Identification of novel loci involved in entry by Legionella pneumophila

Legionella pneumophila is primarily an intracellular pathogen during infect ion; thus, the mechanisms of entry into host cells are likely to be importa nt for pathogenesis. Several L. pneumophila mutants that display an enhance d-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type, In the course of character izing the genetic basis of one of these mutants, C3, a strategy was develop ed for the isolation of laboratory-media-repressed virulence determinants f rom L, pneumophila, Screens for dominant mutations using a genomic DNA libr ary from C3 resulted in the isolation of three cosmids that confer an Enh p henotype to wild-type L. pneumophila, Transposon mutagenesis of these cosmi ds allowed identification of three loci that affect entry, Analysis of the putative proteins encoded by these loci, designated rtxA and enhC, demonstr ated similarity to repeats in the structural toxin protein and the secreted SeI-1 protein from Caenorhabditis elegans, respectively. L, pneumophila rt xA and enhC mutants display significantly reduced entry into host cells, co mpared to wild-type bacteria, The phenotype that the cosmids containing the se loci confer is most likely due to elevated expression resulting from the ir presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth condi tions is generally applicable to the isolation of virulence determinants fr om L. pneumophila and other bacterial pathogens.