Identification of novel loci involved in entry by Legionella pneumophila

Legionella pneumophila is primarily an intracellular pathogen during infection; thus, the mechanisms of entry into host cells are likely to be important for pathogenesis. Several L. pneumophila mutants that display an enhanced-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type. In the course of characterizing the genetic basis of one of these mutants, C3, a strategy was developed for the isolation of laboratory-media-repressed virulence determinants from L. pneumophila. Screens for dominant mutations using a genomic DNA library from C3 resulted in the isolation of three cosmids that confer an Enh phenotype to wild-type L. pneumophila. Transposon mutagenesis of these cosmids allowed identification of three loci that affect entry. Analysis of the putative proteins encoded by these loci, designated rtxA and enhC, demonstrated similarity to repeats in the structural toxin protein and the secreted Sel-1 protein from Caenorhabditis elegans, respectively. L. pneumophila rtxA and enhC mutants display significantly reduced entry into host cells, compared to wild-type bacteria. The phenotype that the cosmids containing these loci confer is most likely due to elevated expression resulting from their presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth conditions is generally applicable to the isolation of virulence determinants from L. pneumophila and other bacterial pathogens.