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RNA silencing is a gene silencing mechanism directed by siRNAs and miRNAs. Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that theLegionella pneumophilatype IV-secreted effector LegK1 suppresses siRNA- and miRNA-activities in human cells. This ability depends on its kinase activity and on a functional tryptophan-dependent Argonaute (Ago)-binding platform. We further show that the capacity of LegK1 to activate NF-κB signaling contributes to silencing suppression, demonstrating a link between effector-mediated NF-κB signaling activation and silencing suppression. LegK1 also promotesL. pneumophilagrowth in both amoeba and human macrophages, supporting a key role of this effector in virulence. In infected macrophages, the latter activity relies on the genetic targeting of human Ago4, highlighting a novel function of this host factor in antibacterial resistance.

Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of

Legionella pneumophila, the causative agent of Legionnaires’ disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced “reverse mode” of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

The composition and dynamics of the lung microbiome during pneumonia are not known, although the lung microbiome might influence the severity and outcome of this infectious disease, similar to what was shown for the microbiome at other body sites. Here we report the findings of a comprehensive analysis of the lung microbiome composition of three patients with long-term pneumonia due to L. pneumophila and its evolution during antibiotic treatment. This work adds to our understanding of how the microbiome changes during disease and antibiotic treatment and points to microorganisms and their interactions that might be beneficial. In addition to bacteria and fungi, our analyses included archaea and eukaryotes (protozoa), showing that both are present in the pulmonary microbiota and that they might also play a role in the response to the microbiome disturbance., Despite the importance of pneumonia to public health, little is known about the composition of the lung microbiome during infectious diseases, such as pneumonia, and how it evolves during antibiotic therapy. To study the possible relation of the pulmonary microbiome to the severity and outcome of this respiratory disease, we analyzed the dynamics of the pathogen and the human lung microbiome during persistent infections caused by the bacterium Legionella pneumophila and their evolution during antimicrobial treatment. We collected 10 bronchoalveolar lavage fluid samples from three patients during long-term hospitalization due to pneumonia and performed a unique longitudinal study of the interkingdom microbiome, analyzing the samples for presence of bacteria, archaea, fungi, and protozoa by high-throughput Illumina sequencing of marker genes. The lung microbiome of the patients was characterized by a strong predominance of the pathogen, a low diversity of the bacterial fraction, and an increased presence of opportunistic microorganisms. The fungal fraction was more stable than the bacterial fraction. During long-term treatment, no genomic changes or antibiotic resistance-associated mutations that could explain the persistent infection occurred, according to whole-genome sequencing analyses of the pathogen. After antibiotic treatment, the microbiome did not recover rapidly but was mainly constituted of antibiotic-resistant species and enriched in bacteria, archaea, fungi, or protozoa associated with pathogenicity. The lung microbiome seems to contribute to nonresolving Legionella pneumonia, as it is strongly disturbed during infection and enriched in opportunistic and/or antibiotic-resistant bacteria and microorganisms, including fungi, archaea, and protozoa that are often associated with infections.

BackgroundLung microbiome analyses have shown that the healthy lung is not sterile but it is colonized like other body sites by bacteria, fungi and viruses. However, little is known about the microbial composition of the lung microbiome during infectious diseases such as pneumonia and how it evolves during antibiotic therapy. To better understand the impact of the composition of the pulmonary microbiome on severity and outcome of pneumonia we analysed the composition and evolution of the human lung microbiome during pneumonia caused by the bacteriumLegionella pneumophila.ResultsWe collected 10 bronchoalveolar lavage (BAL) samples from three patients during long-term hospitalisation due to severe pneumonia and performed a longitudinal in-depth study of the composition of their lung microbiome by high-throughput Illumina sequencing of the 16S rRNA gene (bacteria and archaea), ITS region (fungi) and 18S rRNA gene (eukaryotes). We found that the composition of the bacterial lung microbiome during pneumonia is hugely disturbed containing a very high percentage of the pathogen, a very low bacterial diversity, and an increased presence of opportunistic microorganisms such as species belonging to Staphylococcaceae and Streptococcaceae. The microbiome of antibiotic treated patients cured from pneumonia represented a different perturbation state with a higher abundance of resistant bacteria (mainly Firmicutes) and a significantly different bacterial composition as that found in healthy individuals. In contrast, the mycobiome remains more stable during pneumonia and antimicrobial therapy. Interestingly we identified possible cooperation within and between both communities. Furthermore, archaea (Methanobrevibacter) and protozoa (AcanthamoebaandTrichomonas) were detected.ConclusionsBacterial pneumonia leads to a collapse of the healthy microbiome and a strongly disturbed bacterial composition of the pulmonary microbiome that is dominated by the pathogen. Antibiotic treatment allows some bacteria to regrow or recolonize the lungs but the restoration of a healthy lung microbiome composition is only regained a certain time after the antibiotic treatment. Archaea and protozoa should also be considered, as they might be important but yet overseen members of the lung microbiome. Interactions between the micro- and the mycobiome might play a role in the restoration of the microbiome and the clinical evolution of the disease.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella . Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

SignificanceLegionella pneumophilais a bacterial pathogen causing outbreaks of a lethal pneumonia. The genusLegionellacomprises 65 species for which aquatic amoebae are the natural reservoirs. Using functional and comparative genomics to deconstruct the entire bacterial genus we reveal the surprising parallel evolutionary trajectories that have led to the emergence of human pathogenicLegionella.An unexpectedly large and unique repository of secreted proteins (>16,000) containing eukaryotic-like proteins acquired from all domains of life (plant, animal, fungal, archaea) is contrasting with a highly conserved type 4 secretion system. This study reveals an unprecedented environmental reservoir of bacterial virulence factors, and provides a new understanding of how reshuffling and gene-acquisition from environmental eukaryotic hosts, may allow for the emergence of human pathogens.AbstractThe bacterial genusLegionellacomprises 65 species among, whichLegionella pneumophilais a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80Legionellagenomes spanning 58 species. Uniquely, an immense repository of 16,000 secreted proteins encoding 137 different eukaryotic-like domains and more than 200 eukaryotic-like proteins is paired with a highly conserved T4SS. Specifically, we show that eukaryotic Rho and Rab GTPase domains are found nearly exclusively in eukaryotes andLegionellaspecies. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F/U-box and SET domains were present in >70% of all species suggesting that manipulation of host signal transduction, protein turnover and chromatin modification pathways, respectively are fundamental intracellular replication strategies forLegionellae. In contrast, the Sec-7 domain was restricted toL. pneumophilaand seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by inter-domain gene transfer. Furthermore we revealed the surprising extent to which legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts and provides a new understanding of how dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens.

Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires’ disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic “hotspots” of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila, whereby multiple non-contiguous segments that originate from the same molecule of donor DNA are imported into a recipient genome during a single episode of recombination., Author summary Legionella pneumophila is an environmental bacterium that causes Legionnaires’ disease, a serious and potentially fatal pneumonia. Previous studies have shown that members of this species undergo a process called recombination, whereby DNA is imported from another bacterial cell into the recipient genome. The imported DNA can either replace an equivalent segment of the genome (homologous recombination) or can comprise novel genes that are new to the recipient genome (non-homologous recombination). Whilst recombination plays an undoubtedly important role in L. pneumophila evolution, accounting for more than 96% of the diversity observed within some lineages, little is known about its biological impact. In this study, we performed a detailed characterisation of the dynamics and effect of homologous recombination on L. pneumophila evolution in six clinically important lineages of L. pneumophila. We identified “hotspot” regions of the genome in which an excess of homologous recombination events was observed, which provided important clues to the selection pressures faced by L. pneumophila. By determining the donors of the recombined regions, we also revealed that recombination has occurred most frequently between isolates from the same clade, but also occurred between isolates from different major clades. This demonstrates the possibility of new adaptations arising in one lineage and being transferred to another distantly related lineage, which we predict has been an important factor in the emergence of several major disease-causing strains from diverse genomic backgrounds.

International audience; Histone posttranslational modifications control eukaryotic gene expression and regulate many biological processes including immunity. Pathogens alter host epigenetic control to aid pathogenesis. We find that the intracellular bacterial pathogen Legionella pneumophila uses a Dot/Icm type IV secreted effector, RomA, to uniquely modify the host chromatin landscape. RomA, a SET domain-containing methyltransferase, trimethylates K14 of histone H3, a histone mark not previously described in mammals. RomA localizes to the infected cell nucleus where it promotes a burst of H3K14 methylation and consequently decreases H3K14 acetylation, an activating histone mark, to repress host gene expression. ChIP-seq analysis identified 4,870 H3K14 methylated promoter regions, including innate immune genes. Significantly reduced replication of a RomA-deleted strain in host cells was trans-complemented by wild-type, but not by catalytically inactive, RomA. Thus, a secreted L. pneumophila effector targets the host cell nucleus and modifies histones to repress gene expression and promote efficient intracellular replication. Copyright © 2013 Elsevier Inc. All rights reserved.

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection ( n = 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.

Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires’ disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission.

International audience; The bacterium Legionella pneumophila is found ubiquitously in aquatic environments and can cause a severe pneumonia in humans called Legionnaires' disease. How this bacterium switches from intracellular to extracellular life and adapts to different hosts and environmental conditions is only partly understood. Here we used RNA deep sequencing from exponentially (replicative) and post exponentially (virulent) grown L. pneumophila to analyze the transcriptional landscape of its entire genome. We established the complete operon map and defined 2561 primary transcriptional start sites (TSS). Interestingly, 187 of the 1805 TSS of protein-coding genes contained tandem promoters of which 93 show alternative usage dependent on the growth phase. Similarly, over 60% of 713 here identified ncRNAs are phase dependently regulated. Analysis of their conservation among the seven L. pneumophila genomes sequenced revealed many strain specific differences suggesting that L. pneumophila contains a highly dynamic pool of ncRNAs. Analysis of six ncRNAs exhibiting the same expression pattern as virulence genes showed that two, Lppnc0584 and Lppnc0405 are indeed involved in intracellular growth of L. pneumophila in A. castellanii. Furthermore, L. pneumophila encodes a small RNA named RsmX that functions together with RsmY and RsmZ in the LetA-CsrA regulatory pathway, crucial for the switch to the virulent phenotype. Together our data provide new insight into the transcriptional organization of the L. pneumophila genome, identified many new ncRNAs and will provide a framework for the understanding of virulence and adaptation properties of L. pneumophila.

Legionella pneumophila , a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes ( lpp0831 , wzm , and wzt ) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non- Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.

Streptococcus gallolyticus(formerly known asStreptococcus bovisbiotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer.S. gallolyticusis part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis.S. gallolyticushas the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore,S. gallolyticussynthesizes all 20 amino acids and more vitamins than any other sequencedStreptococcusspecies. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis.

[This corrects the article DOI: 10.1371/journal.pgen.1006855.].

Escherichia coli A0 34/86 (O83:K24:H31) is a commensal strain that has been used for prophylactic and therapeutic colonization of the intestine of newborn infants. To identify traits specific for E. coli A0 34/86, we used a minimal tiling set of 148 BAC clones of A0 34/86 genomic DNA, to construct restriction-digested BAC arrays. Hybridization with genomic DNA from four E. coli strains (CFT073; O157:H7; K12 and Nissle 1917) allowed selection of two BAC clones that were sequenced to identify A0 34/86-specific regions. Genes for the yersiniabactin siderophore system, several proteins homologous to Salmonella enterica serovar Typhimurium vitamin B12 synthesis proteins, as well as genes necessary for the degradation of propanediol, the pix fimbriae determinant and genes coding for a putative phosphoglycerate transport system present also on pathogenicity island V of E. coli strain 536 were all identified in E. coli A0 34/86. This comparative analysis underlines the important genome heterogeneity between E. coli strains.

International audience; The human immunodeficiency virus (HIV) Vif protein blocks incorporation of two host cell cytidine deaminases, APOBEC3F and 3G, into the budding virion. Not surprisingly, on a vif background nascent minus strand DNA can be extensively edited leaving multiple uracil residues. Editing occurs preferentially in the context of TC (GA on the plus strand) and CC (GG) depending on the enzyme. To explore the distribution of APOBEC3F and -3G editing across the genome, a product/substrate ratio (AA + AG)/(GA + GG) was computed for a series of 30 edited genomes present in the data bases. Two highly polarized gradients were noted each with maxima just 5' to the central polypurine tract (cPPT) and LTR proximal polypurine tract (3'PPT). The gradients are in remarkable agreement with the time the minus strand DNA remains single stranded. In vitro analyses of APOBEC3G deamination of nascent cDNA spanning the two PPTs showed no pronounced dependence on the PPT RNA:DNA heteroduplex ruling out the competing hypothesis of a PPT orientation effect. The degree of hypermutation varied smoothly among genomes indicating that the number of APOBEC3 molecules packaged varied considerably.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates as an intracellular parasite of amoebae and persists in the environment as a free-living microbe. Here we have analyzed the complete genome sequences of L. pneumophila Paris (3,503,610 bp, 3,077 genes), an endemic strain that is predominant in France, and Lens (3,345,687 bp, 2,932 genes), an epidemic strain responsible for a major outbreak of disease in France. The L. pneumophila genomes show marked plasticity, with three different plasmids and with about 13% of the sequence differing between the two strains. Only strain Paris contains a type V secretion system, and its Lvh type IV secretion system is encoded by a 36-kb region that is either carried on a multicopy plasmid or integrated into the chromosome. Genetic mobility may enhance the versatility of L. pneumophila. Numerous genes encode eukaryotic-like proteins or motifs that are predicted to modulate host cell functions to the pathogen's advantage. The genome thus reflects the history and lifestyle of L. pneumophila, a human pathogen of macrophages that coevolved with fresh-water amoebae.

Motivation: Contigs-Assembly and Annotation Tool-Box (CAAT-Box) is a software package developed for the computational part of a genome project where the sequence is obtained by a shotgun strategy. CAAT-Box contains new tools to predict links between contigs by using similarity searches with other whole genome sequences. Most importantly, it allows annotation of a genome to commence during the finishing phase using a gene-oriented strategy. For this purpose, CAAT-Box creates an Individual Protein file (IPF) for each ORF of an assembly. The nucleotide sequence reported in an IPF corresponds to the sequence of the ORF with 500 additional bases before the ORF and 200 bases after. For annotation, additional information like Blast results can be added or linked to the IPFs as well as automatic and/or manual annotations. When a new assembly is performed, CAAT-Box creates new IPFs according to the old IPF panel. CAAT-Box recognizes the modified IPFs which are the only ones used for a new automatic analysis after each assembly. Using this strategy, the user works with a group of IPFs independently of the closure phase progression. The IPFs are accessible by a web server and can therefore be modified and commented by different groups. Result: CAAT-Box was used to obtain and to annotate several complete genomes like Listeria monocytogenes or Strepcoccus agalactiae. Availability: The program may be obtained from the authors and is freely available to non-profit organisations.

Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty-five per cent of the predicted genes have an ortholog in the Streptococcus pyogenes genome, representing a conserved backbone between these two streptococci. Among the genes in S. agalactiae that lack an ortholog in S. pyogenes, 50% are clustered within 14 islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared with other pathogenic streptococci, S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.

Background The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. Results We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. Conclusions Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0505-0) contains supplementary material, which is available to authorized users.

This chapter starts with a description of the general characteristics of the genome sequences of Legionella pneumophila and Legionella longbeachae. It then highlights the characteristic features and common traits of the two main human-pathogenic Legionella species. Emphasis is given to putative virulence and Legionella life cycle-related functions. In the second part, the focus is on the comparison of these genome sequences, in order to learn about the plasticity of the Legionella genomes and the possible mechanisms involved. In the third part, the possible evolution of the identified virulence factors are analyzed and discussed. Finally, future perspectives in Legionella genomics are presented. L. longbeachae NSW 150 encodes four T4SS, a rather exceptional number and L. longbeachae D-4968 encodes two T4SS. The authors found that IcmE/DotG of L. longbeachae (1,525 amino acids) is 477 amino acids larger than that of L. pneumophila (1,048 amino acids). Most of the T4SS are located on regions of genome plasticity; some even show plasticity on the gene/protein level, as shown above for DotG. The authors undertook a phylogenetic analysis for the L. longbeachae protein Llo2643, which contains PPR repeats, a protein family typically present in plants. In the last few years, genome analyses, as well as comparative and functional genomics, have demonstrated that genome plasticity plays a major role in differences in host cell exploitation and niche adaptation of Legionella.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

We report the complete 119,443-bp sequence of the pgm locus from Yersinia pestis and its flanking regions. Sequence analysis confirms that the 102-kb unstable pgm locus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system of Bordetella pertussis . Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among different Y. pestis and Yersinia pseudotuberculosis strains, the sequence of selected regions of the pgm locus and flanking regions were compared from 20 different Y. pestis and 10 Y. pseudotuberculosis strains. The results showed that the genes interrupted in Y. pestis are intact in Y. pseudotuberculosis . However, one of these mutations, in the bvgS homologue, is only present in Y. pestis strains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29 Yersinia strains suggest that Y. pestis is a recently emerged pathogen that is probably entering the initial phase of reductive evolution.

International audience; Legionella pneumophila est un bacille à Gram négatif à l'origine d'une infection pulmonaire grave appelée légionellose (ou maladie du légionnaire) [11]. Les légionelles sont des bactéries vivant en milieu aquatique et affectionnant particulièrement les eaux tièdes. Ce sont des bactéries intracellulaires facultatives qui colonisent habituellement les amibes, protozoaires qui prolifèrent dans l'eau, mais elles sont également capables d'infecter l'homme par le biais d'aérosols. Une fois phagocytées, les légionelles survivent et se multiplient au détriment de la cellule hôte : cette réplication intracellulaire semble constituer le moyen prédominant de multiplication de ces bactéries dans l'environnement. La capacité de Legionella à infecter les cellules eucaryotes est intimement liée à la faculté de manipuler les mécanismes de la cellule afin de créer une niche intracellulaire pour sa réplication, et ceci grâce à un arsenal de protéines sécrétées dans la cellule hôte (effecteurs) qui agissent tout au long de l'infection. Plus particulièrement, un système de sécrétion de type IV (appelé Dot/Icm) entre en jeu pour transporter ces effecteurs dans la cellule cible [12]. Ces effecteurs redirigent alors le trafic du phagosome de L. pneumophila, permettant ainsi sa transformation en un organite dérivé du réticulum endoplasmique (RE) [1]. Le séquençage complet du génome de L. pneumophila en 2004 [2, 3] a constitué une avancée importante dans la recherche sur les légionelles. Sa particularité la plus étonnante est la présence d'un grand nombre et d'une grande variété de gènes codant pour des protéines similaires à des protéines eucaryotes, dites eukaryotic-like proteins (ELP), ainsi qu'à des protéines contenant des domaines eucaryotes (EDP). Ces protéines sont les premières candidates à être impliquées dans le détournement des fonctions cellulaires [4].

International audience; BackgroundDuring host specialization, inactivation of genes whose function is no more required is favored by changes in selective constraints and evolutionary bottlenecks. The Gram positive bacteria Streptococcus agalactiae (also called GBS), responsible for septicemia and meningitis in neonates also emerged during the seventies as a cause of severe epidemics in fish farms. To decipher the genetic basis for the emergence of these highly virulent GBS strains and of their adaptation to fish, we have analyzed the genomic sequence of seven strains isolated from fish and other poikilotherms.ResultsComparative analysis shows that the two groups of GBS strains responsible for fish epidemic diseases are only distantly related. While strains belonging to the clonal complex 7 cannot be distinguished from their human CC7 counterparts according to their gene content, strains belonging to the ST260-261 types probably diverged a long time ago. In this lineage, specialization to the fish host was correlated with a massive gene inactivation and broad changes in gene expression. We took advantage of the low level of sequence divergence between GBS strains and of the emergence of sublineages to reconstruct the different steps involved in this process. Non-homologous recombination was found to have played a major role in the genome erosion.ConclusionsOur results show that the early phase of genome reduction during host specialization mostly involves accumulation of small and likely reversible indels, followed by a second evolutionary step marked by a higher frequency of large deletions.

International audience; We report the complete and annotated genome sequence of the animal pathogen Listeria ivanovii subsp. ivanovii strain PAM 55 (serotype 5), isolated in 1997 in Spain from an outbreak of abortion in sheep. The sequence and its analysis are available at an interactive genome browser at the Institut Pasteur (http://genolist.pasteur.fr/LivaList/).

International audience; Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

We sought to examine ADAR-1 editing of measles and influenza virus genomes derived from inactivated seasonal influenza and live attenuated measles virus vaccines grown on chicken cells as the culture substrate. Using highly sensitive 3DI-PCR (R. Suspène et al., Nucleic Acids Res. 36:e72, 2008), it was possible to show that ADAR-1 could hyperdeaminate adenosine residues in both measles virus and influenza virus A genomes. Detailed analysis of the dinucleotide editing context showed preferences for 5′ArA and 5′UrA, which is typical of editing in mammalian cells. The hyperedited mutant frequency, including genomes and antigenomes, was a log greater for influenza virus compared to measles virus, suggesting a greater sensitivity to restriction by ADAR-1.

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species., Author Summary Legionella longbeachae, found in potting soil, and L. pneumophila, present in aquatic environments, are opportunistic human pathogens that cause Legionnaires' disease, a severe and often fatal pneumonia. The analysis and comparison of the genome sequences of four L. longbeachae genomes together with the study of its gene expression program and virulence pattern in different infection models provides important new insight on the organism's lifestyle and virulence strategies. L. longbeachae harbors a unique repertoire of secreted substrates, many of which encode eukaryotic like domains that may help the pathogen to subvert host functions and cause disease. Curiously, L. longbeachae may also be able to interact with plants. Several proteins present mainly in plants and phytopathogenic bacteria and several enzymes that might confer the ability to degrade plant material were identified in its genome. Interestingly, L. longbeachae encodes a chemotaxis system but no flagella, in contrast L. pneumophila encodes flagella but no chemotaxis system. It will be an interesting aspect of future research to understand these peculiarities. Finally, the genome sequence and analysis reported here will aid in understanding how L. longbeachae causes disease and will open new possibilities to develop tools for rapid identification and risk prediction of L. longbeachae infection.

P>Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of Gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.

Legionella pneumophila is a human pathogen that was recognized only about 30 years ago. It is the causative agent of Legionnaires' disease, a severe pneumonia that is transmitted through inhalation of aerosols of contaminated water. Shortly after its discovery, the ability of Legionella to multiply intracellularly in fresh water protozoa was discovered. This long lasting co-evolution between the eukaryotic host and Legionella has led to the selection of a panoply of virulence factors, which allow to exploit important cellular processes during infection. Compelling evidence for the importance of protozoa in the evolution of this bacterium comes from analysis of complete genome sequences. A key feature of the L. pneumophila genomes is the presence of a high number and wide variety of eukaryotic like proteins and protein domains probably acquired through horizontal gene transfer and/or convergent evolution. In the last years several different typing methods aiming in investigating the molecular epidemiology of L. pneumophila have been developed. Furthermore, the access to whole genome sequences of several L. pneumophila strains allowed to apply large scale comparative genomic studies using DNA arrays. A higher genetic diversity among environmental isolates with respect to clinical isolates and the presence of specific clones of L. pneumophila overrepresented in human disease or causing legionellosis world wide, were identified. Further studies analyzing the natural populations of Legionella more in detail will allow to gain a better understanding of the population structure and the ecological diversity of this species. This review describes the latest observations about the structure of L. pneumophila populations, the techniques used to study the molecular epidemiology and evolution of L. pneumophila, the knowledge gained from genome analysis, and discusses future perspectives.

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

International audience; Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10(-2) to 10(-5)in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR ( approximately 10(-4) to 10(-5)). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Deltavif efficiently.

The genome of a clinical isolate of Neisseria meningitidis is described. This and other reannotated Neisseria genomes are compiled in a database., Background Genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. However, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis) and/or genes essential for cell viability, which are necessary to achieve this goal, are often sorely lacking. This holds true for Neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia. Results By determining and manually annotating the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembling a library of defined mutants in up to 60% of its non-essential genes, we have created NeMeSys, a biological resource for Neisseria meningitidis systematic functional analysis. To further enhance the versatility of this toolbox, we have manually (re)annotated eight publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database. The potential of NeMeSys for narrowing the gap between sequence and function is illustrated in several ways, notably by performing a functional genomics analysis of the biogenesis of type IV pili, one of the most widespread virulence factors in bacteria, and by identifying through comparative genomics a complete biochemical pathway (for sulfur metabolism) that may potentially be important for nasopharyngeal colonization. Conclusions By improving our capacity to understand gene function in an important human pathogen, NeMeSys is expected to contribute to the ongoing efforts aimed at understanding a prokaryotic cell comprehensively and eventually to the design of new therapies.

Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae , the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.

Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.