Characterization of the glucosyltransferase activity of Legionella pneumophila effector SetA.

Legionella pneumophila glucosyltransferase SetA, which is introduced into target cells by a type IV secretion system, affects the intracellular traffic of host cells. Here, we characterized the enzyme activity of the Legionella effector. We report that Asp118 and Arg121 of SetA are essential for glucohydrolase and glucotransferase activities. Exchange of Trp36 to alanine reduced the enzyme activity of SetA. All three amino acids were crucial for the cytotoxic effects of SetA in yeast. We observed that phosphatidylinositol-3-phosphate (PI3P) increased the glucosyltransferase activity of SetA severalfold, while the glucohydrolase activity was not affected. In the presence of PI3P, we observed the glucosylation of actin, vimentin and the chaperonin CCT5 in the cytosolic fraction of target cells. Studies on the functional consequences of glucosylation of skeletal muscle α-actin in vitro revealed inhibition of actin polymerization by glucosylation.

A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins.

Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP-N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. (1)H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

Domain organization of Legionella effector SetA.

Legionella pneumophila is a human pathogen causing severe pneumonia called Legionnaires' disease. Multiple Legionella effectors are type IV-secreted into the host cell to establish a specific vesicular compartment for pathogen replication. Recently, it has been reported that the Legionella effector SetA shares sequence similarity with glycosyltransferases and interferes with vesicular trafficking of host cells. Here we show that SetA possesses glycohydrolase and mono-O-glucosyltransferase activity by using UDP-glucose as a donor substrate. Whereas the catalytic activity is located at the N terminus of SetA, the C terminus (amino acids 401-644) is essential for guidance of SetA to vesicular compartments of host cells. EGFP-SetA expressed in HeLa cells localizes to early endosomes by interacting with phosphatidylinositol 3-phosphate. EGFP-SetA, transiently expressed in RAW 264.7 macrophages, associates with early phagosomes after infection with Escherichia coli and L. pneumophila. Only the combined expression of the C- and N-terminal domains induces growth defects in yeast similar to full-length SetA. The data indicate that SetA is a multidomain protein with an N-terminal glucosyltransferase domain and a C-terminal phosphatidylinositol 3-phosphate-binding domain, which guides the Legionella effector to the surface of the Legionella-containing vacuole. Both, the localization and the glucosyltransferase domains of SetA are crucial for cellular functions.