Purification and Characterization of a UDP-Glucosyltransferase Produced by Legionella pneumophila

Abstract

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.Legionella pneumophila is the principal agent of Legionnaires' disease, a pneumonic illness of humans. This bacterium is a facultative intracellular parasite that invades and proliferates in both professional and nonprofessional phagocytes (54). The host cells for L. pneumophila include freshwater protozoans and human alveolar macrophages and epithelial cells. Bacterial factors that have been identified as crucial for intracellular infection include, among others, the major outer membrane protein (25), Mip (13), Hsp60 (17, 18), PilD and type II protein secretion (4, 43), type IV pili (29, 51), flagella (41), the Dot/Icm type IV protein secretion system, and the products of the enh, lvh, mil, and pmi loci (2,11,14,16,35,49). In addition to these virulence factors, a variety of enzymes have been identified in Legionella cultures, including a lowmolecular-mass cytotoxin (30), a metalloprotease cytolysin (8, 9), phospholipases (3, 4, 6), lipases (3), phosphatases (5, 44) and phosphokinases (45). For many of these activities, the role in virulence is unclear and/or the eukaryotic substrate(s) is unknown. Thus, further studies aimed at identifying and characterizing novel L. pneumophila enzymes should be important for understanding both Legionnaires' disease and bacterial interactions with eukary...

Lgt: a Family of Cytotoxic Glucosyltransferases Produced by Legionella pneumophila

“…Our previous data suggested that by inhibiting protein synthesis in target cells, the glucosyltransferase Lgt1 plays an important role in the virulence of L. pneumophila (3,4). Therefore, we studied whether the L. pneumophila genome database contains more than one copy of the corresponding gene or any other homologous sequences.…”

“…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).…”

Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A

“…Eukaryotic cell extracts (proteins at 7-10 mg͞ml) used as substrates in the reaction were prepared by sonication of Caco-2 or EBL cells (17,28). Glucosylation reaction was carried out in 20 l of a mixture consisting of 20 mM Tris⅐HCl (pH 7.5), 150 mM NaCl, 1 mM MnCl 2 , 2-5 g of recombinant Legionella enzyme, and 50 -70 g of crude cell extract or 2-4 g of purified recombinant eEF1A1 or eEF1A2 (the EF-coding plasmids were gifts from Charlotte R. Knudsen, University of Aarhus, Aarhus, Denmark) and 10 M of UDP-[ 14 C]glucose (American Radiolabeled Chemicals, St. Louis, MO).…”

“…Recently, a 60-kDa glucosyltransferase from L. pneumophila was identified that modifies an Ϸ50-kDa protein in eukaryotic cells (17). The corresponding gene in L. pneumophila chromosome (accession no.…”

Elongation Factor 1A Is the Target of Growth Inhibition in Yeast Caused by Legionella pneumophila Glucosyltransferase Lgt1

“…These enzymes use UDPglucose as a cofactor and target eukaryotic substrates by covalent attachment of a glucosyl moiety (7). The crystal structure of Lgt1 has been solved, allowing the characterization of the Legionella enzyme as a GT-A glucosyltransferase structurally related to clostridial glucosylating toxins (8,9).…”