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

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...

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Summary Mono‐glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide‐binding proteins of the Rho family. However, toxin‐induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin‐catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.