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Infect. Immun., 01 1998, 203-212, Vol 66, No. 1
Y Abu Kwaik
The eukaryotic protein synthesis inhibitor cycloheximid has been used by
many investigators to selectively radiolabel intracellular bacteria.
Although cycloheximide has no direct effect on bacterial gene expression,
there are concerns that long-term inhibition of the host cell protein
synthesis may have secondary effects on bacterial gene expression.
Therefore, prior to further identification and cloning of the
macrophage-induced (MI) genes of Legionella pneumophila, the effects of
cycloheximide on L. pneumophila-infected U937 cells were evaluated by
transmission electron microscopy. Inhibition of protein synthesis of the
host cell for 6 h had no major effect on the ultrastructure of the host
cell, on the formation of rough endoplasmic reticulum-surrounded
replicative phagosome, or on initiation of intracellular bacterial
replication. In contrast, by 15 h of cycloheximide treatment, there was
profound deterioration in the host cell as well as in the phagosome. To
examine protein synthesis by L. pneumophila during the intracellular
infection, U937 macrophage-like cells were infected with L. pneumophila,
and intracellular bacteria were radiolabeled during a 2-h cycloheximide
treatment or following 12 h of cycloheximide treatment. Comparison by
two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis
of the protein profile of radiolabeled in vitro-grown L. pneumophila to
that of intracellularly radiolabeled bacteria showed that 23 proteins were
induced in response to the intracellular environment during 2 h of
inhibition of host cell protein biosynthesis. Twelve MI proteins of L.
pneumophila were artifactually induced due to prolonged inhibition of the
host cell protein synthesis. The gene encoding a 20-kDa MI protein was
cloned by a reverse genetics technique. Sequence analysis showed that the
cloned gene encoded a protein that was 80% similar to the enzyme inorganic
pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene
showed that compared to in vitro-grown bacteria, expression of the
pyrophosphatase gene (ppa) was induced fourfold throughout the
intracellular infection. There was no detectable induction in transcription
of the ppa promoter during exposure to stress stimuli in vitro. The ppa
gene of L. pneumophila is the first example of a regulated ppa gene which
is selectively induced during intracellular infection and which may reflect
enhanced capabilities of macromolecular biosynthesis by intracellular L.
pneumophila. The data indicate caution in the long-term use of inhibition
of host cell protein synthesis to selectively examine gene expression by
intracellular bacteria.
Copyright © 1998, American Society for Microbiology
Induced expression of the Legionella pneumophila gene encoding a 20- kilodalton protein during intracellular infection
Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington 40536-0084, USA. yabukw@pop.uky.edu
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