgh Medical Center. Patient isolates from a large outbreak, and nearly half of the contemporaneous environmental isolates showed the same DNA profile. Other isolates derived from non-outbreak patients showed easily distinguishable profiles. Other Legionella isolates collected between 1984 and 1994 were also analysed by this method. Our studies demonstrate that four strains were common among patient and environmental isolates at the four hospitals. These strains were also found to be different from a limited number of isolates from outside the Pittsburgh area. Because of its speed, simplicity and powerful discriminating ability, we believe that the D-RAPD approach provides epidemiologists and hospital infection control teams with a powerful tool in their efforts in analysing and terminating infection outbreaks.
Mol Cell Probes 1995 Dec
PMID:Typing of Legionella pneumophila serogroup 1 isolates by degenerate (D-)RAPD fingerprinting. 880 11

The differential display (DD)-PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells. Several DD-PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post-infection of the U937 macrophage-like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage-induced locus) was induced by intracellular bacteria during the first few hours post-infection of macrophages but the expression was downregulated by 12 h post-infection. Transcription of eml was not growth phase-related in vitro, and was not affected by in vitro stress stimuli. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD-PCR product was cloned. Six mini-Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L. pneumophila chromosome. Compared to the wild-type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100-fold increase in killing by macrophages during the first 5 h of the intracellular infection, and showed a 100-fold increase in killing during the first 24h of infection of the amoeba Hartmanella vermiformis. The 6th mutant had a phenotype indistinguishable from the wild-type strain. The cytopathicity defect of the mutants to the U937 cells was restored to wild-type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragment containing eml is a novel L. pneumophila locus whose expression is uniquely induced by non-stress stimuli during early stages of the intracellular infection of phagocytic cells. Expression of this locus is required for survival of L. pneumophila within macrophages and within amoebae during early stages of the infection.
Mol Microbiol 1996 Aug
PMID:The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. 886 78

Genomic fingerprints from DNA of fifteen environmental and clinical strains of Legionella pneumophila serogroup 1, isolated from diverse geographic areas of Greece, during the period 1986 to 1994, were generated with arbitrarily-primed polymerase chain reaction (AP-PCR) in order to use the discriminatory power of two arbitrary primers, BG2 and M13 Forward to clarify the relationship among the fifteen isolates. Both primers were found to have the ability to discriminate strains of the same serogroup, to identify strains related to each other even though they were isolated at different times. Therefore, AP-PCR using BG2 or M13 Forward as primers, appears to be a useful tool which provides a fast and simple method for epidemiological fingerprinting.
Cell Mol Biol (Noisy-le-grand) 1996 Sep
PMID:Typing of Legionella pneumophila strains isolated in Greece by arbitrarily-primed PCR. 889 50

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.
Mol Microbiol 1996 Sep
PMID:A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression. 889 84

Ribosomal RNA targets from Mycobacterium avium complex (23S), Mycoplasma pneumoniae (16S), Pneumocystis carinii (18S) and Legionella pneumophila (16S) were detected in four separate assays on a model automated Q-beta amplification instrument. Sandwich hybridization, reversible target capture, detector probe amplification and fluorescent signal detection occurred in closed, disposable packs at 38 degrees C. Packs were injected with 0.5 ml samples in 3.06 M guanidine thiocyanate. Ten samples per run were read after 7 h, requiring only 4 min loading time. Synthetic RNA transcripts and purified, natural RNAs from up to four different strains per assay were diluted to 10(6) or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumocystis). All analytes were detected at 10(6) targets. The limits of detection were found at 10(5) to 10(4). Discrimination against competitor RNA was tested using up to 10(9) molecules (1000 X excess) of appropriate test strains. Samples containing either zero targets or 10(7) competitors produced negative results in 95 to 100% of the samples, depending on the assay. Closely related Legionella and Mycoplasma species cross-reacted at high challenge levels of 10(9) molecules as a result of sequence similarities in the target regions. These results demonstrate the utility and versatility of an automated, high sensitivity, closed system for amplified analysis of direct-from-sample testing of respiratory pathogens.
Mol Cell Probes 1996 Oct
PMID:Detection of rRNA from four respiratory pathogens using an automated Q beta replicase assay. 891 Aug 91

Expression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli. Transcription of gspA is regulated by two promoters, one of which is regulated by the sigma 32 heat-shock transcription factor. We utilized a gspA promoter fusion to a promoter less lacZ to probe the phagososmal 'microenvironment' for the kinetics of exposure of intracellular L. pneumophila to stress stimuli. Expression through the gspA promoter was constitutively induced by approx. 16-fold throughout the intracellular infection, and occurred predominantly through the sigma 32-regulated promoter. Expression of the gspA promoter was induced approx. 4.5-fold, 5-, 11- and 9-fold upon exposure of L. pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild-type strain AA200. Compared to in vitro-grown wild-type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wild-type phenotypes were restored to strain AA224 by complementation with a plasmid containing wild-type gspA. There was no difference between the wild-type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to in vitro-grown bacteria, macrophage-grown and amoebae-grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli. Our data showed that regardless of the capacity of L. pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L. pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection. Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage-induced and stress-induced proteins within the intracellular environment.
Mol Microbiol 1997 May
PMID:Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant. 917 55


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