Interactions microsporidies-insectes in vivo : dissémination de Nosema bombycis (Microsporidia) dans son hôte Bombyx mori (Lepidoptera) et caractérisation de protéines structurales majeures de N. bombycis impliquées dans l'invasion - Université Clermont Auvergne Accéder directement au contenu
Thèse Année : 2007

Microsporidian-insect interactions in vivo : dissemination of Nosema bombycis (Microsporidia) in Bombyx mori (Lepidoptera) and characterization of some N. bombycis major invasion-involded structural proteins

Interactions microsporidies-insectes in vivo : dissémination de Nosema bombycis (Microsporidia) dans son hôte Bombyx mori (Lepidoptera) et caractérisation de protéines structurales majeures de N. bombycis impliquées dans l'invasion

Résumé

Nosema bombycis is a spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. It is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about N. bombycis in vivo dissemination mechanism and its interactions with its natural host B. mori at the cellular or molecular level, despite the ongoing genome sequencing projects of both organisms. In this study, we investigate N. bombycis (Zhenjiang isolate ISC-ZJ) dissemination in per os infected B. mori larvae (Nistari). At different times post-infestation, larvae were harvested and B. mori larva tissues, hemocytes and hemolymph were analysed by histochemistry and immunocytochemistry microscopy (optic and transmission electron microscopy). This allowed us to document not only N. bombycis ISC-ZJ in vivo life cycle at the ultrastructural level, but also the hitherto unreported N. bombycis dissemination route in silkworm larvae. N. bombycis infestation first settles in the anterior epithelial midgut. The infection spreads to the muscles and tracheae closely surrounding the anterior midgut. Distant tissues and organs are then infected concomitantly with the apparition of the parasite in the hemolymph or internalized in hemocytes, before the systemic infestation with all tissues parasitized. Our results strongly suggest that hemolymph and hemocytes are vectors for the parasite dissemination in its host. Moreover the silkworm innate immune response, although existing -we identified N. bombycis melanization and its phagocytosis by B. mori hemocytes - seem to be strongly overtaken by the microsporidian development with underlying mechanisms still unknown. To further study the N. bombycis/B. mori interactions at the molecular level, we focused on major microsporidian structural proteins from the spore wall and the polar tube which are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by mono- and bi-dimensionnal electrophoresis to establish complementary proteomic profiles. Three proteins (71, 48 and 30 kDa) were shown to be located at the parasite spore wall using a polyclonal antibody raised against formaldehyde-fixed spores. Moreover seventeen polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots, spot PEL1 (54 kDa, pI 5.4), spot PEL2 (36 kDa, pI 10) and spot PEL3 (150 kDa, pI 7.1), were shown to correspond to polar tube proteins (PTPs) by IFA and transmission electron microscopy immunocytochemistry on cryosections. These PTPs were respectively named NbPTP1, NbPTP2 and NbPTP3 according to their global physio-chemical properties. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS mass spectrometry. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by Mascot and Sequest searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a non-genomic context.
Nosema bombycis est un parasite obligatoire intracellulaire et eukaryoitique microsporidia apparenté aux champignons. Cette microsporidie est l'agent responsable de la pébrine, maladie du ver à soie Bombyx mori qui inflige de sévères pertes économiques à la sériciculture mondiale. Nous avons étudié l'interactions N. bombycis-B. mori in vivo : l'infestation par N. bombycis démarre au niveau de l'épithélium intestinal antérieur, puis s'étend aux muscles et trachées adjacents. Les tissus plus distants sont ensuite infectés. Cependant, les réponses immune mélanisation et phagocytose, l'hémolymphe et les hémocytes sont les vecteurs de la dissémination de N. bombycis dans son hôte. Nous avons développé une approche protéomique pour identifier des protéines de tube polaire (PTP). Trois PTPs ont été caracterisés par immunocytochimie MET et MS/MS. Des motifs de séquence peptidique ont pu en être déduits par les programmes Peaks Online et DeNovoX, puis évalués par algorithmes Mascot et Sequest
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Dates et versions

tel-00717814 , version 1 (13-07-2012)

Identifiants

  • HAL Id : tel-00717814 , version 1

Citer

Jian-Yang Wang. Interactions microsporidies-insectes in vivo : dissémination de Nosema bombycis (Microsporidia) dans son hôte Bombyx mori (Lepidoptera) et caractérisation de protéines structurales majeures de N. bombycis impliquées dans l'invasion. Biochimie, Biologie Moléculaire. Université Blaise Pascal - Clermont-Ferrand II; Université d'Auvergne - Clermont-Ferrand I, 2007. Français. ⟨NNT : 2007CLF21736⟩. ⟨tel-00717814⟩
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