A 22-kb DNA locus of Legionella pneumophila is described that contains 18 genes, 16 of which are required for macrophage killing (icm genes). In this paper two previously described icm loci were linked by the discovery of five genes located between the two loci. Four of the newly described genes are required for macrophage killing (icmMLKE) and one is dispensable. The 16 icm genes appeared to be organized as six individual genes (icmR, icmQ, icmG, icmC, icmD, and icmF), and four operons (icmTS, icmPO, icmMLKE, and icmJB). Four icm genes (icmP, icmO, icmL, and icmE) show significant sequence similarity to plasmid genes involved in conjugation, whereas the other icm genes were found not to bear any sequence similarity to database entries. We found that L. pneumophila can mediate plasmid DNA transfer at a frequency of 10 ؊3 to 10 ؊4 per donor. Strains containing null mutations in two icm genes (icmT and icmR) showed a severe reduction in conjugation frequency and macrophage killing. Strains containing an insertion in four other icm genes (icmF, icmE, icmC, and dotA) were shown to have a less severe defect in conjugation. Mutations in the other 11 icm genes had no effect on conjugation frequency. We currently do not know whether conjugation itself plays a role in macrophage killing. It is possible either that small plasmids can take advantage of an existing secretion system to be mobilized or that DNA transfer is required for human macrophage killing by L. pneumophila.Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen with a broad host range. The bacteria are able to infect, multiply within, and kill human macrophages, as well as free-living amoebae (1, 2). When inside host cells, L. pneumophila are found within a specialized phagosome that does not fuse with lysosomes (3). The bacteria multiply within the specialized phagosome, until the cell eventually lyses, releasing bacteria that can start new rounds of infection.Several years ago, a collection of 55 L. pneumophila mutants defective for macrophage killing were isolated from a large (n ϭ 4,500) pool of Tn903dIIlacZ insertions, and classified into 16 DNA hybridization groups (4). One of these groups (group 1), which contains 10 of the insertion mutants, was previously characterized as the icmA-dotA region (5, 6). More recently, five additional DNA hybridization groups were characterized as two separate regions (ref. 7; M.P. and H.A.S., unpublished results). One of the regions (6.5 kb), was shown to contain nine insertion mutations located on a single DNA hybridization group (group 3), that contains six icm genes (7). The second region (11 kb) was shown to contain 18 insertion mutations located on four contiguous DNA hybridization groups (groups 2, 6, 4, and 17), which contain an additional six icm genes (M.P. and H.A.S., unpublished results).The aim of this study was to complete the characterization of the additional DNA hybridization groups. We found that the two regions described above are conne...
We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.
Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.
A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for the identification and characterization of bacterial pathogenesis determinants.
Legionella pneumophila, the bacterial agent of legionnaires' disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot-dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen.
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