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Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL ). An insertion mutation was constructed in pilEL and introduced into theL. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that thepilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.

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SummaryExpression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli. Transcription of gspA is regulated by two promoters, one of which is regulated by the 32 heat-shock transcription factor. We utilized a gspA promoter fusion to a promoterless lacZ to probe the phagososmal 'microenvironment' for the kinetics of exposure of intracellular L. pneumophila to stress stimuli. Expression through the gspA promoter was constitutively induced by approx. 16-fold throughout the intracellular infection, and occurred predominantly through the 32-regulated promoter. Expression of the gspA promoter was induced approx. 4.5-fold, 5-, 11-and 9-fold upon exposure of L. pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild-type strain AA200. Compared to in vitrogrown wild-type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wildtype phenotypes were restored to strain AA224 by complementation with a plasmid containing wild-type gspA. There was no difference between the wild-type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to in vitro-grown bacteria, macrophage-grown and amoebae-grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli. Our data showed that regardless of the capacity of L. pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L. pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection. Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage-induced and stress-induced proteins within the intracellular environment.

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