Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila.
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.
Belyi
Iouri
I
Gamaleya Research Institute of Epidemiology and Microbiology, Moscow 123098, Russia.
Popoff
Michel R
MR
Cianciotto
Nicholas P
NP
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007875:Legionella / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014530:Uridine Diphosphate / Q000378:metabolism*
D014530:Uridine Diphosphate; D005964:Glucosyltransferases