RESUMO
Vertebrate females transfer antibodies via the placenta, colostrum and milk or via the egg yolk to protect their immunologically immature offspring against pathogens. This evolutionarily important transfer of immunity is poorly documented in invertebrates and basic questions remain regarding the nature and extent of parental protection of offspring. In this study, we show that a lipopolysaccharide binding protein/bactericidal permeability increasing protein family member from the invertebrate Biomphalaria glabrata (BgLBP/BPI1) is massively loaded into the eggs of this freshwater snail. Native and recombinant proteins displayed conserved LPS-binding, antibacterial and membrane permeabilizing activities. A broad screening of various pathogens revealed a previously unknown biocidal activity of the protein against pathogenic water molds (oomycetes), which is conserved in human BPI. RNAi-dependent silencing of LBP/BPI in the parent snails resulted in a significant reduction of reproductive success and extensive death of eggs through oomycete infections. This work provides the first functional evidence that a LBP/BPI is involved in the parental immune protection of invertebrate offspring and reveals a novel and conserved biocidal activity for LBP/BPI family members.
Assuntos
Proteínas de Fase Aguda/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biomphalaria , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Imunidade Materno-Adquirida , Infecções/imunologia , Glicoproteínas de Membrana/metabolismo , Oomicetos , Zigoto , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biomphalaria/genética , Biomphalaria/imunologia , Biomphalaria/metabolismo , Biomphalaria/parasitologia , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Feminino , Imunidade Materno-Adquirida/genética , Infecções/genética , Infecções/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacologia , Testes de Sensibilidade Microbiana , Oomicetos/efeitos dos fármacos , Oomicetos/imunologia , Oomicetos/patogenicidade , Proteínas Recombinantes/farmacologia , Zigoto/imunologia , Zigoto/metabolismo , Zigoto/parasitologiaRESUMO
LBPs (lipopolysaccharide binding proteins) and BPIs (bactericidal permeability increasing proteins) are important proteins involved in defense against bacterial pathogens. We recently discovered a novel biocidal activity of a LBP/BPI from the gastropod Biomphalaria glabrata and demonstrated its role in parental immune protection of eggs, highlighting the importance of LBP/BPIs in invertebrate immunity. Here we characterize four additional LBP/BPI from B. glabrata, presenting conserved sequence architecture and exon-intron structure. Searches of invertebrate genomes revealed that existence of LBP/BPIs is not a conserved feature since they are absent from phyla such as arthropods and platyhelminths. Analyses of LBP/BPI transcripts from selected mollusk species showed recent parallel duplications in some species, including B. glabrata. In this snail species, LBP/BPI members vary in their expression tissue localization as well as their change in expression levels after immune challenges (Gram-negative bacterium; Gram-positive bacterium or yeast). These results, together with the predicted protein features provide evidences of functional specialization of LBP/BPI family members in molluscs.
Assuntos
Proteínas de Fase Aguda/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções Bacterianas/imunologia , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Micoses/imunologia , Caramujos/imunologia , Proteínas de Fase Aguda/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Evolução Biológica , Proteínas Sanguíneas/genética , Proteínas de Transporte/genética , Sequência Conservada , Duplicação Gênica , Imunidade , Invertebrados , Glicoproteínas de Membrana/genética , Especificidade de Órgãos , Especificidade da Espécie , TranscriptomaRESUMO
Aphids attack virtually all plant species and cause serious crop damages in agriculture. Despite their dramatic impact on food production, little is known about the molecular processes that allow aphids to exploit their host plants. To date, few aphid salivary proteins have been identified that are essential for aphid feeding, and their nature and function remain largely unknown. Here, we show that a macrophage migration inhibitory factor (MIF) is secreted in aphid saliva. In vertebrates, MIFs are important pro-inflammatory cytokines regulating immune responses. MIF proteins are also secreted by parasites of vertebrates, including nematodes, ticks, and protozoa, and participate in the modulation of host immune responses. The finding that a plant parasite secretes a MIF protein prompted us to question the role of the cytokine in the plant-aphid interaction. We show here that expression of MIF genes is crucial for aphid survival, fecundity, and feeding on a host plant. The ectopic expression of aphid MIFs in leaf tissues inhibits major plant immune responses, such as the expression of defense-related genes, callose deposition, and hypersensitive cell death. Functional complementation analyses in vivo allowed demonstrating that MIF1 is the member of the MIF protein family that allows aphids to exploit their host plants. To our knowledge, this is the first report of a cytokine that is secreted by a parasite to modulate plant immune responses. Our findings suggest a so-far unsuspected conservation of infection strategies among parasites of animal and plant species.
Assuntos
Afídeos/genética , Herbivoria , Proteínas de Insetos/genética , Fatores Inibidores da Migração de Macrófagos/genética , Imunidade Vegetal , Vicia faba/imunologia , Sequência de Aminoácidos , Animais , Afídeos/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Dados de Sequência Molecular , Folhas de Planta , Saliva/química , Alinhamento de SequênciaRESUMO
Our present understanding of the functioning and evolutionary history of invertebrate innate immunity derives mostly from studies on a few model species belonging to ecdysozoa. In particular, the characterization of signaling pathways dedicated to specific responses towards fungi and Gram-positive or Gram-negative bacteria in Drosophila melanogaster challenged our original view of a non-specific immunity in invertebrates. However, much remains to be elucidated from lophotrochozoan species. To investigate the global specificity of the immune response in the fresh-water snail Biomphalaria glabrata, we used massive Illumina sequencing of 5'-end cDNAs to compare expression profiles after challenge by Gram-positive or Gram-negative bacteria or after a yeast challenge. 5'-end cDNA sequencing of the libraries yielded over 12 millions high quality reads. To link these short reads to expressed genes, we prepared a reference transcriptomic database through automatic assembly and annotation of the 758,510 redundant sequences (ESTs, mRNAs) of B. glabrata available in public databases. Computational analysis of Illumina reads followed by multivariate analyses allowed identification of 1685 candidate transcripts differentially expressed after an immune challenge, with a two fold ratio between transcripts showing a challenge-specific expression versus a lower or non-specific differential expression. Differential expression has been validated using quantitative PCR for a subset of randomly selected candidates. Predicted functions of annotated candidates (approx. 700 unisequences) belonged to a large extend to similar functional categories or protein types. This work significantly expands upon previous gene discovery and expression studies on B. glabrata and suggests that responses to various pathogens may involve similar immune processes or signaling pathways but different genes belonging to multigenic families. These results raise the question of the importance of gene duplication and acquisition of paralog functional diversity in the evolution of specific invertebrate immune responses.