Characterization of an Axenic Strain of <i>Hartmannella vermiformis</i> Obtained from an Investigation of Nosocomial Legionellosis

Fields, Barry S.; Nerad, Thomas A.; Sawyer, Thomas K.; King, Christopher H.; Barbaree, James M.; Martin, W. J.; Morrill, William E.; Sanden, Gary N.

doi:10.1111/j.1550-7408.1990.tb01269.x

Cited by 79 publications

(66 citation statements)

References 20 publications

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“…This species of amoeba is known to support the growth of L. pneumophila and was isolated from a clinical case of legionellosis (29). Both the rcp mutant and 130b were recovered in similar numbers from the supernatants overlying the infected monolayers until 24 h p.i.…”

Section: Resultsmentioning

confidence: 99%

“…Intracellular infection by L. pneumophila strains was assessed in human U937 cells (1,54,65,66,76) differentiated into macrophage-like cells by treatment with phorbol myristate acetate and in Hartmannella vermiformis amoebae (17,19,29,54,71). Assessment of the intracellular growth kinetics of L. pneumophila in U937 macrophages was performed as previously reported (54).…”

Section: Methodsmentioning

confidence: 99%

“…In the natural environment, Legionella species occupy the niches of soil and water, either free-living, in biofilms, or, more commonly, residing within amoebal hosts (29,69). Problems occur when legionellae enter and replicate in human-made water systems, from which inhalation of bacterium-ladened aerosols leads to the establishment of a pneumonia referred to as Legionnaires' disease (28).…”

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confidence: 99%

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Identification of Legionella pneumophila rcp , a pagP -Like Gene That Confers Resistance to Cationic Antimicrobial Peptides and Promotes Intracellular Infection

2001

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In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg 2؉ medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Identification of Legionella pneumophila rcp , a pagP -Like Gene That Confers Resistance to Cationic Antimicrobial Peptides and Promotes Intracellular Infection

2001

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“…vermiformis strain CDC-19(ATCC 50237) has been cloned and grown in axenic culture as a model for the study of the pathogenesis of L. pneumophila . This strain was isolated from a water source of an outbreak of nosocomial Legionnaires' disease in a hospital in South Dakota, and its presence in the potable water sites correlated with the presence of the epidemic strain of L. pneumophila (Breiman et al, 1990;Fields et al, 1990). The amoebae were maintained in ATCC culture medium 1034.…”

Section: Construction Of An Insertion Mutation In Gspamentioning

confidence: 99%

Transcriptional regulation of the macrophage‐induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant

Kwaik

Gao

Harb

et al. 1997

Molecular Microbiology

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SummaryExpression 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 32 heat-shock transcription factor. We utilized a gspA promoter fusion to a promoterless 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 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 vitrogrown wild-type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wildtype 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.

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“…(Adeleke et al, 1996). These bacteria derived their name from their ability to infect and multiply in amoebae in an identical way to members of the genus Legionella (Rowbotham, 1986 ;Fields et al, 1990). LLAP-3 was isolated from the sputum of a pneumonia patient, following co-culture of the sputum with amoebae.…”

Section: Introductionmentioning

confidence: 99%

Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species.

Adeleke¹,

Fields²,

Benson³

et al. 2001

International Journal of Systematic and Evolutionary Microbiology

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Characterization of an Axenic Strain of Hartmannella vermiformis Obtained from an Investigation of Nosocomial Legionellosis

Cited by 79 publications

References 20 publications

Identification of Legionella pneumophila rcp , a pagP -Like Gene That Confers Resistance to Cationic Antimicrobial Peptides and Promotes Intracellular Infection

Identification of Legionella pneumophila rcp , a pagP -Like Gene That Confers Resistance to Cationic Antimicrobial Peptides and Promotes Intracellular Infection

Transcriptional regulation of the macrophage‐induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant

Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species.

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