Sequencing of the rpoB gene in Legionella pneumophila and characterization of mutations associated with rifampin resistance in the Legionellaceae.
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Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae. (+info)
Differential effects of virulent versus avirulent Legionella pneumophila on chemokine gene expression in murine alveolar macrophages determined by cDNA expression array technique.
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The cDNA expression array technique is a powerful tool to determine, at one time from many genes, specific gene messages modulated by infection. In the present study, we identified genes modulated in response to virulent versus avirulent Legionella pneumophila infection of the alveolar macrophage cell line MH-S by the cDNA expression array technique. Many macrophage genes were found to be modulated after 5 h of in vitro infection with L. pneumophila. In particular, it was found that the monocyte chemotactic protein 3 (MCP-3) gene expression was significantly induced by infection with virulent L. pneumophila but not with avirulent L. pneumophila. In contrast, other chemokine genes, such as macrophage inflammatory protein (MIP) 1alpha, were induced by both virulent and avirulent L. pneumophila. Reverse transcription (RT)-PCR assay of total RNA isolated from macrophages infected with the bacteria for 5 or 24 h confirmed the differential induction of the chemokine genes by virulent versus avirulent L. pneumophila. Thus, the cDNA expression array technique readily revealed differential induction by L. pneumophila infection of select chemokine genes of macrophages from more than 1,100 genes. These results also indicate that certain chemokine genes may be selectively induced by virulent bacteria. (+info)
Evaluation of a human monocytic cell line THP-1 model for assay of the intracellular activities of antimicrobial agents against Legionella pneumophila.
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We examined the intracellular activities of 11 antimicrobial agents against Legionella pneumophila using a human monocyte-derived cell line, THP-1. Colony counting and microscopic examination of L. pneumophila co-incubated with THP-1 cells (5 x 105 cells/well) were performed. Both extra- and intra-cellular multiplication of L. pneumophila were observed and were dependent on the inoculum of L. pneumophila in the culture; L. pneumophila did not grow in the cell culture medium alone. Light microscopic examination confirmed that extracellular L. pneumophila originated from THP-1 cells disrupted by bacterial multiplication. L. pneumophila multiplied by 3-4 logs after 24 h incubation with THP-1 cells and their number remained stable at 106-107 cfu/mL until 72 h. The results of viability studies using four antimicrobial agents-ciprofloxacin, erythromycin, minocycline and rifampicin-demonstrated that our system was suitable for the intracellular activity assay. We used a concept of 'minimum extracellular concentration inhibiting intracellular multiplication' (MIEC) to evaluate the intracellular activity of antimicrobial agents. The MIECs of three beta-lactams were markedly higher than their conventional MICs while those of macrolides, quinolones, rifampicin and minocycline were similar to their MICs. Our results suggest that evaluation of the clinical efficacy of drugs against L. pneumophila should include determination of their intracellular activity against the bacteria, which could be measured using our assay system in THP-1 cells. (+info)
Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila.
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Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79-90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are "trapped" within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo. (+info)
Detection of Legionella pneumophila using a real-time PCR hybridization assay.
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A real-time PCR hybridization assay for Legionella pneumophila is described; the assay uses LightCycler (Idaho Technology) methodology to specifically detect 2.5 CFU/reaction, equivalent to 1,000 CFU/liter of starting water sample. The assay, including DNA extraction and confirmation of product identity, is completed within 90 min of receipt of a sample. (+info)
Persistent Legionella infection in a patient after bone marrow transplantation.
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We report on a patient who developed Legionella pneumonia after bone marrow transplantation. Despite appropriate antibiotic treatment, disease progressed. The patient developed a lung abscess from which Legionella and Prevotella were isolated. Cure was achieved by surgical resection. The resected material was sterile, but 16S ribosomal DNA analysis revealed Legionella DNA. (+info)
Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles.
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After ingestion by macrophages, Legionella pneumophila inhibits acidification and maturation of its phagosome. After a 6-10-h lag period, the bacteria replicate for 10-14 h until macrophage lysis releases dozens of progeny. To examine whether the growth phase of intracellular L. pneumophila determines the fate of its phagosome, interactions between the endosomal network and pathogen vacuoles were analyzed throughout the primary infection period. Surprisingly, as L. pneumophila replicated exponentially, a significant proportion of the vacuoles acquired lysosomal characteristics. By 18 h, 70% contained lysosomal-associated membrane protein 1 (LAMP-1) and 40% contained cathepsin D; 50% of the vacuoles could be labeled by endocytosis, and the pH of this population of vacuoles averaged 5.6. Moreover, L. pneumophila appeared to survive and replicate within lysosomal compartments: vacuoles harboring more than five bacteria also contained LAMP-1, inhibition of vacuole acidification and maturation by bafilomycin A1 inhibited bacterial replication, bacteria within endosomal vacuoles responded to a metabolic inducer by expressing a gfp reporter gene, and replicating bacteria obtained from macrophages, but not broth, were acid resistant. Understanding how L. pneumophila first evades and then exploits the endosomal pathway to replicate within macrophages may reveal the mechanisms governing phagosome maturation, a process also manipulated by Mycobacteria, Leishmania, and Coxiella. (+info)
Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and studies of KatA function.
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Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L. pneumophila and Escherichia coli and compared with prior studies of katB in this laboratory. katA is induced during exponential growth and is the predominant peroxidase in stationary phase. When katA is inactivated, L. pneumophila is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L. pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen. (+info)