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.

FIG. 1.

Identification of a new UDP-glucosyltransferase activity. Ultrasonic extracts from different Legionella species were incubated in the presence of HeLa cell extract and [¹⁴C]UDP-glucose. After this, the samples were subjected to SDS-PAGE, dried, and exposed to X-ray film. Lane 1, L. pneumophila Philadelphia I; lane 2, L. pneumophila 130b; lane 3, L. pneumophila ATCC 33823; lane 4, L. pneumophila ATCC 35096; lane 5, L. longbeachae ATCC 33462; lane 6, L. gormanii ATCC 33297; lane 7, L. steigerwaltii ATCC 35302; lane 8, L. pneumophila Philadelphia I without eukaryotic substrate; lane 9, HeLa cell extract without bacterial extract. The data represent data obtained in several experiments.

FIG. 2.

Identification of a substrate for L. pneumophila UDP-glucosyltransferase in different eukaryotic cell extracts. Ultrasonic extracts from the L. pneumophila Philadelphia I strain were incubated in the presence of HeLa (lane 1), Vero (lane 2), CHO (lane 3), and THP-1 (lane 4) cell preparations and [¹⁴C]UDP-glucose. After this, the samples were subjected to SDS-PAGE, dried, and exposed to X-ray film.

FIG. 3.

Purification of a UDP-glucosyltransferase from L. pneumophila. Samples were subjected to SDS-PAGE and stained with Coomassie brilliant blue R250. Lanes 1 and 5, molecular mass markers; (from top to bottom, 94, 67, 43, and 30 kDa; Amersham Biosciences); lanes 2 and 3, ultrasonic lysate of L. pneumophila Philadelphia I containing 30 and 15 μg of protein, respectively; lane 4, purified, 60-kDa UDP-glucosyltransferase (ca. 0.5 μg).

FIG. 4.

Enzymatic activity of UDP-glucosyltransferase purified from L. pneumophila and recombinant E. coli. Approximately 0.5 μg of purified native protein (lanes 1 and 3) or recombinant protein (lanes 2 and 4) was incubated in the presence of [¹⁴C]UDP-glucose with (lanes 1 and 2) or without (lanes 3 and 4) HeLa cell extract. After this, the samples were subjected to SDS-PAGE, dried, and exposed to X-ray film.

FIG. 5.

PCR detection of the L. pneumophila UDP-glucosyltransferase gene. Based on nucleotide sequence data for L. pneumophila Philadelphia I, specific primers were used to PCR amplify DNA fragments from purified genomic preparations. Following amplification, the DNA mixtures were subjected to agarose gel electrophoresis and stained with ethidium bromide. Lane 1, molecular mass markers (from top to bottom, 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1 [threefold intensity], and 0.5 kb; Amersham Biosciences); lane 2, L. pneumophila Philadelphia I; lane 3, L. pneumophila 130b; lane 4, L. pneumophila ATCC 33823; lane 5, L. pneumophila ATCC 35096; lane 6, L. longbeachae ATCC 33462, lane 7, L. gormanii ATCC 33297; lane 8, L. steigerwaltii ATCC 35302.

FIG. 6.

SDS-PAGE analysis of recombinant L. pneumophila UDP-glucosyltransferase. Lane 1, crude lysate of E. coli BL21(p28b-13); lane 2, purified recombinant UDP-glucosyltransferase; lane 3, UDP-glucosyltransferase purified from L. pneumophila; lane 4, molecular mass markers (94, 67, 43, 30, 20, and 14 kDa). Because of its N-terminal polyhistidine tag, the recombinant enzyme migrated more slowly than the native enzyme.

FIG. 7.

Immunoblot analysis of different Legionella strains. Ultrasonic extracts were obtained from various legionellae grown on BCYE agar, and then samples containing approximately 5 μg of protein were subjected to SDS-PAGE, transferred onto a nitrocellulose filter, and probed with an antiserum raised against purified L. pneumophila UDP-glucosyltransferase. Lane 1, UDP-glucosyltransferase purified from L. pneumophila; lane 2, purified recombinant UDP-glucosyltransferase; lane 3, L. pneumophila Philadelphia I; lane 4, L. pneumophila 130b; lane 5, L. pneumophila ATCC 33823; lane 6, L. pneumophila ATCC 35096; lane 7, L. longbeachae ATCC 33462; lane 8, L. gormanii ATCC 33297; lane 9, L. steigerwaltii ATCC 35302. Minor bands observed in recombinant protein and some L. pneumophila preparations probably resulted from proteolytic degradation of a major 60-kDa enzyme.