Etude des adhésines HBHA et LBP impliquées dans l'interaction de Mycobacterium avium ssp. paratuberculosis avec les cellules épithéliales intestinales, cibles privilégiées de la bactérie in vivo

Mycobacterium avium ssp. paratuberculosis (Map), agent étiologique de la paratuberculose, a évolué en deuxtypes dénommés, S pour Sheep et C pour Cattle . L intestin grêle est le site primaire de l infection à Map mais les mécanismes moléculaires impliqués dans l implantation du bacille restent largement méconnus. L objectif de mon projet de thèse visait à identifier et caractériser les adhésines exprimées par Map par des approches génétiques et biochimiques. J ai ainsi purifié la HBHA et la LBP par chromatographie d affinité puis les ai identifiés en spectrométrie de masse. L originalité de ce travail repose sur le polymorphisme de ces adhésines observé entre les souches de type C et S. Cette variabilité a été mise en évidence sur le domaine d interaction avec les sucres sulfatés de la cellule hôte influençant l affinité des adhésines pour l héparine. Ce travail de thèse a permis de caractériser pour la première fois ces deux adhésines produites par Map. Le polymorphisme de la HBHA et de la LBP, discriminant les types C et S, ouvre de nombreuses perspectives sur l évolution de l espèce M. avium et le rôle de ces adhésines sur le tropisme intestinal, la préférence d hôte de Map ou encore leur potentiel diagnostic.Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of paratuberculosis, has evolved into two types called, S for "Sheep" and C for "Cattle." The small intestine is the primary site of Map infection but the molecular mechanisms involved in the establishment of bacilli are still unknown. The aim of my thesis was to identify and characterize the adhesins expressed by Map by genetic and biochemical approaches. I purified HBHA and LBP by affinity chromatography then identified them by mass spectrometry. The originality of this work is based on the polymorphism of these adhesins observed between strains of type C and S. This variability has been demonstrated in the binding domain involved in interaction with sulfated sugars of host cell influences adhesins affinity for heparin. This thesis has characterized for the first time these two adhesins produced by Map. Specific polymorphism highlighted related to the evolution of the species avium, opens large number questions on their role on the pathogenesis of Map including the cellular tropism, host preference or interest of these antigens to improve diagnostic.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Environmental Mycobacterium avium subsp. paratuberculosis Hosted by Free-Living Amoebea

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

Supersymmetric structure of the induced W gravities

We derive the supersymmetric structure present in W-gravities which has been already observed in various contexts as Yang-Mills theory, topological field theories, bosonic string and chiral W_{3}-gravity. This derivation which is made in the geometrical framework of Zucchini, necessitates the introduction of an appropriate new basis of variables which replace the canonical fields and their derivatives. This construction is used, in the W_{2}-case, to deduce from the Chern-Simons action the Wess-Zumino-Polyakov action.Comment: 17 pages, Latex. To appear in Class. Quantum. Gravit

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

International audienceBACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays

Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae

<p>Abstract</p> <p>Background</p> <p>The outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism <it>Mycobacterium smegmatis</it>, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb.</p> <p>Results</p> <p>We have recently sequenced the complete genomes of two fast-growers causing human infections, <it>Mycobacterium abscessus </it>(CIP 104536T) and <it>M. chelonae </it>(CIP 104535T). We show here that these two species contain genes corresponding to all those of the <it>M. smegmatis </it>"GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in <it>M. chelonae </it>and <it>M. abscessus</it>. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (<it>atf1 </it>and <it>atf2</it>) in <it>M. abscessus </it>and <it>M. chelonae </it>has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (<it>M. chelonae</it>, 900 kb; <it>M. abscessus</it>, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins.</p> <p>Conclusion</p> <p>Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in <it>M. smegmatis </it>represents the ancestral form and that evolution has scattered various pieces throughout the genome in <it>M. abscessus </it>and <it>M. chelonae</it>.</p

Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne's disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods. RESULTS: Single nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species. We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains. WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year. CONCLUSIONS: This study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections

A geometrical approach to super W-induced gravities in two dimensions

A geometrical study of supergravity defined on (1|1) complex superspace is presented. This approach is based on the introduction of generalized superprojective structures extending the notions of super Riemann geometry to a kind of super W-Riemann surfaces. On these surfaces a connection is constructed. The zero curvature condition leads to the super Ward identities of the underlying supergravity. This is accomplished through the symplectic form linked to the (super)symplectic manifold of all super gauge connections. The BRST algebra is also derived from the knowledge of the super W-symmetries which are the gauge transformations of the vector bundle canonically associated to the generalized superprojective structures. We obtain the possible consistent BRST (super)anomalies and their cocycles related by the descent equations. Finally we apply our considerations to the case of supergravity.Comment: 29 pages, latex, no figures, to appear in Nucl. Phys.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(<it>Map</it>) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of <it>Map </it>is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of <it>Map </it>isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of <it>Map</it>, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.</p> <p>Results</p> <p>164 <it>Map </it>isolates from seven European countries representing 19 different host species were genotyped by standardized IS<it>900 </it>- restriction fragment length polymorphism (IS<it>900</it>-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS<it>900</it>-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.</p> <p>Conclusion</p> <p>The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of <it>Map </it>infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from <it>Map </it>isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of <it>Map </it>between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.</p

Survie environnementale de M. Bovis : un obstacle écologique au contrôle et à l’éradication de la tuberculose bovine

National audienceDes réémergences sporadiques de la tuberculose bovine, principalement en Bourgogne et en Nouvelle-Aquitaine, compliquent l’éradication de cette zoonose,dont la France est indemne depuis 2001. Cet article fait le point sur les sources de contamination des bovins. Réservoir dans la faune sauvage, contamination de l’environnement dans lequel Mycobacterium bovis est capable de survivre ou transmission possible via des vecteurs de la microfaune tels que les amibes sont autant de facteurs susceptibles d’expliquer la situation endémique dans certaines zones

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