INDUCED EXPRESSION OF THE LEGIONELLA-PNEUMOPHILA GENE ENCODING A 20-KILODALTON PROTEIN DURING INTRACELLULAR INFECTION

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 expres sion, there are concerns that long-term inhibition of the host cell pr otein synthesis may have secondary effects on bacterial gene expressio n. Therefore, prior to further identification and cloning of the macro phage-induced (MI) genes of Legionella pneumophila, the effects of cyc loheximide on L. pneumophila-infected U937 cells were evaluated by tra nsmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the hos t cell, on the formation of rough endoplasmic reticulum-surrounded rep licative phagosome, or on initiation of intracellular bacterial replic ation. In contrast, by 15 h of cycloheximide treatment, there aas prof ound deterioration in the host cell as well as in the phagosome. To ex amine protein synthesis by L. pneumophila during the intracellular inf ection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximid e treatment or following 12 h of cycloheximide treatment. Comparison b y two-dimensional sodium dodecyl sulfate-polyacrylamide gel electropho resis of the protein profile of radiolabeled in vitro grown L. pneumop hila to that of intracellularly radiolabeled bacteria showed that 23 p roteins were induced in response to the intracellular environment duri ng 2 h of inhibition of host cell protein biosynthesis. Twelve MI prot eins of L. pneumophila were artifactually induced due to prolonged inh ibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analy sis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion t o a promoterless lacZ gene showed that compared to in vitro-grown bact eria, expression of the pyrophosphatase gene (ppa) was induced fourfol d throughout the intracellular infection. There was no detectable indu ction in transcription of the ppa promoter during exposure to stress s timuli in vitro. The ppa gene of L. pneumophila is the first example o f a regulated ppa gene which is selectively induced during intracellul ar infection and,which may reflect enhanced capabilities of macromolec ular biosynthesis by intracellular L, pneumophila. The data indicate c aution in the long-term use of inhibition of host cell protein synthes is to selectively examine gene expression by intracellular bacteria.