Biomphalysin, a new β pore-forming toxin involved in Biomphalaria glabrata immune defense against Schistosoma mansoni.

International audienceAerolysins are virulence factors belonging to the β pore-forming toxin (β-PFT) superfamily that are abundantly distributed in bacteria. More rarely, β-PFTs have been described in eukaryotic organisms. Recently, we identified a putative cytolytic protein in the snail, Biomphalaria glabrata, whose primary structural features suggest that it could belong to this β-PFT superfamily. In the present paper, we report the molecular cloning and functional characterization of this protein, which we call Biomphalysin, and demonstrate that it is indeed a new eukaryotic β-PFT. We show that, despite weak sequence similarities with aerolysins, Biomphalysin shares a common architecture with proteins belonging to this superfamily. A phylogenetic approach revealed that the gene encoding Biomphalysin could have resulted from horizontal transfer. Its expression is restricted to immune-competent cells and is not induced by parasite challenge. Recombinant Biomphalysin showed hemolytic activity that was greatly enhanced by the plasma compartment of B. glabrata. We further demonstrated that Biomphalysin with plasma is highly toxic toward Schistosoma mansoni sporocysts. Using in vitro binding assays in conjunction with Western blot and immunocytochemistry analyses, we also showed that Biomphalysin binds to parasite membranes. Finally, we showed that, in contrast to what has been reported for most other members of the family, lytic activity of Biomphalysin is not dependent on proteolytic processing. These results provide the first functional description of a mollusk immune effector protein involved in killing S. mansoni

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway

Babesia divergens glycosylphosphatidylinositols modulate blood coagulation and induce Th2-biased cytokine profiles in antigen presenting cells

This work was supported by the the University of Tours (to IDP and FDG), the University of Montpellier (to SD and EC), the Deutsche Forschungsgemeinschaft (to RTS), the Wellcome Trust project grant 093228 (to TKS) and the Campus France/DAAD PHC PROCOPE 24931RE (to RTS and EC). The funding source has no involvement in the conduct of the research and preparation of the article.Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.Publisher PDFPeer reviewe

Dynamique conformationnelle chez les protéines d'adhésion de Babesia (mythe ou réalité ?)

L'une des infections parasitaires les plus courantes chez les animaux à travers le monde est la babésiose ou piroplasmose. Causée par le développement intraérythrocytaire d'un parasite du genre Babesia, elle présente de nombreux signes cliniques semblables à ceux du paludisme. Ce parasite, du phylum des Apicomplexes, est transmis via le vecteur tique et effectue son cycle de reproduction dans les cellules rouges du sang de l'hôte vertébré. En Europe B. divergens et B. canis sont les espèces majoritairement responsables respectivement de la babésiose bovine et la babésiose canine. Dans une stratégie de recherche vaccinale, l'étude de protéines parasitaires en contact avec la circulation sanguine est primordiale pour comprendre les interactions hôte-parasite et identifier des candidats vaccins à haut potentiel. Les protéines à ancrage GPI (glycosylphosphatidylinositol) font partie de ces protéines. La première protéine à ancrage GPI décrite chez B. divergens est Bd37.1. Elle induit une protection totale contre une infection à B. divergens à la condition qu'une séquence hydrophobe soit ajoutée en C-terminale. La résolution de la structure RMN de cette protéine a permis de mettre en évidence un probable mécanisme de changement conformationnel en fonction du pH. La structure composée de 3 sous domaines montre que celle-ci n'est maintenue que par des ponts salins qui peuvent se rompre en milieu acide. Or l'environnement membranaire dans lequel évolue Bd37.1 ancrée à la surface du parasite et/ou à l'approche du globule rouge lors de l'invasion est acide. Cette dynamique conformationnelle de la protéine -Bd37, liée à l'environnement membranaire, pourrait être à l'origine du mécanisme qui confère une immunité en fonction de la présence ou non de la séquence hydrophobe en C-terminale de Bd37.1. Nous avons cherché à estimer les implications d'une telle dynamique dans les interactions hôtes-parasites à travers l'étude structurale de 2 protéines parasitaires (Bd37.1 et Bc28.1). Dans le premier cas nous étudions la dynamique conformationnelle de la protéine d'adhésion Bd37.1. Nous avons exploré les différentes conformations que pourrait adopter la protéine Bd37.1 par une approche de biophysique et nous avons stabilisé ces différentes conformations en solution par le biais de mutations pour les étudier. Parmi ces mutants, le mutant EDK- -Bd37 dont les ponts salins ont été rompus montre des caractéristiques différentes de -Bd37. Les données enregistrées sur ce mutant nous ont amené à résoudre sa structure et à tester son pouvoir vaccinant. Dans une seconde partie, nous caractérisons biochimiquement et fonctionnellement une autre protéine Bc28.1, l'orthologue de Bd37.1. chez B. canis, accompagnée de la résolution de sa structure. Nous montrons que Bc28.1 est une protéine d'adhésion localisée à la surface du parasite et nous comparons les structures de Bd37.1 et Bc28.1. Ces deux structures sont finalement très différentes tandis que localisation et fonction sont similaires.One of the most common parasitic infections in animals worldwide is babesiosis or piroplasmosis. Caused by the intraerythrocytic development of Babesia parasite, it has many clinical signs similar to those of malaria. This parasite of the phylum Apicomplexa, is transmitted via the tick vector and performs its reproductive cycle in red blood cells of the vertebrate host. B. In Europe divergens and B. canis species are mainly responsible respectively for bovine babesiosis and canine babesiosis. A strategy of vaccine research, the study of parasite proteins in contact with the bloodstream is essential for understanding host-parasite interactions and identify vaccine candidates with high potential. Anchored protein GPI (glycosylphosphatidylinositol) are part of these proteins. The first protein GPI anchors described in B. divergens is Bd37.1. It induces complete protection against infection with B. divergens provided a hydrophobic sequence is added at the C-terminus. Resolution NMR structure of this protein has highlighted a probable mechanism of conformational change as a function of pH. The structure consists of three sub areas shows that it is only maintained by salt bridges which can break in acidic medium. However, the environment within which Bd37.1 membrane anchored to the surface of the parasite and / or approach the red blood cell during the invasion is acidic. This conformational dynamics of the protein- Bd37 linked to the membrane environment, could be at the origin of the mechanism that confers immunity depending on the presence or absence of the hydrophobic sequence at the C-terminus of Bd37.1. We sought to assess the implications of such dynamics in host-parasite interactions through structural study of two parasite proteins (Bd37.1 and Bc28.1). In the first case we study the conformational dynamics of the adhesion protein Bd37.1. We explored the different conformations that may be adopted by a protein Bd37.1 biophysical approach and we have stabilized in different conformations in solution through mutations to study. Among these mutants, the mutant -Bd37-EDK including salt bridges were broken shows different characteristics -Bd37. The data on this mutant led us to solve the structure and to test its power vaccinating. In a second part, we characterize biochemically and functionally Bc28.1 another protein, the ortholog Bd37.1. in B. canis, accompanied with the resolution of its structure. We show that Bc28.1 is an adhesion protein localized to the parasite surface and compare the structures and Bd37.1 Bc28.1. These two structures are ultimately very different while location and function are similar.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Glycosylphosphatidylinositols of <em>Babesia divergens</em> have unique effects on host cells of the innate immune system

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Bioaccumulated provitamin A in black soldier fly larvae is bioavailable and capable of improving vitamin A status of gerbils

International audienceThe aim was to study whether provitamin A (proVA), which can bioaccumulate in black 27 soldier fly larvae (BSFL), is bioavailable and can restore VA status in mammals. A model for 28 studying the metabolism of this vitamin, the gerbil, was either fed a standard diet (C+ group), a 29 diet without VA (C-), a diet in which VA was provided by β-carotene (β-C) from sweet 30 potatoes (SP), or a diet in which VA was provided by β-C from BSFL that had been fed sweet 31 potatoes (BSFL). The animals were killed at the end of the supplementation period and β-C, 32 retinol and retinyl esters were measured in plasma and liver. As expected β-C was not detected 33 in plasma and liver of the C+ and C- groups. β-C concentrations were lower (p < 0.05) in 34 plasma and liver of the BSFL group as compared to the SP group. Liver retinol and retinyl ester 35 concentrations were lower in the C- group than in all the other groups (p < 0.05). These 36 concentrations were not significantly different in the C+ and SP groups while they were lower 37 in the BSFL group (p < 0.05 for retinyl oleate and retinyl linoleate). In total, the liver stock of 38 retinol equivalent was almost twice lower in the BSFL group than in the SP group. Thus, β-C 39 present in the BSFL matrix is bioavailable and capable of improving VA status, but this matrix 40 decreases its effectiveness by a factor of around two compared to the sweet potato matrix

Structure of the Mycobacterium tuberculosis OmpATb protein: A model of an oligomeric channel in the mycobacterial cell wall

International audienceThe pore-forming outer membrane protein OmpATb from Mycobacterium tuberculosis is a virulence factor required for acid resistance in host phagosomes. In this study, we determined the 3D structure of OmpATb by NMR in solution. We found that OmpATb is composed of two independent domains separated by a proline-rich hinge region. As expected, the high-resolution structure of the C-terminal domain (OmpATb(198-326)) revealed a module structurally related to other OmpA-like proteins from Gram-negative bacteria. The N-terminal domain of OmpATb (73-204), which is sufficient to form channels in planar lipid bilayers, exhibits a fold, which belongs to the alpha+beta sandwich class fold. Its peculiarity is to be composed of two overlapping subdomains linked via a BON (Bacterial OsmY and Nodulation) domain initially identified in bacterial proteins predicted to interact with phospholipids. Although OmpATb(73-204) is highly water soluble, current-voltage measurements demonstrate that it is able to form conducting pores in model membranes. A HADDOCK modeling of the NMR data gathered on the major monomeric form and on the minor oligomeric populations of OmpATb(73-204) suggest that OmpATb(73-204) can form oligomeric rings able to insert into phospholipid membrane, similar to related proteins from the Type III secretion systems, which form multisubunits membrane-associated rings at the basal body of the secretion machinery. Proteins 2011; 79:645-661. (C) 2010 Wiley-Liss, Inc

Monitoring Unfolding of Titin I27 Single and Bi Domain with High-Pressure NMR Spectroscopy

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