Lgt: a Family of Cytotoxic Glucosyltransferases Produced by Legionella pneumophila

Abstract

Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the ϳ60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila, which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella-host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella.The protein synthesis machinery of eukaryotic cells is a wellknown target for pathogenic microorganisms during hostpathogen interaction. Examples of bacterial protein toxins targeting host protein synthesis include Shiga-and Shiga-like toxins, which act as rRNA N-glycosidases, and diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A, which ADPribosylate the modified histidine residue diphthamide in eukaryotic elongation factor 2 (eEF2). Both types of enzymatic activities result in inhibition of protein synthesis and death of target cells (26,34).Recently, an ϳ60-kDa glucosyltransferase, referred to here as Lgt1 (Legionella pneumophila glucosyltransferase 1), was identified in L. pneumophila cultures. Well-studied examples of bacterial glucosylating enzymes targeting eukaryotic proteins are the large clostridial cytotoxins. They glucosylate 20-to 25-kDa small GTPases of the Rho family, thereby inhibiting the regulatory functions of these switch proteins (1, 16). In contrast, the Legionella enzyme modified an ϳ50-kDa component in mammalian cell extracts, which was identified subsequently as eEF1A. This elongation factor also represents a GTP-binding protein, possessing GTPase activity. Lgt1 modifies serine-53 of eEF1A, located in the GTPase domain near the switch 1 region of the GTPase. This modification results in inhibition of protein synthesis both in vitro and in vivo and causes death of intoxicated eukaryotic cells (3, 4).Many Legionella proteins occur in a set of redundant molecules, executing apparently closely related functions (6,12,18,27,28). Therefore, we screened Legionella genome databases for Lgt1 analogs. Here we report that L. pneumophila strains Philadelphia-1, Lens, Paris, and Corby (GenBank accession numbers NC_002942, NC_006369, NC_006368...

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.…”

“…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.…”

Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors

“…Modification by Lgt leads to arrest in protein synthesis. Although all 3 Lgt proteins appear functionally equivalent in vitro, they are differentially regulated, suggesting specific roles at different time points during infection (Belyi et al 2008). Similar to these glucosyltransferases, the effector SidI inhibits protein translation by targeting eEF1A and, with lower affinity, eEF1B␥ (Shen et al 2009).…”