Legionella pneumophila - Host Interactions: Insights Gained from Comparative Genomics and Cell Biology.
ABSTRACT Legionella pneumophila is the etiological agent of Legionnaires' disease and of the less acute disease Pontiac fever. It is a Gram-negative bacterium present in fresh and artificial water environments that replicates in protozoan hosts and is also found in biofilms. Replication within protozoa is essential for the survival of the bacterium. The last years have seen a giant step forward in the genomics of L. pneumophila. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates in 2004 and a fourth in 2007 has paved the way for major breakthroughs in understanding the biology of L. pneumophila in particular and Legionella in general. Sequence analysis identified several specific features of Legionella: (i) an extraordinary genetic diversity among the different isolates and (ii) the presence of an unexpected high number and variety of eukaryotic-like proteins, predicted to be involved in the exploitation of the host cellular processes by mimicking specific eukaryotic functions. In this chapter, we will first discuss the insights gained from genomics by highlighting the characteristic features and common traits of the four L. pneumophila genomes obtained through genome analysis and comparison and then we will focus on the newest results obtained by functional analysis of different eukaryotic-like proteins and describe their involvementin the pathogenicity of L. pneumophila.
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ABSTRACT: L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires’ disease.International Journal of Medical Microbiology 08/2014; 304(8). DOI:10.1016/j.ijmm.2014.08.010 · 3.42 Impact Factor
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ABSTRACT: The detection of Legionella pneumophila in environmental and clinical samples is frequently performed by PCR amplification of the mip and/or 16S rRNA genes. Combined with DNA sequencing, these two genetic loci can be used to distinguish different species of Legionella and identify L. pneumophila. However, the recent Legionella genome sequences have opened up hundreds of possibilities for the development of new molecular targets for detection and diagnosis. Ongoing comparative genomics has the potential to fine tune the identification of Legionella species and serogroups by combining specific and general genetic targets. For example, the coincident detection of LPS biosynthesis genes and virulence genes may allow the differentiation of both pathogen and serogroup without the need for nucleotide sequencing. We tested this idea using data derived from a previous genomic subtractive hybridization we performed between L. pneumophila serogroup 1 and L. micdadei. Although not yet formally tested, these targets serve as an example of how comparative genomics has the potential to improve the scope and accuracy of Legionella molecular detection if embraced by laboratories undertaking Legionella surveillance.Frontiers in Microbiology 11/2010; 1:123. DOI:10.3389/fmicb.2010.00123 · 3.94 Impact Factor
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ABSTRACT: Background Legionnaires¿ disease is a severe form of pneumonia caused by the environmental bacterium Legionella pneumophila. Outbreaks commonly affect people with known risk factors, but the genetic and pathogenic complexity of L. pneumophila within an outbreak is not well understood. Here, we investigate the etiology of the major Legionnaires¿ disease outbreak that occurred in Edinburgh, UK, in 2012, by examining the evolutionary history, genome content, and virulence of L. pneumophila clinical isolates.ResultsOur high resolution genomic approach reveals that the outbreak was caused by multiple genetic subtypes of L. pneumophila, the majority of which had diversified from a single progenitor through mutation, recombination, and horizontal gene transfer within an environmental reservoir prior to release. In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution. Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes, correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.Conclusions Taken together, our study indicates previously cryptic levels of pathogen heterogeneity within a Legionnaires¿ disease outbreak, a discovery that impacts on source attribution for future outbreak investigations. Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.Genome Biology 11/2014; 15(11):504. DOI:10.1186/PREACCEPT-1675723368141690 · 10.47 Impact Factor