Identification of genes involved in interactions between Biomphalaria glabrata and Schistosoma mansoni by suppression subtractive hybridization.
ABSTRACT: Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, a medically important schistosome. In order to identify transcripts involved in snail-schistosome interactions, subtractive cDNA libraries were prepared, using suppression subtractive hybridization (SSH) between a parasite-exposed schistosome-resistant and a susceptible strain of B. glabrata, and also between schistosome-exposed and unexposed snails from the resistant snail line. Separate libraries were made from both haemocytes and the haemopoietic organ. Subtraction was performed in both directions enriching for cDNAs differentially expressed between parasite-exposed resistant and susceptible samples and up or down-regulated in the resistant line after challenge. The resulting eight libraries were screened and eight genes, differentially expressed between the haemocytes of resistant and susceptible snail strains, were identified and confirmed with reverse transcriptase PCR, including two transcripts expected to be involved in the stress response mechanism for regulating the damaging oxidative burst pathways involved in cytotoxic killing of the parasite: the iron-storage and immunoregulatory molecule, ferritin, and HtrA2, a serine protease involved in the cellular stress response. Transcripts with elevated levels in the resistant strain, had the same expression patterns in the subtracted libraries and unsubtracted controls; higher levels in exposed resistant snails compared to susceptible ones and down-regulated in exposed compared with unexposed resistant snails. Differential expression of two of the transcripts with no known function from the susceptible strain, was independently confirmed in a repeat exposure experiment.
Project description:The outcome of infection in the host snail Biomphalaria glabrata with the digenean parasite Schistosoma mansoni is determined by the initial molecular interplay occurring between them. The mechanisms by which schistosomes evade snail immune recognition to ensure survival are not fully understood, but one possibility is that the snail internal defence system is manipulated by the schistosome enabling the parasite to establish infection. This study provides novel insights into the nature of schistosome resistance and susceptibility in B. glabrata at the transcriptomic level by simultaneously comparing gene expression in haemocytes from parasite-exposed and control groups of both schistosome-resistant and schistosome-susceptible strains, 2 h post exposure to S. mansoni miracidia, using an novel 5K cDNA microarray. Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the two snail strains and tests for asymmetric distributions of gene function also identified immune-related gene expression in resistant snails, but not in susceptible. Gene set enrichment analysis revealed that genes involved in mitochondrial electron transport, ubiquinone biosynthesis and electron carrier activity were consistently up-regulated in resistant snails but down-regulated in susceptible. This supports the hypothesis that schistosome-resistant snails recognize schistosomes and mount an appropriate defence response, while in schistosome-susceptible snails the parasite suppresses this defence response, early in infection.
Project description:During its life cycle, the helminth parasite Schistosoma mansoni uses the freshwater snail Biomphalaria glabrata as an intermediate host to reproduce asexually generating cercariae for infection of the human definitive host. Following invasion of the snail, the parasite develops from a miracidium to a mother sporocyst and releases excretory-secretory products (ESPs) that likely influence the outcome of host infection. To better understand molecular interactions between these ESPs and the host snail defence system, we determined gene expression profiles of haemocytes from S. mansoni-resistant or -susceptible strains of B. glabrata exposed in vitro to S. mansoni ESPs (20 ?g/ml) for 1 h, using a 5K B. glabrata cDNA microarray. Ninety-eight genes were found differentially expressed between haemocytes from the two snail strains, 57 resistant specific and 41 susceptible specific, 60 of which had no known homologue in GenBank. Known differentially expressed resistant-snail genes included the nuclear factor kappa B subunit Relish, elongation factor 1?, 40S ribosomal protein S9, and matrilin; known susceptible-snail specific genes included cathepsins D and L, and theromacin. Comparative analysis with other gene expression studies revealed 38 of the 98 identified genes to be uniquely differentially expressed in haemocytes in the presence of ESPs, thus identifying for the first time schistosome ESPs as important molecules that influence global snail host-defence cell gene expression profiles. Such immunomodulation may benefit the schistosome, enabling its survival and successful development in the snail host.
Project description:Continuing transmission of human intestinal schistosomiasis depends on the parasite's access to susceptible snail intermediate hosts (often Biomphalaria glabrata). Transmission fails when parasite larvae enter resistant individuals in wild snail populations. The genetic basis for differences in snail susceptibility/resistance is being intensively investigated as a means to devise novel control strategies based on resistance genes. Reactive oxygen species produced by the snail's defence cells (haemocytes) are effectors of resistance. We hypothesised that genes relevant to production and consumption of reactive oxygen species would be expressed differentially in the haemocytes of snail hosts with different susceptibility/resistance phenotypes. By restricting the genetic diversity of snails, we sought to facilitate identification of resistance genes. By inbreeding, we procured from a 13-16-R1 snail population with both susceptible and resistant individuals 52 lines of B. glabrata (expected homozygosity ~87.5%), and determined the phenotype of each in regard to susceptibility/resistance to Schistosoma mansoni. The inbred lines were found to have line-specific differences in numbers of spreading haemocytes; these were enumerated in both juvenile and adult snails. Lines with high cell numbers were invariably resistant to S. mansoni, whereas lines with lower cell numbers could be resistant or susceptible. Transcript levels in haemocytes were quantified for 18 potentially defence-related genes. Among snails with low cell numbers, the different susceptibility/resistance phenotypes correlated with differences in transcript levels for two redox-relevant genes: an inferred phagocyte oxidase component and a peroxiredoxin. Allograft inflammatory factor (potentially a regulator of leucocyte activation) was expressed at higher levels in resistant snails regardless of spread cell number. Having abundant spreading haemocytes is inferred to enable a snail to kill parasite sporocysts. In contrast, snails with fewer spreading haemocytes seem to achieve resistance only if specific genes are expressed constitutively at levels that are high for the species.
Project description:Resistance or susceptibility of the snail host Biomphalaria glabrata to Schistosoma mansoni is determined by the genetics of both the snail and parasite. Although Mendelian genetics governs adult resistance to infection, juvenile resistance and susceptibility are complex traits. In this study, suppression subtractive hybridization was used to construct forward and reverse cDNA libraries to identify genes involved in the immediate response of juvenile resistant (BS-90), non-susceptible (LAC2) snails, and susceptible (NMRI) snails after early exposure to S. mansoni. Expressed Sequence Tags (ESTs) were generated from the repertoire of enriched transcripts. In resistant snails, several ESTs corresponded to transcripts involved in immune regulation/defense response. While no defense related transcripts were found among juvenile susceptible snail ESTs, we detected transcripts involved in negative regulation of biological process/morphogenesis/proliferation. Differential gene expression and temporal regulation of representative transcripts were compared among snails pre- and post-exposure to either normal or attenuated miracidia using quantitative real time RT-PCR. Results showed that several transcripts, such as fibrinolytic C terminal domain, cytidine deaminase, macrophage expressed gene 1, protein kinase C receptor, anti-microbial peptide; theromacin and Fas remained up-regulated regardless of whether or not snails were exposed to normal or attenuated miracidia. While ESTs related to C-type lectin and low-density lipoprotein receptor were induced only by exposure to normal miracidia. By comparing changes in gene expression between resistant and susceptible juvenile snails responding either to normal or attenuated parasites, we can conclude that the transcription of genes associated with the intra-dermal penetration process of the snail host by invading miracidia may need to be taken into account when assessing differential gene expression between resistant and susceptible strains of B.glabrata in relation to S. mansoni exposure.
Project description:Biomphalaria glabrata snails that display either resistant or susceptible phenotypes to the parasitic trematode, Schistosoma mansoni provide an invaluable resource towards elucidating the molecular basis of the snail-host/schistosome relationship. Previously, we showed that induction of stress genes either after heat-shock or parasite infection was a major feature distinguishing juvenile susceptible snails from their resistant counterparts. In order to examine this apparent association between heat stress and snail susceptibility, we investigated the effect of temperature modulation in the resistant snail stock, BS-90. Here, we show that, incubated for up to 4 hrs at 32°C prior to infection, these resistant snails became susceptible to infection, i.e. shedding cercariae at 5 weeks post exposure (PE) while unstressed resistant snails, as expected, remained resistant. This suggests that susceptibility to infection by this resistant snail phenotype is temperature-sensitive (ts). Additionally, resistant snails treated with the Hsp 90 specific inhibitor, geldanamycin (GA) after heat stress, were no longer susceptible to infection, retaining their resistant phenotype. Consistently, susceptible snail phenotypes treated with 100 mM GA before parasite exposure also remained uninfected. These results provide direct evidence for the induction of stress genes (heat shock proteins; Hsp 70, Hsp 90 and the reverse transcriptase [RT] domain of the nimbus non-LTR retrotransposon) in B. glabrata susceptibility to S. mansoni infection and characterize the resistant BS-90 snails as a temperature-sensitive phenotype. This study of reversing snail susceptibility phenotypes to S. mansoni provides an opportunity to directly track molecular pathway(s) that underlie the B. glabrata snail's ability to either sustain or destroy the S. mansoni parasite.
Project description:Schistosomes develop successfully in susceptible snails but are encapsulated and killed in resistant ones. Mechanism(s) shaping these outcomes involves the parasites ability to evade the snail's defenses. RNA analysis from resistant (BS-90), non-susceptible (LAC2) and susceptible (NMRI) juvenile Biomphalaria glabrata to Schistosoma mansoni revealed that stress-related genes, heat shock protein 70 (Hsp 70) and reverse transcriptase (RT), were dramatically co-induced early in susceptible snails, but not in resistant/non-susceptible ones. These transcripts were, however, down regulated upon exposure to irradiated parasites although penetration behavior of irradiated vs. normal parasites were the same, indicating that Hsp 70 and RT regulation was elicited by infection and not injury. Understanding molecular events involved in stress response transcriptional regulation of Hsp 70 in juvenile snails could pave a way towards the identification of genes involved in schistosome/snail interactions.
Project description:Biomphalaria glabrata is the major intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. Much remains to be discovered concerning specific molecules mediating the defence events in these intermediate hosts, triggered by invading schistosomes. An expressed sequence tag (EST) gene discovery strategy known as ORESTES has been employed to identify transcripts that might be involved in snail-schistosome interactions in order to examine gene expression patterns in infected B. glabrata. Over 3930 ESTs were sequenced from cDNA libraries made from both schistosome-exposed and unexposed snails using different tissue types, producing a database of 1843 non-redundant clones. The non-redundant set has been assessed for gene ontology and KEGG pathway assignments. This approach has revealed a number of signalling, antioxidant and immune-related gene homologues that, based on current understanding of molluscan and other comparative systems, might play an important role in the molluscan defence response towards infection.
Project description:BACKGROUND:Schistosomiasis is a harmful neglected tropical disease caused by infection with Schistosoma spp., such as Schistosoma mansoni. Schistosoma must transition within a molluscan host to survive. Chemical analyses of schistosome-molluscan interactions indicate that host identification involves chemosensation, including naïve host preference. Proteomic technique advances enable sophisticated comparative analyses between infected and naïve snail host proteins. This study aimed to compare resistant, susceptible and naïve Biomphalaria glabrata snail-conditioned water (SCW) to identify potential attractants and deterrents. METHODS:Behavioural bioassays were performed on S. mansoni miracidia to compare the effects of susceptible, F1 resistant and naïve B. glabrata SCW. The F1 resistant and susceptible B. glabrata SCW excretory-secretory proteins (ESPs) were fractionated using SDS-PAGE, identified with LC-MS/MS and compared to naïve snail ESPs. Protein-protein interaction (PPI) analyses based on published studies (including experiments, co-expression, text-mining and gene fusion) identified S. mansoni and B. glabrata protein interaction. Data are available via ProteomeXchange with identifier PXD015129. RESULTS:A total of 291, 410 and 597 ESPs were detected in the susceptible, F1 resistant and naïve SCW, respectively. Less overlap in ESPs was identified between susceptible and naïve snails than F1 resistant and naïve snails. F1 resistant B. glabrata ESPs were predominately associated with anti-pathogen activity and detoxification, such as leukocyte elastase and peroxiredoxin. Susceptible B. glabrata several proteins correlated with immunity and anti-inflammation, such as glutathione S-transferase and zinc metalloproteinase, and S. mansoni sporocyst presence. PPI analyses found that uncharacterised S. mansoni protein Smp_142140.1 potentially interacts with numerous B. glabrata proteins. CONCLUSIONS:This study identified ESPs released by F1 resistant, susceptible and naïve B. glabrata to explain S. mansoni miracidia interplay. Susceptible B. glabrata ESPs shed light on potential S. mansoni miracidia deterrents. Further targeted research on specific ESPs identified in this study could help inhibit B. glabrata and S. mansoni interactions and stop human schistosomiasis.
Project description:Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to ?-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which shed S. mansoni cercariae 1-week before the susceptible controls. Our results represent the first functional characterization of a gastropod TLR, and demonstrate that BgTLR is an important snail immune receptor that is capable of influencing infection outcome following S. mansoni challenge.