Effector Glycosyltransferases in Legionella

Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP–glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells.In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA (subversion of eukaryotic vesicle trafficking A), which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure–function relationship of Lgt glucosyltransferases

Aminoacyl-tRNA-Charged Eukaryotic Elongation Factor 1A Is the Bona Fide Substrate for Legionella pneumophila Effector Glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNAPhe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNAHis and Phe-tRNAPhe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's

Protein glutaminylation is a yeast-specific posttranslational modification of elongation factor 1A

Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the α amino group of mono-l-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae. However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe but not in various other eukaryotic organisms tested despite strict conservation of the Glu45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification that appears to influence protein translation

Glycosylating Effectors of Legionella pneumophila: Finding the Sweet Spots for Host Cell Subversion

Work over the past two decades clearly defined a significant role of glycosyltransferase effectors in the infection strategy of the Gram-negative, respiratory pathogen Legionella pneumophila. Identification of the glucosyltransferase effectors Lgt1-3, specifically modifying elongation factor eEF1A, disclosed a novel mechanism of host protein synthesis manipulation by pathogens and illuminated its impact on the physiological state of the target cell, in particular cell cycle progression and immune and stress responses. Recent characterization of SetA as a general O-glucosyltransferase with a wide range of targets including the proteins Rab1 and Snx1, mediators of membrane transport processes, and the discovery of new types of glycosyltransferases such as LtpM and SidI indicate that the vast effector arsenal might still hold more so-far unrecognized family members with new catalytic features and substrates. In this article, we review our current knowledge regarding these fascinating biomolecules and discuss their role in introducing new or overriding endogenous post-translational regulatory mechanisms enabling the subversion of eukaryotic cells by L. pneumophila

Glycosylating Effectors of Legionella pneumophila: Finding the Sweet Spots for Host Cell Subversion

Work over the past two decades clearly defined a significant role of glycosyltransferase effectors in the infection strategy of the Gram-negative, respiratory pathogen Legionella pneumophila. Identification of the glucosyltransferase effectors Lgt1-3, specifically modifying elongation factor eEF1A, disclosed a novel mechanism of host protein synthesis manipulation by pathogens and illuminated its impact on the physiological state of the target cell, in particular cell cycle progression and immune and stress responses. Recent characterization of SetA as a general O-glucosyltransferase with a wide range of targets including the proteins Rab1 and Snx1, mediators of membrane transport processes, and the discovery of new types of glycosyltransferases such as LtpM and SidI indicate that the vast effector arsenal might still hold more so-far unrecognized family members with new catalytic features and substrates. In this article, we review our current knowledge regarding these fascinating biomolecules and discuss their role in introducing new or overriding endogenous post-translational regulatory mechanisms enabling the subversion of eukaryotic cells by L. pneumophila

Lgt: a Family of Cytotoxic Glucosyltransferases Produced by Legionella pneumophila▿ †

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 ∼60-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

Isotopic composition of oxygen, carbon, and sulfur in interstitial water and cores at DSDP Leg 59 Holes

Comprehensive isotopic studies based on data from the Deep Sea Drilling Project have elucidated numerous details of the low- and high-temperature mechanisms of interaction between water and rocks of ocean crustal seismic Layers 1 and 2. These isotopic studies have also identified climatic changes during the Meso-Cenozoic history of oceans. Data on the abundance and isotopic composition of sulfur in the sedimentary layer as well as in rocks of the volcanic basement are more fragmentary than are oxygen and carbon data. In this chapter we specifically concentrate upon isotopic data related to specific features of the mechanisms of low-temperature interaction of water with sedimentary and volcanogenic rocks. The Leg 59 data provide a good opportunity for such lithologic and isotopic studies, because almost 600 meters of basalt flows and sills interbedded with tuffs and volcaniclastic breccias were cored during the drilling of Hole 448A. Moreover, rocks supposedly exposed to hydrothermal alteration play an important role at the deepest horizons of that mass. Sulfur isotopic studies of the character of possible biogenic processes of sulfate reduction in sediments are another focus, as well as the nature and origin of sulfide mineralization in Layer-2 rocks of remnant island arcs. Finally, oxygen and carbon istopic analyses of biogenic carbonates in the cores also enabled us to investigate the effects of changing climatic conditions during the Cenozoic. These results are compared with previous data from adjacent regions of the Pacific Ocean. Thus this chapter describes results of isotopic analyses of: oxygen and sulfur of interstitial water; oxygen and carbon of sedimentary carbonates and of calcite intercalations and inclusions in tuffs and volcaniclastic breccias interbedded with basalt flows; and sulfur of sulfides in these rocks