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Schistosoma mansoni employs immune evasion and immunosuppression to overcome immune responses mounted by its snail and human hosts. Myriad immunomodulating factors underlie this process, some of which are proteases. Here, we demonstrate that one protease, an invadolysin we have termed SmCI-1, is released from the acetabular glands of S. mansoni cercaria and is involved in creating an immunological milieu favorable for survival of the parasite. The presence of SmCI-1 in the cercarial stage of S. mansoni is released during transformation into the schistosomula. SmCI-1 functions as a metalloprotease with the capacity to cleave collagen type IV, gelatin and fibrinogen. Additionally, complement component C3b is cleaved by this protease, resulting in inhibition of the classical and alternative complement pathways. Using SmCI-1 knockdown cercariae, we demonstrate that SmCI-1 protects schistosomula from complement-mediated lysis in human plasma. We also assess the effect of SmCI-1 on cytokine release from human peripheral blood mononuclear cells, providing compelling evidence that SmCI-1 promotes an anti-inflammatory microenvironment by enhancing production of IL-10 and suppressing the production of inflammatory cytokines like IL-1B and IL-12p70 and those involved in eosinophil recruitment and activation, like Eotaxin-1 and IL-5. Finally, we utilize the SmCI-1 knockdown cercaria in a mouse model of infection, revealing a role for SmCI-1 in S. mansoni survival.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis., Author summary Schistosomiasis is one of the most prevalent parasitic diseases in the world. Though treatments for schistosomiasis infection exist, there is no vaccine, and reinfection is common in areas where the parasite occurs. One possible way to mitigate schistosomiasis is by controlling the transmission of the parasite larvae from the snails that carry them. Understanding the snail-parasite relationship is essential for the development of new means to interrupt transmission of the parasite from snails to humans. Snails possess immune mechanisms for fighting infection, most of which are based in hemolymph tissue. Here we characterize a novel protein, Grctm6, in a snail host of schistosome parasites. Grctm6 is structurally similar to certain other immune proteins and is present in snail hemolymph. Importantly, we demonstrate that the release of schistosomes by infected snails is exacerbated when this protein is experimentally suppressed in live snails. These results support the suitability of Grctm6 as a possible target for reducing the transmission of this human disease.

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 β-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., Author Summary The freshwater snail Biomphalaria glabrata is the subject of intensive research, primarily due to its biomedical importance as an intermediate host for the parasitic flatworm Schistosoma mansoni, which is a causative agent of the disease schistosomiasis–a chronic, debilitating condition that affects over 260 million people worldwide. Studies of this snail have led to the identification of many factors, some with high homology to known immune molecules in other organisms and many more that are unique with no known homology. However, research into the functional and mechanistic roles of these factors has only recently progressed. In this study, we have demonstrated the functional relevance of a Toll-like receptor (TLR) that we identified in B. glabrata. Transcriptional expression of this TLR was rapidly induced in a snail strain that is resistant to S. mansoni, in a pattern that is consistent with their phenotype. Furthermore, knockdown of the TLR resulted in 43% of resistant snails becoming infected with S. mansoni to patency. This advances our understanding of the mechanistic basis of snail-schistosome compatibility significantly, and may one day facilitate the development of tools for improving the control of schistosomiasis. It also contributes novel functionality to TLRs, one of the most evolutionarily conserved pattern-recognition receptors and cognate signalling pathways in immunity.

Metalloproteases (MPs) have demonstrated roles in immune modulation. In some cases, these enzymes are produced by parasites to influence host immune responses such that parasite infection is facilitated. One of the best examples of parasite-mediated immune modulation is the matrix metalloprotease (MMP) leishmanolysin (Gp63), which is produced by species of the genus Leishmania to evade killing by host macrophages. Leishmanolysin-like proteins appear to be quite common in many invertebrates, however our understanding of the functions of these non-leishmania enzymes is limited. Numerous proteomic and transcriptomic screens of schistosomes, at all life cycle stages of the parasite, have identified leishmanolysin-like MPs as being present in abundance; with the highest levels being found during the intramolluscan larval stages and being produced by cercaria. This study aims to functionally characterize a Schistosoma mansoni variant of leishmanolysin that most resembles the enzyme produced by Leishmania, termed SmLeish. We demonstrate that SmLeish is an important component of S. mansoni excretory/secretory (ES) products and is produced by the sporocyst during infection. The presence of SmLeish interferes with the migration of Biomphalaria glabrata haemocytes, and causes them to present a phenotype that is less capable of sporocyst encapsulation. Knockdown of SmLeish in S. mansoni miracidia prior to exposure to susceptible B. glabrata reduces miracidia penetration success, causes a delay in reaching patent infection, and lowers cercaria output from infected snails., Author summary Parasitic flatworms, or digenetic trematodes, cause a wide range of diseases of both medical and agricultural importance. Nearly all species of digenea require specific species of snail for their larval development and transmission. The factors underpinning snail host specificity and how they dictate infection establishment and maintenance are interesting areas of research, both from the perspective of evolutionary immunology and potential application in the design of tools that aim to prevent trematode transmission. Currently, our understanding of snail-trematode associations is one-sided, being predominantly derived from studies that have focused on the snail immune response, with almost nothing known about how the parasite facilitates infection. Metalloproteases, such as leishmanolysin, are proteolytic enzymes; some of which are produced by parasites to influence host immune responses and facilitate parasite success upon encountering the host defense response. Here, we have functionally characterized a leishmanolysin-like metalloprotease (SmLeish) from Schistosoma mansoni, a causative agent of human schistosomiasis, which afflicts over 260 million people globally. We demonstrate that SmLeish is associated with developing sporocysts and is also located in S. mansoni excretory/secretory products and interferes with snail haemocyte morphology and migration. Knockdown of SmLeish in S. mansoni miracidia prior to exposure to Biomphalaria glabrata snails reduces miracidia penetration success, delays attainment of patent infections, and lowers cercaria output from infected snails.

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished., Author Summary Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Understanding the factors important for snail resistance to schistosome infection will facilitate new lines of investigation to 1) understand the underlying basis of compatibility between schistosomes and snails in endemic areas and how this affects transmission dynamics and control efforts; and 2) to reveal ways to manipulate natural snail populations to enhance their resistance to schistosome infections. Here, we present the first evidence that a snail immune molecule, fibrinogen related protein 3 (FREP3), is important for successful defense against schistosome infections in Biomphalaria snails. In addition, we demonstrate that FREP3 is a target suppressed by trematode parasites to facilitate their establishment within the snail.