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Intracellular survival and expression of virulence determinants of Legionella pneumophila
Abstract:
Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as well as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human embryo lung fibroblastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further studies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella.
Insights
Legionella pneumophila, the cause of Legionnaires' disease, loses virulence in lab cultures. Researchers identified five potential virulence factors, including novel toxins, aiding intracellular survival studies.
Area of Science:
- Microbiology
- Cell Biology
- Pathogenesis
Background:
- Legionella pneumophila causes Legionnaires' disease by surviving within host cells.
- The bacterium inhibits phagosome-lysosome fusion and acidification.
- In vitro cultivation leads to a loss of virulence in Legionella strains.
Purpose of the Study:
- To investigate the mechanisms of intracellular survival of Legionella.
- To identify virulence factors of Legionella pneumophila.
- To clone and characterize genes involved in Legionella pathogenesis.
Main Methods:
- Construction of a genomic library of Legionella pneumophila in Escherichia coli.
- Cosmid cloning technique to isolate putative virulence factors.
- Cell culture systems using human macrophages and lung fibroblasts.
Main Results:
- Five putative virulence factors were cloned.
- Two cloned factors exhibited hemolytic activity; one is legiolysin, a novel toxin lysing human erythrocytes.
- Three cloned factors were membrane-associated proteins (19, 26, 60 kDa).
- A second hemolysin showed proteolytic and cytolytic properties.
Conclusions:
- Legionella pneumophila virulence factors have been identified.
- Novel toxins and membrane proteins involved in pathogenesis were cloned.
- Further research is needed to elucidate the precise roles of these factors in Legionnaires' disease.