Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A

Abstract

Legionella pneumophila, the causal agent of Legionnaires' disease, is an intracellular parasite and invades and proliferates within different eukaryotic cells, including human alveolar macrophages. After several 100-fold multiplication within host cells, the pathogens are released for new invasion by induction of apoptosis or necrosis. Here we report that L. pneumophila produces a glucosyltransferase, which selectively modifies an Ϸ50-kDa mammalian protein by using UDP-glucose as a cosubstrate. MS analysis identified the protein substrate as the mammalian elongation factor (EF)1A. Legionella glucosyltransferase modifies its eukaryotic protein substrate at serine-53, which is located in the GTPase domain of the EF. Glucosylation of EF1A results in inhibition of eukaryotic protein synthesis and death of target cells. Our findings show a mode of inhibition of protein synthesis by microbial pathogens and offer a perspective for understanding of the host-pathogen interaction of L. pneumophila.host-pathogen interaction ͉ coralent modification ͉ protein synthesis inhibition

Elongation Factor 1A Is the Target of Growth Inhibition in Yeast Caused by Legionella pneumophila Glucosyltransferase Lgt1

“…Addition of purified glucosyltransferase to S. cerevisiae translational extract inhibited protein synthesis in a dose-dependent manner. These results were similar to those obtained with mammalian reticulocyte lysates recently (12,13). Also similar to experiments with mammalian cells, expression of lgt1 in the cytosol of S. cerevisiae resulted in severe toxic effects.…”

“…Using a reticulocyte-based translation system, we recently showed that Lgt1 effectively blocks mammalian protein synthesis in vitro (12,13). Similar results were obtained, when translation extracts from S. cerevisiae were programmed with mRNA coding for firefly luciferase.…”

“…As shown in our previous studies, substitution of serine 53 by alanine turned elongation factor into a protein not modifiable in vitro by L. pneumophila glucosyltransferases Lgt (12,13). In the current study, we engineered S. cerevisiae strains, which contained such non-modifiable Tef1-S53A as the only elongation factor present.…”

“…As shown in our previous investigations with mammalian eEF1A, substitution of the glucose-accepting serine 53 of the elongation factor by an alanine residue resulted in a variant protein, which is not glucosylated by Lgt1 in vitro (12). Moreover, F54A, Y56A, and W58A substitutions severely affected glucosylation efficiency of mutated eEF1A-derived peptides, whereas G50A, K51A, G52A/K55A, and V59A produced only minor effects (10).…”

“…Glucosylation by Lgt1 parallels inhibition of protein synthesis both in vitro and in vivo and leads to death of target mammalian cells (12,13). Thus far, it is not clear whether the lethal effect of Lgt1 is linked solely to modification of eEF1A or glucosylation of Hbs1 also contributes to toxic effects caused by the Legionella enzyme.…”