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

ABSTRACT: Aerolysins are virulence factors belonging to the ? pore-forming toxin (?-PFT) superfamily that are abundantly distributed in bacteria. More rarely, ?-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 ?-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 ?-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.

SUBMITTER: Galinier R

PROVIDER: S-EPMC3605176 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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

Galinier Richard R Portela Julien J Moné Yves Y Allienne Jean François JF Henri Hélène H Delbecq Stéphane S Mitta Guillaume G Gourbal Benjamin B Duval David D

PLoS pathogens 20130321 3

Aerolysins 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 Bi ...[more]

PMID: 23555242

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Project description:Schistosomiasis is an infectious disease infecting mammals as the definitive host and fresh water snails as the intermediate host. Understanding the molecular and biochemical relationship between the causative schistosome parasite and its hosts will be key to understanding and ultimately treating and/or eradicating the disease. There is increasing evidence that pathogens that have co-evolved with their hosts can manipulate their hosts' behaviour at various levels to augment an infection. Bacteria, for example, can induce beneficial chromatin remodelling of the host genome. We have previously shown in vitro that Biomphalaria glabrata embryonic cells co-cultured with schistosome miracidia display genes changing their nuclear location and becoming up-regulated. This also happens in vivo in live intact snails, where early exposure to miracidia also elicits non-random repositioning of genes. We reveal differences in the nuclear repositioning between the response of parasite susceptible snails as compared to resistant snails and with normal or live, attenuated parasites. Interestingly, the stress response gene heat shock protein (Hsp) 70 is only repositioned and then up-regulated in susceptible snails with the normal parasite. This movement and change in gene expression seems to be controlled by the parasite. Other differences in the behaviour of genes support the view that some genes are responding to tissue damage, for example the ferritin genes move and are up-regulated whether the snails are either susceptible or resistant and upon exposure to either normal or attenuated parasite. This is the first time host genome reorganisation has been seen in a parasitic host and only the second time for any pathogen. We believe that the parasite elicits a spatio-epigenetic reorganisation of the host genome to induce favourable gene expression for itself and this might represent a fundamental mechanism present in the human host infected with schistosome cercariae as well as in other host-pathogen relationships.

Project description:Circulating hemocytes of the snail Biomphalaria glabrata, a major intermediate host for the blood fluke Schistosoma mansoni, represent the primary immune effector cells comprising the host's internal defense system. Within hours of miracidial entry into resistant B. glabrata strains, hemocytes infiltrate around developing sporocysts forming multi-layered cellular capsules that results in larval death, typically within 24-48 h post-infection. Using an in vitro model of hemocyte-sporocyst encapsulation that recapitulates in vivo events, we conducted a comparative proteomic analysis on the responses of hemocytes from inbred B. glabrata strains during the encapsulation of S. mansoni primary sporocysts. This was accomplished by a combination of Laser-capture microdissection (LCM) to isolate sections of hemocyte capsules both in the presence and absence of sporocysts, in conjunction with mass spectrometric analyses to establish protein expression profiles. Comparison of susceptible NMRI snail hemocytes in the presence and absence of sporocysts revealed a dramatic downregulation of proteins in during larval encapsulation, especially those involved in protein/CHO metabolism, immune-related, redox and signaling pathways. One of 4 upregulated proteins was arginase, competitor of nitric oxide synthetase and inhibitor of larval-killing NO production. By contrast, when compared to control capsules, sporocyst-encapsulating hemocytes of resistant BS-90 B. glabrata exhibited a more balanced profile with enhanced expression of shared proteins involved in protein synthesis/processing, immunity, and redox, and unique expression of anti-microbial/anti-parasite proteins. A final comparison of NMRI and BS-90 host hemocyte responses to co-cultured sporocysts demonstrated a decrease or downregulation of 77% of shared proteins by NMRI cells during encapsulation compared to those of the BS-90 strain, including lipopolysaccharide-binding protein, thioredoxin reductase 1 and hemoglobins 1 and 2. Overall, using this in vitro model, results of our proteomic analyses demonstrate striking differences in proteins expressed by susceptible NMRI and resistant BS-90 snail hemocytes to S. mansoni sporocysts during active encapsulation, with NMRI hemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed immune-relevant proteins (e.g., FREP2) compared to BS-90. Our data suggest that snail strain differences in hemocyte protein expression during the encapsulation process account for observed differences in their cytotoxic capacity to interact with and kill sporocysts.

Project description:The World Health Organization (WHO) estimates that approximately 240 million people in 78 countries require treatment for schistosomiasis, an endemic disease caused by trematodes of the genus Schistosoma. In Brazil, Schistosoma mansoni is the only species representative of the genus whose passage through an invertebrate host, snails of the genus Biomphalaria, is obligatory before infecting a mammalian host, including humans. The availability of the genome and transcriptome of B. glabrata makes studying the regulation of gene expression, particularly the regulation of miRNA and piRNA processing pathway genes, possible. This might assist in better understanding the biology of B. glabrata as well as its relationship to the parasite S. mansoni. Some aspects of this interaction are still poorly explored, including the participation of non-coding small RNAs, such as miRNAs and piRNAs, with lengths varying from 18 to 30 nucleotides in mature form, which are potent regulators of gene expression. Using bioinformatics tools and quantitative PCR, we characterized and validated the miRNA and piRNA processing pathway genes in B. glabrata. In silico analyses showed that genes involved in miRNA and piRNA pathways were highly conserved in protein domain distribution, catalytic site residue conservation and phylogenetic analysis. Our study showed differential expression of putative Argonaute, Drosha, Piwi, Exportin-5 and Tudor genes at different snail developmental stages and during infection with S. mansoni, suggesting that the machinery is required for miRNA and piRNA processing in B. glabrata at all stages. These data suggested that the silencing pathway mediated by miRNAs and piRNAs can interfere in snail biology throughout the life cycle of the snail, thereby influencing the B. glabrata/S. mansoni interaction. Further studies are needed to confirm the participation of the small RNA processing pathway proteins in the parasite/host relationship, mainly the effective participation of small RNAs in regulating their target genes.

Project description:Background: The full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used.Principal findings: Transcripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity.Conclusions/significance: This in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated.

Dataset Information

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

Publications

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

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