Ligionella pneumophila: Avirulent to virulent conversion through passage in cultured human embryonic lung fibroblasts

Wong, Mae C.; Peacock, William L.; McKinney, Roger M.; Wong, K. H.

doi:10.1007/bf01566594

Cited by 30 publications

(17 citation statements)

References 9 publications

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“…C and D) . Phagosomes of wild-type L. pneumophila could not be examined at 24 h after ingestion because monocyte monolayers containing these bacteria were destroyed by this time as a result of intracellular multiplication of these bacteria.…”

Section: Mutant L Pneumophila Do Not Form the Ribosome-lined Replicamentioning

confidence: 99%

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Horwitz

1987

The Journal of Experimental Medicine

157

122

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Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.

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Section: Mutant L Pneumophila Do Not Form the Ribosome-lined Replicamentioning

confidence: 99%

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Horwitz

1987

The Journal of Experimental Medicine

157

122

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“…In addition, the streptomycin resistance determinant on TnS was expressed in L. pneumophila.Legionella pneumophila and related species are fastidious facultative intracellular parasites for which determinants of virulence have not been clearly established. Continued propagation of virulent strains on laboratory media leads to a loss of virulence (15), which can be restored by passage of the avirulent strains in cell culture (24). Despite transitions between virulence and avirulence, no phenotypic markers have been found which correlate with loss of virulence.…”

mentioning

confidence: 99%

Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome

Keen¹,

Street²,

Hoffman³

1985

J Bacteriol

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Transposon TnS was introduced into Legionella pneumophila on plasmid pRK340, which is temperature sensitive for plasmid maintenance. The presence of plasmid DNA was confirmed by agarose gel electrophoresis and by coijugal transfer of the plasmid to Escherichia coli. TnS insertions were obtaied by culturing L. pneumophia at the nonpermissive temperature (43°C) on buffered charcoal-yeast extract agar containing kanamycin. Of the 260 kanamycin-resistant colonies picked, 220 failed to conjugate pRK340 to E. coli. Plasmid DNA was not visualized from eight randomly picked TnS-containing strains, and Southern hybridizations indicated that TnS, but not pRK340, inserted into multiple sites in the Legioneila chromosome. In addition, the streptomycin resistance determinant on TnS was expressed in L. pneumophila.Legionella pneumophila and related species are fastidious facultative intracellular parasites for which determinants of virulence have not been clearly established. Continued propagation of virulent strains on laboratory media leads to a loss of virulence (15), which can be restored by passage of the avirulent strains in cell culture (24). Despite transitions between virulence and avirulence, no phenotypic markers have been found which correlate with loss of virulence. Although plasmid-encoded virulence factors have been reported for other gram-negative bacteria (18,19), no correlation between plasmids and virulence has been made for the legionellae (1, 5). Genetic regulation of the transition from virulence to avirulence in the legionellae may be under control mechanisms similar to those recently described for Bordetella pertussis (23). Weiss and Falkow (23) demonstrated that the phase change (reversible conversion from virulence to avirulence) of B. pertussis was controlled by a chromosomal trans-acting gene product. However, before progress can be made towards resolving mechanisms of pathogenesis for the legionellae, it will be necessary both to develop a workable genetic system and to identify specific markers associated with virulence.Since transposon mutagenesis has proved to be an effective tool in studies of pathogenesis, we set out to develop a means for delivering transposons into the chromosome of L. pneumophila. It has recently been reported that Pseudomonas plasmids of the IncP-1 incompatibility group can be transferred to several Legionella species via conjugation (8). Chen et al. (6) have introduced TnS into several Legionella species on the broad-host-range suicide plasmid pJB4JI, but they did not demonstrate transposition. In this study we show that pRK340, a temperature-sensitive derivative of the IncP-1 plasmid RK2, can effectively deliver Tn5 to the chromosome of L. pneumophila.All strains and plasmids used in this study are listed in Table 1. L. pneumophila strains were maintained on ACES (N-(2-acetamido)-2-aminoethanesulfonic acid)-buffered charcoal-yeast extract (BCYE) agar (17), and batch cultures were grown in BYE medium (without charcoal) and harvested as previously described (12). All cultu...

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“…In order to study the factors and conditions which influence the ability of Legionella strains to survive in eukaryotic cells, different cell culture systems were established [6][7][8]. The macrophage-like cellline U937 which was derived from human histiocytic cells and HL60, which originate in human peripheral blood cells, are widely used for such studies.…”

Section: Intracellular Growth Of Legionellamentioning

confidence: 99%

Intrazelluläres Überleben und Expression der Virulenzdeterminanten von Legionella pneumophila

et al. 1991

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Ligionella pneumophila: Avirulent to virulent conversion through passage in cultured human embryonic lung fibroblasts

Cited by 30 publications

References 9 publications

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome

Intrazelluläres Überleben und Expression der Virulenzdeterminanten von Legionella pneumophila

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