Abstract
Bioinformatic analysis of the Legionella pneumophila Philadelphia 1 genome revealed four novel open reading frames (ORFs) that have no known prokaryotic homologues but are predicted to encode products with significant domain homology to eukaryotic proteins. The authors postulated that the gene products of these eukaryotic-like ORFs may interact with eukaryotic host cell proteins and thus play a role in the subversion of host cellular trafficking pathways by L. pneumophila and the establishment of the replicative niche inside host cells. RNA was extracted from stationary phase broths of L. pneumophila 130b using the Epicentre Masterpure RNA Purification Kit (MCR85102). cDNA was synthesized, and specific primers for each gene were used to amplify 200 to 1,100 base pair portions of each ORF. This revealed that all four ORFs were transcribed in stationary phase. To investigate the potential role that each eukaryotic-like gene under investigation may play in interactions with host cells, insertional mutants were constructed for each ORF. The chapter focuses on localization of the protein products of eukaryotic-like ORFs. To characterize the products of lpg1905 and lpg2644, a combination of epitope-tagging and specific polyclonal antibodies were used to localize the proteins within the bacteria.
Published Version
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