THE USE OF DIFFERENTIAL DISPLAY-PCR TO ISOLATE AND CHARACTERIZE A LEGIONELLA-PNEUMOPHILA LOCUS INDUCED DURING THE INTRACELLULAR INFECTION OF MACROPHAGES

The differential display (DD)-PCR technique has been modified to ident ify prokaryotic cDNA fragments that are differentially induced by facu ltative intracellular bacteria in response to the intracellular enviro nment of eukaryotic cells. Several DD-PCR fragments identified from th e intracellular bacterium Legionella pneumophila were induced at 4 h p ost-infection of the U937 macrophage-like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the pr edicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosoma l locus containing the 700 bp fragment (eml, for early stage macrophag e-induced locus) was induced by intracellular bacteria during the firs t few hours post-infection of macrophages but the expression was downr egulated by 12 h post-infection. Transcription of eml was not growth p hase-related if, vitro, and was not affected by in vitro stress stimul i. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD-PCR produc t was cloned. Six mini-Tn 10 insertions in the 3.7 kb EcoRI fragment w ere recombined into the L. pneumophila chromosome. Compared to the wil dtype strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100-fold increas e in killing by macrophages during the first 5 h of the intracellular infection, and showed a 100-fold increase in killing during the first 24 h of infection of the amoeba Hartmanella vermiformis. The 6th mutan t had a phenotype indistinguishable from the wild-type strain. The cyt opathicity defect of the mutants to the U937 cells was restored to wil d-type levels by complementation of the mutants with a plasmid contain ing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragm ent containing eml is a novel L. pneumophila locus whose expression is uniquely induced by nonstress stimuli during early stages of the intr acellular infection of phagocytic cells. Expression of this locus is r equired for survival of L. pneumophila with in macrophages and within amoebae during early stages of the infection.