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Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

ABSTRACT: Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA(-) strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.

SUBMITTER: Jager J

PROVIDER: S-EPMC3911869 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

Jäger Jens J Marwitz Sebastian S Tiefenau Jana J Rasch Janine J Shevchuk Olga O Kugler Christian C Goldmann Torsten T Steinert Michael M

Infection and immunity 20131028 1

Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replicati ...[more]

PMID: 24166955

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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.

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Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

Publications

Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

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