Abstract
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.
Highlights
Legionella pneumophila is responsible for severe pneumonia referred to as Legionnaires disease [1]
It was shown that a 45aa peptide, comprising aa29-72 of eukaryotic elongation factor 1A (eEF1A), is more efficiently modified than full length eEF1A [36], suggesting that a specific conformation of eEF1A is essential for glucosylation
Extensive search for additional factors in yeast cell lysate, which enhanced the efficiency of Legionella glucosyltransferase family (Lgt)-catalyzed glucosylation of eEF1A, revealed non-proteinaceous cytosolic components, comprising aminoacyl-tRNA and GTP
Summary
Legionella pneumophila is responsible for severe pneumonia referred to as Legionnaires disease [1]. After entering phagocytic host cells such as alveolar macrophages, Legionella survives and replicates intracellularly [4] To this end, Legionella produces a plethora of effector proteins, which are injected into the host cell cytosol via a type IVb secretion system (Dot/Icm system) [5,6,7] to subvert the phagosome into a specialized compartment known as ‘‘Legionella containing vacuole’’ (LCV) [8,9,10]. A few were biochemically validated and even less were characterized at the molecular level [8] Some of these effectors are known to target distinct regulatory host cell factors such as GTPases or ATPases [16,17,18,19,20,21,22,23] in order to transform the host cell into a replication permissive environment [8].
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