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MOTIVATION: Understanding the molecular evolutionary history of organisms usually requires visual comparison of genomic regions from related species or strains. Although several applications already exist to achieve this task, they are either too old, too limited, or too complex for most user's needs. RESULTS: GenoFig is a graphical application for the visualization of prokaryotic genomic regions, intended to be as easy to use as possible and flexible enough to adapt to a variety of needs. GenoFig allows the personalized representation of annotations extracted from GenBank files in a consistent way across sequences, using regular expressions. It also provides several unique options to optimize the display of homologous regions between sequences, as well as other more classical features such as sequence GC percent or GC-skew representations. In summary, GenoFig is a simple, free, and highly configurable tool to explore the evolution of specific genomic regions in prokaryotes and to produce publication-ready figures. AVAILABILITY AND IMPLEMENTATION: Genofig is fully available at https://forgemia.inra.fr/public-pgba/genofig under a GPL 3.0 license.
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Genômica , Software , Genômica/métodos , Evolução Molecular , Interface Usuário-Computador , Gráficos por ComputadorRESUMO
Salmonella enterica is an important foodborne pathogen. Here, we present the construction and characterization of a high-density transposon sequencing library of the Salmonella Typhimurium ATCC 14028 strain. Essential, advantageous, and disadvantageous genes for growth in rich culture medium were identified on the chromosome and the pSLT plasmid.
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Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 M. avium subsp. paratuberculosis strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.
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Escherichia coli/genética , Lipopolissacarídeos/classificação , Lipopolissacarídeos/genética , Filogenia , Sistemas de Secreção Tipo VI/genética , DNA Bacteriano , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Genes Bacterianos , Fenótipo , Sorogrupo , Virulência/genéticaRESUMO
Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.
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Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of Johne's disease in ruminants. The IS900 insertion sequence (IS) has been used widely as an epidemiological marker and target for PCR diagnosis. Updated DNA sequencing technologies have led to a rapid increase in available Map genomes, which makes it possible to analyze the distribution of IS900 in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS900 element and how it affects genomic evolution and gene function of Map. A secondary goal is to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS900. Complete genomes from the major phylogenetic lineages known as C-type and S-type (including subtypes I and III), were chosen to represent the genetic diversity of Map. IS900 elements were located in these genomes using BLAST software and the relevant fragments extracted. An in silico RFLP analysis using the BstEII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS900 and the resulting RFLP profiles were analyzed and compared by digital visualization of the separated restriction fragments. The program developed for this study allowed automated localization of IS900 sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS900 copies ranged from 16 in the C-type isolate to 22 in the S-type subtype I isolate. A loci-by-loci sequence alignment of all IS900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS900 insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. Preferential insertion motif sequences were identified for IS900 along with genes bordering all IS900 insertions. Rarely did IS900 insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS900 distribution in Map genomes to enrich knowledge on the distribution dynamics of this IS for epidemiological purposes, for understanding Map evolution and for studying the biological implications of IS900 insertions.
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The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.
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Mycobacterium avium comprises four subspecies that contain both human and veterinary pathogens. At the inception of this study, twenty-eight M. avium genomes had been annotated as RefSeq genomes, facilitating direct comparisons. These genomes represent strains from around the world and provided a unique opportunity to examine genome dynamics in this species. Each genome was confirmed to be classified correctly based on SNP genotyping, nucleotide identity and presence/absence of repetitive elements or other typing methods. The Mycobacterium avium subspecies paratuberculosis (Map) genome size and organization was remarkably consistent, averaging 4.8 Mb with a variance of only 29.6 kb among the 13 strains. Comparing recombination events along with the larger genome size and variance observed among Mycobacterium avium subspecies avium (Maa) and Mycobacterium avium subspecies hominissuis (Mah) strains (collectively termed non-Map) suggests horizontal gene transfer occurs in non-Map, but not in Map strains. Overall, M. avium subspecies could be divided into two major sub-divisions, with the Map type II (bovine strains) clustering tightly on one end of a phylogenetic spectrum and Mah strains clustering more loosely together on the other end. The most evolutionarily distinct Map strain was an ovine strain, designated Telford, which had >1,000 SNPs and showed large rearrangements compared to the bovine type II strains. The Telford strain clustered with Maa strains as an intermediate between Map type II and Mah. SNP analysis and genome organization analyses repeatedly demonstrated the conserved nature of Map versus the mosaic nature of non-Map M. avium strains. Finally, core and pangenomes were developed for Map and non-Map strains. A total of 80% Map genes belonged to the Map core genome, while only 40% of non-Map genes belonged to the non-Map core genome. These genomes provide a more complete and detailed comparison of these subspecies strains as well as a blueprint for how genetic diversity originated.
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Mycobacterium bovis strain Mb3601 was isolated from the lymph node of an infected bovine in a bovine tuberculosis highly enzoonotic area of Burgundy, France. It was selected to obtain a complete genome for a new clonal complex, mainly constituted by SB0120-spoligotype strains that we propose to name "European 3". It was recently described as "clonal group I" based on whole-genome SNP analysis of 87 French strains. Here we describe the 4,365,068 bp complete genome obtained by the combination of PacBio and Illumina technologies. This genome of 65.64% G + C content includes 4024 predicted protein-coding genes, 52 tRNA, 3 rRNA and 11 copies of IS6110.
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Genoma Bacteriano , Mycobacterium bovis/genética , Animais , Bovinos , França , Tuberculose Bovina/microbiologia , Sequenciamento Completo do GenomaRESUMO
Streptococcus agalactiae is a major pathogen and is the leading cause of neonatal infections in industrialized countries. The diversity of strains isolated from two pregnant women was investigated. Here, we present the draft genome sequences of strains W8A2, W8A6, W10E2, and W10F3, obtained in order to ascertain their phylogenetic affiliation.
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Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for surveillance of this significant veterinary pathogen. For Map, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable number variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called "MAC-INMV-SSR database" was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods.
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Biologia Computacional/métodos , Técnicas de Genotipagem/normas , Complexo Mycobacterium avium/classificação , Marcadores Genéticos , Técnicas de Genotipagem/métodos , Internet , Repetições de Microssatélites , Repetições Minissatélites , Tipagem de Sequências Multilocus/métodos , Tipagem de Sequências Multilocus/normas , Complexo Mycobacterium avium/genética , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/genética , SoftwareRESUMO
Bovine tuberculosis (TB) is a zoonotic disease, mainly caused by Mycobacterium bovis. France was declared officially TB free in 2001, however, the disease persists in livestock and wildlife. Among wild animals, deer are particularly susceptible to bovine TB. Here, a whole genome sequence (WGS) analysis was performed on strains with the same genetic profile-spoligotype SB0121, Multiple Loci VNTR Analysis (MLVA) 6 4 5 3 11 2 5 7-isolated from different types of outbreaks, including from deer or cattle herds, or zoological or hunting parks where the presence of infected deer was a common trait in most of them. The results of the phylogeny based on the SNP calling shows that two sub-clusters co-exist in France, one related to deer bred to be raised as livestock, and the other to hunting parks and zoos. The persistence over almost 30 years of sporadic cases due to strains belonging to these clusters highlights the deficiency in the surveillance of captive wildlife and the need for better monitoring of animals, especially before movement between parks or herds.
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In recent years the diversity of the French Mycobacterium bovis population responsible for bovine tuberculosis (bTB) outbreaks since 1970 has been described in detail. To further understand bTB evolution in France, we used single nucleotide polymorphisms (SNPs) based on whole genome sequence versus classical genotyping methods in order to identify accurate phylogenetic relationships between M. bovis strains. Whole genome sequencing was carried out on a selection of 87 strains which reflect the French M. bovis population's genetic diversity. Sequences were compared to the M. bovis reference genome AF2122/97. Comparison among the 87 genomes revealed 9,170 sites where at least one strain shows a SNP with respect to the reference genome; 1,172 are intergenic and 7,998 in coding sequences, of which 2,880 are synonymous and 5,118 non-synonymous. SNP-based phylogenetic analysis using these 9,170 SNP is congruent with the cluster defined by spoligotyping and multilocus variable number of tandem repeat analysis typing. In addition, some SNPs were identified as specific to genotypic groups. These findings suggest new SNP targets that can be used for the development of high-resolving methods for genotyping as well as for studying M. bovis evolution and transmission patterns. The detection of non-synonymous SNPs on virulence genes enabled us to distinguish different clusters. Our results seem to indicate that genetically differentiated clusters could also display distinctive phenotypic traits.
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Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of M. avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae (FLA) might be a potential environmental host. FLA are protozoa found in water and soil that are described as reservoirs of pathogenic and non-pathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of M. avium subsp. paratuberculosis and Acanthamoeba castellanii. The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of M. avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A M. avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various M. avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of M. avium subsp. paratuberculosis.
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Amoeba/microbiologia , Microbiologia Ambiental , Mycobacterium avium subsp. paratuberculosis/classificação , Paratuberculose/microbiologia , Animais , Genoma Bacteriano , Genótipo , Tipagem de Sequências Multilocus , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Fagossomos/metabolismo , Fagossomos/microbiologia , Sequências de Repetição em TandemRESUMO
Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease affecting livestock, wild animals, and sometimes humans. We report here three draft genome sequences of Mycobacterium bovis strains of spoligotypes SB0821 and SB0134, isolated from wildlife but circulating in wildlife-livestock multihost systems, and SB0121, circulating exclusively in cattle.
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Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.
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Parede Celular/genética , Lipídeos de Membrana/genética , Peptídeo Sintases/genética , Sequência de Aminoácidos , Parede Celular/metabolismo , Parede Celular/fisiologia , Lipídeos de Membrana/química , Mycobacterium avium/genética , Mycobacterium avium/metabolismo , Peptídeos/genética , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease, affecting livestock, wild animals, and sometimes humans. We report the draft genome sequence of a Mycobacterium bovis strain isolated from wild boar of spoligotype SB0120 (or BCG-like) also present in wildlife-livestock multi-host systems.
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The description of the population of M. bovis strains circulating in France from 1978 to 2013 has highlighted the discriminating power of the MLVA among predominant spoligotype groups. In the present study we aimed to characterize clonal groups via MLVA and to better understand the strain's population structure. MLVA was performed with eight MIRU-VNTR loci, most of them defined by the Venomyc European consortium. The discriminatory index of each MLVA loci was calculated for SB0120, SB0134, SB0121 and the "F4-family", the main spoligotype groups in France. Differences in global DI per spoligotype, but also by locus within each spoligotype, were observed, which strongly suggest the clonal complex nature of these major groups. These MLVA results were compared to those of other European countries where strain collections had been characterized (Spain, Portugal, Italy, Northern Ireland and Belgium). Overall, QUB 3232 and ETR D are respectively the most and the least discriminative loci, regardless of the strains geographical origin. However, marked DI differences are observed in the rest of the MIRU-VNTR loci, again highlighting that strain genetic variability in a country depends on the dominant existing clonal complexes. A web application for M. bovis, including spoligotyping and MIRU-VNTR typing data, was developed to allow inter-laboratory comparison of field isolates. In conclusion, combination of typing methods is required for M. bovis optimum discrimination and differentiation of groups of strains. Thus, the loci employed for MLVA in a country should be those which are the most discriminative for the clonal complexes which characterize their M. bovis population.
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Repetições Minissatélites/genética , Tipagem Molecular/métodos , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Animais , Bovinos , Europa (Continente)/epidemiologia , Variação Genética , Genótipo , Mamíferos/microbiologiaRESUMO
After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.
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Antígenos de Bactérias/imunologia , Doenças dos Bovinos/imunologia , Interferon gama/metabolismo , Mycobacterium avium subsp. paratuberculosis/metabolismo , Paratuberculose/imunologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Imunidade Celular/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/microbiologia , TuberculinaRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis disease affecting ruminants worldwide. The aim of this study was to identify potential candidate antigens and epitopes by bio and immuno-informatic tools which could be later evaluated as vaccines and/or diagnosis. 110 protein sequences were selected from MAP K-10 genome database: 48 classified as putative enzymes involved in surface polysaccharide and lipopolysaccharide synthesis, as membrane associated and secreted proteins, 32 as conserved membrane proteins, and 30 as absent from other mycobacterial genomes. These 110 proteins were preliminary screened for Major Histocompatibility Complex (MHC) class II affinity and promiscuity using ProPred program. In addition, subcellular localization and host protein homology was analyzed. From these analyses, 23 MAP proteins were selected for a more accurate inmunoinformatic analysis (i.e. T cell and B cell epitopes analysis) and for homology with mycobacterial proteins. Finally, eleven MAP proteins were identified as potential candidates for further immunogenic evaluation: six proteins (MAP0228c, MAP1239c, MAP2232, MAP3080, MAP3131 and MAP3890) were identified as presenting potential T cell epitopes, while 5 selected proteins (MAP0232c, MAP1240c, MAP1738, MAP2239 and MAP3641c) harbored a large numbers of epitopes predicted to induce both cell- and antibody-mediated immune responses. Moreover, immunogenicity of selected epitopes from MAP1239c were evaluated in IFN-γ release assay. In summary, eleven M. avium subsp. paratuberculosis proteins were identified by in silico analysis and need to be further evaluated for their immunodiagnostic and vaccine potential in field and mice model.