Identification of Legionella pneumophila -Specific Genes by Genomic Subtractive Hybridization with Legionella micdadei and Identification of lpnE , a Gene Required for Efficient Host Cell Entry

Abstract

Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5 end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for "L. pneumophila entry." In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.

Attenuation and protective efficacy of an O-antigen-deficient mutant of Francisella tularensis LVS

“…One approach to identify such virulence factors is to identify genes present in virulent F. tularensis that are absent or have low homology to genes in closely related F. novicida. SSH has been used to successfully identify virulence genes present in pathogens that are absent in closely related species (Bernier & Sokol, 2005;DeShazer et al, 2001;Harakava & Gabriel, 2003;Liu et al, 2003;Newton et al, 2006;Parsons et al, 2003;Winstanley, 2002), and was therefore used to identify genes uniquely expressed by the more virulent type A and B strains. Seventy-six LVS-specific genes with hypothetical or known functions were identified in eight genomic regions in multiple SSH clones (Ahmed & Inzana, 2004).…”

Enzymatic Properties of an Ecto-nucleoside Triphosphate Diphosphohydrolase from Legionella pneumophila

“…Intracellular Replication of L. pneumophila in THP-1 Macrophages-The ability of L. pneumophila 130b and recombinant derivatives to replicate over a 72-h time period in the monocytic cell line THP-1 was assayed as described previously (37). Briefly, THP-1 cells were seeded into 24-well tissue culture trays (Sarstedt, Leicestershire, UK) at a density of 5ϫ10 5 cells/ well and pretreated with 10 Ϫ8 M phorbol 12-myristate 13-acetate for 36 -48 h to induce differentiation into adherent macrophage-like cells.…”

“…Genes for the expression of site-directed Lpg1905 mutant proteins in L. pneumophila lpg1905::km were reconstructed to include the native ribosome-binding site and signal peptide and cloned into the Legionella expression vector pMIP (37). Because the large size of pMIP precluded use of the QuikChange II site-directed mutagenesis kit (Stratagene), primers 5Ј-CGGGATCCGACAACAAAATATACACAATG-TACCATTCG-3Ј and 5Ј-CCCAAGCTTTCAAGCGCGGTG-AAGCACGACTC-3Ј were used to amplify full-length lpg1905 including the ribosome-binding site for cloning into pGEM-TEasy (Promega).…”

Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

“…In bacteria, proteins containing TPR repeats are thought to have a role in gene regulation, flagellar motor function and virulence (Newton et al, 2007). Moreover in Legionella pneumophila, two genes encoding TPR-containing proteins, lpnE and enhC, have been shown to be associated with entry into human tissue culture cell lines (Cirillo et al, 2000;Newton et al, 2006).…”