The use of differential display‐PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages

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Abstract
The differential display (DD)-PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells. Several DD-PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post-infection of the U937 macrophage-like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage-induced locus) was induced by intracellular bacteria during the first few hours post-infection of macrophages but the expression was downregulated by 12 h post-infection. Transcription of eml was not growth phase-related in vitro, and was not affected by in vitro stress stimuli. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD-PCR product was cloned. Six mini-Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L. pneumophila chromosome. Compared to the wild-type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100-fold increase in killing by macrophages during the first 5 h of the intracellular infection, and showed a 100-fold increase in killing during the first 24h of infection of the amoeba Hartmanella vermiformis. The 6th mutant had a phenotype indistinguishable from the wild-type strain. The cytopathicity defect of the mutants to the U937 cells was restored to wild-type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragment containing eml is a novel L. pneumophila locus whose expression is uniquely induced by non-stress stimuli during early stages of the intracellular infection of phagocytic cells. Expression of this locus is required for survival of L. pneumophila within macrophages and within amoebae during early stages of the infection.