Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model

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Abstract
Legionella pneumophila is the cause of Legionnaires’ disease, which is a form of potentially fatal pneumonia. To identify genes required for virulence of the bacterium, a library of 1,386 L. pneumophila signature tagged transposon mutants was studied for guinea pig virulence. The mutants were screened in pools of 96 each in a guinea pig model of L. pneumophila pneumonia. Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model. All 16 mutants failed to multiply in both lungs and spleens. Four of the sixteen had no apparent defect for intracellular multiplication in macrophages. Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L. pneumophila virulence genes: dotB, dotF/icmG, dotO/icmB, icmX, and proA. Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized. With this methodology, we demonstrate the existence of L. pneumophila genes responsible for non-macrophage-related virulence. The discovery of L. pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens.