A Large Repertoire of Parasite Epitopes Matched by a Large Repertoire of Host Immune Receptors in an Invertebrate Host/Parasite Model

For many decades, invertebrate immunity was believed to be non-adaptive, poorly specific, relying exclusively on sometimes multiple but germ-line encoded innate receptors and effectors. But recent studies performed in different invertebrate species have shaken this paradigm by providing evidence for various types of somatic adaptations at the level of putative immune receptors leading to an enlarged repertoire of recognition molecules. Fibrinogen Related Proteins (FREPs) from the mollusc Biomphalaria glabrata are an example of these putative immune receptors. They are known to be involved in reactions against trematode parasites. Following not yet well understood somatic mechanisms, the FREP repertoire varies considerably from one snail to another, showing a trend towards an individualization of the putative immune repertoire almost comparable to that described from vertebrate adaptive immune system. Nevertheless, their antigenic targets remain unknown. In this study, we show that a specific set of these highly variable FREPs from B. glabrata forms complexes with similarly highly polymorphic and individually variable mucin molecules from its specific trematode parasite S. mansoni (Schistosoma mansoni Polymorphic Mucins: SmPoMucs). This is the first evidence of the interaction between diversified immune receptors and antigenic variant in an invertebrate host/pathogen model. The same order of magnitude in the diversity of the parasite epitopes and the one of the FREP suggests co-evolutionary dynamics between host and parasite regarding this set of determinants that could explain population features like the compatibility polymorphism observed in B. glabrata/S. mansoni interaction. In addition, we identified a third partner associated with the FREPs/SmPoMucs in the immune complex: a Thioester containing Protein (TEP) belonging to a molecular category that plays a role in phagocytosis or encapsulation following recognition. The presence of this last partner in this immune complex argues in favor of the involvement of the formed complex in parasite recognition and elimination from the host

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

Bases moléculaires du polymorphisme de compatibilité dans l'interaction Schistosoma mansoni / Biomphalaria glabrata

The coevolutionary dynamic playing in the host-parasite interaction leads to an arm race between host and parasite. In certain models, this arms race results in a compatibility polymorphism for which the molecular bases remain largely unknown. The aim of this PhD thesis was to identify the molecular determinants of the compatibility polymorphism that exists in the Biomphalaria glabrata / Schistosoma mansoni interaction. First, we developed a comparative proteomics approach between compatible and incompatible strains of the parasite. This approach allows us to identify molecules that could play a key role in this interaction. They consist in highly polymorphic mucin-like proteins, the "Schistosoma mansoni Polymorphic Mucin" (SmPoMucs), and scavengers of reactive oxygen species (ROS scavengers). In order to study more completely the arms race that takes place in the B. glabrata / S. mansoni interaction, we investigate the molluscan counterparts of these molecules. Co-precipitation approaches allow us to show that SmPoMucs interact with diversified immune receptors from the mollusk, the Fibrinogen-related Proteins (FREPs). This is the first evidence of the interaction between an individual repertoire of polymorphic potential parasite antigens (SmPoMucs) and an individual repertoire of diversified potential immune receptors (FREPs) from an invertebrate host. We found a third partner associated with FREPs and SmPoMucs, a thioester-containing Protein (TEP). TEP belongs to a class of molecules involved in the phagocytosis or in encapsulation. The presence of TEP in this immune complex argues in favor of the involvement of the formed complex in parasite immune recognition and elimination. We are also interested in the effector mechanisms responsible for the destruction of the parasite, in B. glabrata they rely essentially on the production of Reactive Oxygen Species (ROS). We show that a phenotypic concordance exists between the levels of host ROS production and parasite ROS scavengers' production. The compatibility polymorphism int he B. glabrata / S. mansoni interaction seems to be based on the confrontation of polymorphic and/or diversified molecules concerning immune recognition mechanisms and on quantitative reciprocal adaptations concerning immune effector mechanismsLa dynamique co-évolutive qui joue dans les systèmes hôte-parasite conduit à une véritable course aux armements entre les deux protagonistes qui se traduit, dans certaines interactions comme celle qui est traitée dans cette thèse, par un polymorphisme de compatibilité dont les bases moléculaires sont méconnues. L'objectif de cette thèse était de progresser dans la connaissance des mécanismes moléculaires sous-jacents à ce polymorphisme de compatibilité dans l'interaction Biomphalaria glabrata/Schistosoma mansoni. Une approche protéomique comparative entre des souches de parasites compatibles et incompatibles nous a permis d'identifier des déterminants moléculaires clés de l'interaction exprimés par le parasite. Il s'agit d'une part de mucines hautement polymorphes potentiellement antigéniques, les "Schistosoma mansoni Polymorphic Mucin" (SmPoMucs), et d'autre part de molécules anti-oxydantes ("ROS scavengers"). Afin d'aborder la question de la course aux armements de manière complète, nous avons également recherché la "contre-partie moléculaire" exprimée par le mollusque et susceptible d'exprimer ce polymorphisme de compatibilité. Dans ce but, des approches de co-précipitation ont été menées. Elles ont permis de montrer que les SmPoMucs interagissaient avec des récepteurs immunitaires diversifiés du mollusque, les Fibrinogen-related Proteins (FREPs). Nous montrons ainsi pour la première fois dans une interaction parasite/hôte invertébré l'intervention d'un "système de type antigène-anticorps" impliquant un répertoire individuel polymorphe d'antigènes potentiels du parasite (les SmPoMucs) et un répertoire individuel diversifié de récepteurs immunitaires de son hôte (les FREPs). Nous avons également montré que le complexe immun formé par les deux dernières molécules citées incluait un troisième partenaire, une thioester-containing protein (TEP) qui appartient à une classe de molécules connue pour son rôle dans la fagocytose ou l'encapsulation. La présence de ce troisième partenaire au sein d'un même complexe renforce le rôle potentiellement immunitaire de ce complexe dans la reconnaissance et l'élimination du parasite. Au travers de cette thèse, nous nous sommes également intéressés à la course aux armements jouant sur les mécanismes effecteurs de l'immunité du mollusque. Dans notre modèle, les effecteurs responsables de la destruction du parasite sont principalement des espèces réactives de l'oxygène (ROS). Dans ce cas aussi, nous avons montré qu'il existe une concordance phénotypique entre la production de ROS par l'hôte et le niveau de "ROS scavengers" produits par le parasite pour contrecarrer la réaction de l'hôte. Ainsi, les mécanismes moléculaires responsables du polymorphisme de compatibilité dans l'interaction B. glabrata/S. mansoni s'appuieraient au moins sur deux facteurs d'une part sur la confrontation de répertoires de molécules polymorphes et/ou diversifiées en ce qui concerne les mécanismes de reconnaissance immunitaire, et d'autre part sur une adaptation réciproque quantitative en ce qui concerne certains mécanismes effecteurs de l'immunité

. Epigenetic varaitions associated to host-plant adaptation of Spodoptera frugiperda

. Epigenetic varaitions associated to host-plant adaptation of [i]Spodoptera frugiperda[/i]. SMBE Satellite meeting SMBEBA 2015 "Investigating biological adaptation with NGS: data and models

Characterization and expression profiling of microRNAs in response to plant feeding in two host-plant strains of the lepidopteran pest Spodoptera frugiperda

International audienceBackground - A change in the environment may impair development or survival of living organisms leading them to adapt to the change. The resulting adaptation trait may reverse, or become fixed in the population leading to evolution of species. Deciphering the molecular basis of adaptive traits can thus give evolutionary clues. In phytophagous insects, a change in host-plant range can lead to emergence of new species. Among them, Spodoptera frugiperda is a major agricultural lepidopteran pest consisting of two host-plant strains having diverged 3 MA, based on mitochondrial markers. In this paper, we address the role of microRNAs, important gene expression regulators, in response to host-plant change and in adaptive evolution. Results - Using small RNA sequencing, we characterized miRNA repertoires of the corn (C) and rice (R) strains of S. frugiperda, expressed during larval development on two different host-plants, corn and rice, in the frame of reciprocal transplant experiments. We provide evidence for 76 and 68 known miRNAs in C and R strains and 139 and 171 novel miRNAs. Based on read counts analysis, 34 of the microRNAs were differentially expressed in the C strain larvae fed on rice as compared to the C strain larvae fed on corn. Twenty one were differentially expressed on rice compared to corn in R strain. Nine were differentially expressed in the R strain compared to C strain when reared on corn. A similar ratio of microRNAs was differentially expressed between strains on rice. We could validate experimentally by QPCR, variation in expression of the most differentially expressed candidates. We used bioinformatics methods to determine the target mRNAs of known microRNAs. Comparison with the mRNA expression profile during similar reciprocal transplant experiment revealed potential mRNA targets of these host-plant regulated miRNAs. Conclusions - In the current study, we performed the first systematic analysis of miRNAs in Lepidopteran pests feeding on host-plants. We identified a set of the differentially expressed miRNAs that respond to the plant diet, or differ constitutively between the two host plant strains. Among the latter, the ones that are also deregulated in response to host-plant are molecular candidates underlying a complex adaptive trait

Immunoprecipitation and Coimmunoprecipitation experiments.

<p><b>A.</b> CoImmunoPrecipitated (CoIP) and Immunoprecipitated (IP) extracts were separated by SDS-PAGE (12.5% gels) and silver-stained. Lanes 2 and 4 correspond to CoIP extracts. Lane 2: CoIP material obtained after incubation of sporocyst extracts from <i>S. mansoni</i> incompatible (IC) strain incubated with extracts from <i>B. glabrata</i> plasma (P). Lane 4: CoIP material obtained after incubation of sporocyst extracts from <i>S. mansoni</i> compatible (C) strain incubated with extracts from <i>B. glabrata</i> plasma (P). Lanes 1, 3 and 5 represent controls of Immunoprecipitated material. Lane 1: IP extracts from sporocyst of <i>S. mansoni</i> incompatible (IC) strain. Lane 3: IP extracts from <i>B. glabrata</i> plasma (P). Lane 5: IP extracts from sporocyst of <i>S. mansoni</i> Compatible (C) strain. <b>B.</b> Western-blot of immunoprecipitated (IP) and coImmunoPrecipitated (coIP) samples probed with anti-r<i>Sm</i>PoMuc antibody. Lane 6: CoIP material obtained after incubation of sporocyst extracts from <i>S. mansoni</i> incompatible (IC) strain incubated with extracts from <i>B. glabrata</i> plasma (P). Lane 7: IP extracts from <i>B. glabrata</i> plasma (P). Lane 8: CoIP material obtained after incubation of sporocyst extracts from <i>S. mansoni</i> compatible (C) strain incubated with extracts from <i>B. glabrata</i> plasma (P). Black arrow heads indicate the position of <i>Sm</i>PoMuc. Bands differentially represented between control and coIP samples are numbered (1 to 4). These four bands were cut and submitted to digestion and mass spectrometry analysis for identification.</p

Amino acid sequences alignment of the C-terminal part of <i>Sm</i>PoMucs from the three identified groups.

<p>The peptides identified by LC-MSMS are underlined. Conserved positions are indicated by an asterisk. GenBank accession numbers: group 1 (EU042600), group 2 (EU042602) and group 3 (EU042633).</p