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- Title
Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant.
- Authors
Kwaik, Yousef Abu; Lian-Yong Gao; Herb, Omar S.; Stone, Barbara J.
- Abstract
Expression of the global stress protein gene (<em>gspA</em>) is induced during the intracellular infection of macrophages and upon exposure of <em>Legionella pneumophila</em> to <em>in vitro</em> stress stimuli. Transcription of <em>gspA</em> is regulated by two promoters, one of which is regulated by the σ32 heat-shock transcription factor. We utilized a <em>gspA</em> promoter fusion to a promoterless lacZ to probe the phagososmal 'microenvironment' for the kinetics of exposure of intracellular <em>L. pneumophila</em> to stress stimuli. Expression through the <em>gspA</em> promoter was constitutively induced by approx. 16-fold throughout the intracellular infection, and occurred predominantly through the σ32-regulated promoter. Expression of the <em>gspA</em> promoter was induced approx. 4.5-fold, 5-, 11- and 9-fold upon exposure of <em>L. pneumophila</em> to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in <em>gspA</em> (strain AA224) was constructed by allelic exchange in the wild-type strain AA200. Compared to <em>in vitro</em>-grown 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 <em>gspA</em>. There was no difference between the wild-type strain and the <em>gspA</em> mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to <em>in vitro</em>-grown bacteria, macrophage-grown and amoebae-grown AA200 and AA224 showed an equal and dramatic increase in resistance to <em>in vitro</em> stress stimuli. Our data showed that regardless of the capacity of <em>L. pneumophila</em> to subvert the microbicidal mechanisms of the macrophage, intracellular <em>L. pneumophila</em> 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 <em>in vitro</em> 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.
- Subjects
HEAT shock proteins; GENETIC transcription; LEGIONELLA pneumophila; MACROPHAGES; AMOEBIDA
- Publication
Molecular Microbiology, 1997, Vol 24, Issue 3, p629
- ISSN
0950-382X
- Publication type
Article
- DOI
10.1046/j.1365-2958.1997.3661739.x