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Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

ABSTRACT: With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625-positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625-positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS.

SUBMITTER: Luneberg E

PROVIDER: S-EPMC2525541 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

Lüneberg E E Zähringer U U Knirel Y A YA Steinmann D D Hartmann M M Steinmetz I I Rohde M M Köhl J J Frosch M M

The Journal of experimental medicine 19980701 1

With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625-positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement com ...[more]

PMID: 9653083

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Project description:Several novel Legionella pneumophila virulence genes were previously discovered by use of signature-tagged mutagenesis (P. H. Edelstein, M. A. Edelstein, F. Higa, and S. Falkow, Proc. Natl. Acad. Sci. 96:8190-8195, 1999). One of these mutants appeared to be defective in multiplication in guinea pig lungs and spleens, yet it multiplies normally in guinea pig alveolar macrophages. Here we report further characterization of the mutated gene and its protein and the virulence role of the gene. The complete sequence of the gene, now called lvgA, is 627 bp long, and its protein product is approximately 27 kDa in size. lvgA was present in all 50 strains of L. pneumophila tested. No significant nucleic acid or protein homology was found in the GenBank database for the gene, nor were any distinctive motifs discovered in a search of other databases. The expression of both DotA and IcmX in the lvgA mutant was normal. Subcellular fractionation studies localized LvgA to the outer membrane fraction, and protease digestion studies suggested that at least some of the protein is surface expressed. No change in bacterial lipopolysaccharide composition or reactivity to serogroup-specific antisera was detected in the mutant. Growth competition studies with alveolar macrophages showed that the mutant was outcompeted by its parent 3-fold in 24 h and 24-fold in 48 h, in contrast to what was observed with the null phenotype in parallel testing with alveolar macrophages or with the A549 alveolar epithelial cell line. This macrophage defect of the mutant bacterium was due to slower growth, as the mutant invaded alveolar macrophages normally. Electron microscopy showed that the mutant bacterium resided in a ribosome-studded phagosome in alveolar macrophages, with no distinction from its parent. The lvgA mutant was outcompeted by its parent about sixfold in guinea pig lungs and spleens; prolonged observation of infected animals showed no late-onset virulence of the mutant. Transcomplementation of the mutant restored the parental phenotype in guinea pigs. The lvgA mutant was twofold more susceptible to killing by human beta-defensin 2 but not to killing by other cationic peptides, serum complement, or polymorphonuclear neutrophils. lvgA is a novel virulence gene that is responsible for pleiotropic functions involving both extracellular and intracellular bacterial resistance mechanisms.

Project description:Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different MLVA-8 (multilocus variable-number tandem-repeat analysis using 8 loci) genotypes dominated different sites of a drinking-water distribution system. Each genotype displayed a unique temperature-dependent growth behavior. Here we compared the pathogenicity potentials of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity toward amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity toward amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity toward macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strains showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective toward Acanthamoeba castellanii Moreover, Gt4 strains exhibited increased cytotoxicity toward macrophages and demonstrated a broader temperature range of amoebal lysis than Gt6 and Gt15 strains. Understanding the virulence traits of Legionella genotypes may improve the assessment of public health risks of Legionella in drinking water.IMPORTANCELegionella pneumophila is the causative agent of a severe form of pneumonia. Here we demonstrated that clinical strains were significantly more cytotoxic toward red blood cells than environmental strains, while their cytotoxicity toward macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic toward amoebae and macrophages and lysed amoebae in a broader temperature range than to the other studied genotypes. The results can explain the relatively high success of Gt4 in the environment and in clinical samples; thus, Gt4 strains should be considered a main factor for the assessment of public health risks of Legionella in drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of different L. pneumophila genotypes, which can be a valuable parameter for future modeling and quantitative microbial risk assessment of Legionella in drinking-water systems.

Project description:The Legionella pneumophila TF3/1 mutant of the Corby strain, which possesses a point mutation in the active site of the O-acetyltransferase, synthesized the polysaccharide chain with a reduced degree of substitution with O-acetyl groups. The mutant did not produce a high-molecular-weight lipopolysaccharide (LPS) fraction above 12 kDa. The disturbances in LPS synthesis have an effect on the composition of other macromolecules (lipids and proteins), as indicated by differences in the infrared absorption spectra between the L. pneumophila Corby strain and its TF3/1 mutant. The wild-type strain contained less N+-CH3 and C-N groups as well as more CH3 groups than the mutant. The fatty acid composition showed that the wild type strain synthesized more branched acyl residues (a15:0, i16:0, and a17:0), a less unsaturated acid (16:1), and a straight-chain acid (18:0) than the mutant. The mutant synthesized approximately twice more a long-chain fatty acid (20:0) than the wild type. The main differences in the phospholipids between both strains were found in the classes of phosphatidylcholines and phosphatidylglycerols (PG). Substantial differences in the cell surface topography of these bacteria and their nanomechanical properties were shown by atomic force microscopy (AFM). The wild type strain had no undulated surface and produced numerous vesicles. In the case of the mutant type, the vesicles were not numerous, but there were grooves on the cell surface. The average roughness of the cell surface of the mutant was approximately twofold higher than in the wild-type strain. In turn, the wild-type strain exhibited much better adhesive properties than the mutant. The kinetic study of the interaction between the L. pneumophila strains and Acanthamoeba castellanii monitored by Förster resonance energy transfer revealed a pronounced difference, i.e., almost instantaneous and highly efficient binding of the L. pneumophila Corby strain to the amoeba surface, followed by penetration into the amoeba cells. This process was clearly not as efficient in the case of the mutant. The results point to LPS and, in particular, to the length of the polysaccharide fraction as an important L. pneumophila determinant involved in the process of adhesion to the host cell.

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Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

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Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

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