The Conserved nhaAR Operon Is Drastically Divergent between B2 and Non-B2 Escherichia coli and Is Involved in Extra-Intestinal Virulence

The Escherichia coli species is divided in phylogenetic groups that differ in their virulence and commensal distribution. Strains belonging to the B2 group are involved in extra-intestinal pathologies but also appear to be more prevalent as commensals among human occidental populations. To investigate the genetic specificities of B2 sub-group, we used 128 sequenced genomes and identified genes of the core genome that showed marked difference between B2 and non-B2 genomes. We focused on the gene and its surrounding region with the strongest divergence between B2 and non-B2, the antiporter gene nhaA. This gene is part of the nhaAR operon, which is in the core genome but flanked by mobile regions, and is involved in growth at high pH and high sodium concentrations. Consistently, we found that a panel of non-B2 strains grew faster than B2 at high pH and high sodium concentrations. However, we could not identify differences in expression of the nhaAR operon using fluorescence reporter plasmids. Furthermore, the operon deletion had no differential impact between B2 and non-B2 strains, and did not result in a fitness modification in a murine model of gut colonization. Nevertheless, sequence analysis and experiments in a murine model of septicemia revealed that recombination in nhaA among B2 strains was observed in strains with low virulence. Finally, nhaA and nhaAR operon deletions drastically decreased virulence in one B2 strain. This effect of nhaAR deletion appeared to be stronger than deletion of all pathogenicity islands. Thus, a population genetic approach allowed us to identify an operon in the core genome without strong effect in commensalism but with an important role in extra-intestinal virulence, a landmark of the B2 strains

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species

<p>Abstract</p> <p>Background</p> <p>Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of <it>Escherichia coli</it>. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B₂variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B₁variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene.</p> <p>Results</p> <p>We identified the gene encoding esterase B as the acetyl-esterase gene (<it>aes</it>) using gene disruption. The analysis of <it>aes </it>nucleotide sequences in a panel of 78 reference strains, including the <it>E. coli </it>reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from <it>aes </it>sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B₁and B₂by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between <it>aes </it>and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of <it>aes </it>to be associated with virulence.</p> <p>Conclusion</p> <p>Our findings suggest that <it>aes </it>does not play a direct role in the virulence of <it>E. coli </it>extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.</p

Performances of the rapid polymyxin Acinetobacter and Pseudomonas tests for colistin susceptibility testing

Owing to the emergence of colistin resistance in nonfermenting Gram negative bacteria, reliable and rapid techniques for testing colistin susceptibility are needed. We evaluated the performances of the Rapid Polymyxin Acinetobacter and Pseudomonas tests using a collection of Acinetobacter baumannii and Pseudomonas aeruginosa clinical isolates.Methods: Colistin susceptibility of A. baumannii and P. aeruginosa isolates (colistin susceptible and colistin resistant) was tested with the Rapid Polymyxin Acinetobacter and Pseudomonas tests and compared with the broth microdilution method.Results: The Rapid Polymyxin Acinetobacter and Pseudomonas tests were able to detect all colistin-resistant and all colistin-susceptible A. baumannii and P. aeruginosa isolates within 4 hours.Conclusion: The Rapid Polymyxin Acinetobacter and Pseudomonas tests are reliable techniques for detecting colistin resistance. Overall, both techniques allow an accurate and a rapid screening (<4  hours) of colistin resistance in A. baumannii and P. aeruginosa

Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance

Recherche des escherichia coli entérovirulents par PCR multiplex au cours des diarrhées infectieuses

PARIS-BIUP (751062107) / SudocSudocFranceF

1. De la main harmonique au solfège : l’apprentissage de la lecture chantée du XVIIe au XVIIIe siècle

Lescat Philippe. 1. De la main harmonique au solfège : l’apprentissage de la lecture chantée du XVIIe au XVIIIe siècle. In: Vibrations, N. 6, 1988. Apprendre la musique, sous la direction de Antoine Hennion. pp. 145-168

Characterization and study of the role of lamp2a in fish

L’Autophagie médiée par les protéines chaperonnes (ou CMA pour Chaperone-Mediated Autophagy) est une voie majeure du catabolisme lysosomal considérée aujourd’hui comme un acteur central de contrôle de nombreuses fonctions cellulaires, et dont les défauts sont associés à plusieurs pathologies humaines, dont des maladies neurodégénératives, des cancers et des troubles du système immunitaire. Selon l’idée actuellement admise, cette fonction cellulaire n’existerait que chez les mammifères ou les oiseaux, qui seraient les seuls à exprimer la protéine LAMP2A, une protéine nécessaire au fonctionnement de la CMA. Or, récemment, nous avons pu mettre en évidence l’existence de séquences exprimées présentant une forte homologie avec LAMP2A de mammifères chez plusieurs espèces de poissons, remettant ainsi en question ce point de vue et suggérant que la CMA soit apparue beaucoup plus tôt au cours de l'évolution qu'on ne l'avait initialement cru. Dans cette thèse, nous retraçons l’histoire évolutive du gène LAMP2 chez les vertébrés. Nous démontrons que ce gène est apparu après la seconde duplication complète du génome survenue chez l'ancêtre commun des vertébrés il y a environ 500 millions d'années. En outre, en adaptant une méthode récemment décrite pour mesurer l’activité de la CMA dans des cellules de mammifères à une lignée de fibroblastes de medaka (Oryzias latipes), nous apportons la preuve de l’existence de cette fonction cellulaire chez cette espèce de poisson. Enfin, afin de caractériser le rôle physiologique de cette fonction chez les poissons, nous avons procédé à l’invalidation par crispR-cas9 de lamp2a chez le medaka. Les poissons générés présentaient de sévères perturbations du métabolisme intermédiaire, comme précédemment décrit chez des souris dont LAMP2A a été invalidée dans le foie. Dans l’ensemble, ces résultats démontrent clairement, et pour la toute première fois, qu’il existe bien une activité CMA fonctionnelle chez les poissons, et apportent ainsi de nouvelles perspectives dans ce domaine de recherche, notamment en autorisant l'utilisation de modèles génétiques complémentaires, tels que le poisson zèbre ou le medaka, pour faire avancer nos connaissances sur les mécanismes régissant cette fonction cellulaire.Chaperone-Mediated Autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player in the control of numerous cellular functions, and whose defects have been associated to several human pathologies, including neurodegenerative diseases, cancers and immune disorders. To date, this cellular function was presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in non-tetrapod species. However, we recently identified the existence of expressed sequences displaying high homology with the mammalian LAMP2A in several fish species, challenging that view and suggesting that CMA appeared much earlier during evolution than initially thought. In the present thesis, we first present new evidences about the evolutionary history of the gene LAMP2 in vertebrates. We demonstrate that LAMP2 appeared after the second whole genome duplication that occurred at the root of the vertebrate lineage approximately 500 million years ago. By using a fluorescent reporter previously used to track CMA in mammalian cells, we then revealed the existence of a CMA-like pathway in a fibroblast cell line of the fish medaka (Oryzias latipes). Finally, to address the physiological role of Lamp2a in fish, we generated, medaka knockout for the splice variant lamp2a, and found severe alterations in the intermediary metabolism, as previously demonstrated in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective