Identification of protein antigens of Legionella pneumophila serogroup 1

Pearlman, Eric; Engleberg, N. Cary; Eisenstein, Barry I.

doi:10.1128/iai.47.1.74-79.1985

Cited by 19 publications

(10 citation statements)

References 26 publications

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“…In this report we present an additional biochemical characterization of the 24-kDa antigen, including confirmation of its separate identity from the previously recognized major outer membrane protein (MOMP) with which it comigrates (5,11,16,23,35). We also present the DNA sequence of mip and the inferred amino acid sequence of the 24-kDa antigen.…”

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confidence: 66%

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DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity

et al. 1989

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In a previous study, a 24-kilodalton (kDa) protein surface antigen of Legionella pneumophila was cloned into Escherichia coli and found to be expressed on the host cell surface. Subsequently, a site-directed mutation in this gene (designated mip) in L. pneumophila was found to impair the capacity of this bacterium to initiate intracellular infection in human macrophages. The work presented here indicates that the antigenic gene product is distinct from the 24to 29-kDa major outer membrane protein of L. pneumophila. In addition,.the antigen was identified as a highly basic protein on two-dimensional nonequilibrium polyacrylamide gels and on two-dimensional monoclonal antibody immunoblots. When the DNA fragment encoding this protein was sequenced, a long open reading frame of 699 base pairs was identified within a region to which antigen expression was previously mapped. mip mRNA isolated from both L. pneumophila and transformed E. coli had the same 5' end, as determined by primer extension analysis, indicating that the same promoter sequences are used in both species. A likely factor-independent transcriptional terminator was found 20 residues downstream of the stop codon, suggesting that mip is encoded on a moniocistronic message. The inferred polypeptide began with a possible 20to 24-residue signal sequence, and, as predicted by two-dimensional electrophoresis, had a molecular weight of 24,868 and was a potent polycation with an estimated pl of 9.8. In previous reports we have described the identification and molecular cloning of a major, 24,000-dalton (Da) surface antigen of Legionella pneumophila (14, 35). This protein is expressed from a cloned fragment on a multicopy plasmid in Escherichia coli and is localized to the surface of the host E. coli strain (15). Recently, we exchanged a mutated copy of this gene for the wild-type gene in L. pneumophila to yield a mutant strain that produced no detectable 24-kDa antigen (8). Although this mutant replicated as well as its isogenic parent within macrophages, it was approximately 80-fold * Corresponding author.

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confidence: 66%

“…We previously characterized the L. pneumophila 24-kDa antigen as a surface protein that induced strong antigenic reactivity in rabbits immunized with killed bacteria (35). Given the observation of others that the purified MOMP is also an important antigen (16), we inferred that these molecules are one and the same.…”

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confidence: 97%

DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity

et al. 1989

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“…The identification was based on colonial and cellular morphology, biochemical reactions, fermentation patterns, use of chromogenic substrates (An-Ident; API, Plainville, N.Y.), and gas-liquid chromatographic analysis of metabolic end products (9,16,18). Selected strains were additionally characterized by SDSpolyacrylamide gel electrophoresis of precipitated and soluble proteins by the method of Pearîman et al (26) with modifications (S. A. Kinder, K. S. Kornman, and S. C.…”

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confidence: 99%

Penicillin resistance in the subgingival microbiota associated with adult periodontitis

Kinder¹,

Holt²,

Korman³

1986

J Clin Microbiol

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In this investigation, the penicillin-resistant and beta-lactamase-producing subgingival microbiota associated with adult periodontitis was identified, and the impact of a recent exposure to penicillin on the recovery of resistant organisms from this microbiota was assessed. Subjects with adult periodontitis were examined clinically and microbiologically. Twenty-one subjects had a documented history of penicillin therapy within the previous 6 months whereas an additional 21 subjects had no history of antibiotic use within 1 year. Subgingival plaque samples were cultured anaerobically on nonselective and penicillin-containing elective media. MICs and beta-lactamase production were determined for the isolates from the elective medium. The penicillin-resistant microbiota consisted primarily of gram-negative organisms, including Bacteroides, Veillonella, Haemophilus, Eikenella, and Capnocytophaga species. The prevalence (P < 0.05) and proportions (P < 0.005) of both penicillin-resistant pigmented Bacteroides and Veillonella species were significantly greater in subjects with recent penicillin exposure. Of the penicillin-resistant genera identified, beta-lactamase production was detected in species of pigmented Bacteroides, Capnocytophaga, and Streptococcus. The prevalence of beta-lactamaseproducing Bacteroides species was significantly greater in subjects with recent penicillin exposure (P < 0.05). Of the antibiotics examined, no single agent was uniformly effective against all of the penicillin-resistant strains, but metronidazole and clindamycin were active against all of the penicillin-resistant pigmented Bacteroides strains.

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“…To identify targets for mutagenesis, we cloned genes encoding surface protein antigens specific to the pathogenic L. pneumophila species (6,(9)(10)(11)24), reasoning that among the factors necessary for macrophage infection some would have these characteristics. Based solely on these immunochemical data (i.e., no functional information), we chose the gene encoding a 24,000-dalton (Da) L. pneumophila-specific antigen as a target for a site-specific mutation.…”

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A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection

et al. 1989

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To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intraceilularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.

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Identification of protein antigens of Legionella pneumophila serogroup 1

Cited by 19 publications

References 26 publications

DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity

DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity

Penicillin resistance in the subgingival microbiota associated with adult periodontitis

A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection

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