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Etude de la susceptibilité des cellules eucaryotes à l'injection de toxines par le système de sécrétion de type 3 de Pseudomonas aeruginosa

Abstract : The pathogenesis of Pseudomonas aeruginosa (P.a) implies multiple virulence factors among which the type III secretion system (T3SS). This multiprotein complex is composed of a needle through which four exotoxins are exported. The protein PopB and PopD form an oligomeric structure (translocon) at the end of the needle that inserts into the host cell membrane and translocates the exotoxins into the cytoplasm. Synthesis and toxin secretion is induced on contact with eukaryotic cell. In this work, we examined the influence of host cell elements on exotoxin translocation efficiency. The delivery of T3SS toxins was investigated using a CCF2/β-lactamase fluorescent reporter system In parallel, the association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. Promyelocytic HL-60 cells and promonocytic U937 cells were found to be resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to neutrophil- or macrophage-like cells resulted in an injection-sensitive phenotype without any significant change in the level of membrane-inserted translocon proteins. Treatment of sensitive HL-60 cells with a cholesterol-depleting agent, resulted in a diminished injection of toxin. Moreover, the PopB translocator was found in the membrane fraction obtained from sucrose-gradient purifications and containing lipid-raft marker flotillin. Through a pharmacological approach, we brought evidence that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore.
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Submitted on : Saturday, March 17, 2012 - 12:47:13 PM
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  • HAL Id : tel-00680045, version 1



Julien Verove. Etude de la susceptibilité des cellules eucaryotes à l'injection de toxines par le système de sécrétion de type 3 de Pseudomonas aeruginosa. Autre [q-bio.OT]. Université de Grenoble, 2011. Français. ⟨NNT : 2011GRENV071⟩. ⟨tel-00680045⟩



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