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.