CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.

ARGONAUTE2 Localizes to Sites of Sporocysts in the Schistosome-Infected Snail, Biomphalaria glabrata.

MicroRNAs (miRNAs) are a class of small regulatory RNA that are generated via core protein machinery. The miRNAs direct gene-silencing mechanisms to mediate an essential role in gene expression regulation. In mollusks, miRNAs have been demonstrated to be required to regulate gene expression in various biological processes, including normal development, immune responses, reproduction, and stress adaptation. In this study, we aimed to establishment the requirement of the miRNA pathway as part of the molecular response of exposure of Biomphalaria glabrata (snail host) to Schistosoma mansoni (trematode parasite). Initially, the core pieces of miRNA pathway protein machinery, i.e., Drosha, DGCR8, Exportin-5, Ran, and Dicer, together with the central RNA-induced silencing complex (RISC) effector protein Argonaute2 (Ago2) were elucidated from the B. glabrata genome. Following exposure of B. glabrata to S. mansoni miracidia, we identified significant expression up-regulation of all identified pieces of miRNA pathway protein machinery, except for Exportin-5, at 16 h post exposure. For Ago2, we went on to show that the Bgl-Ago2 protein was localized to regions surrounding the sporocysts in the digestive gland of infected snails 20 days post parasite exposure. In addition to documenting elevated miRNA pathway protein machinery expression at the early post-exposure time point, a total of 13 known B. glabrata miRNAs were significantly differentially expressed. Of these thirteen B. glabrata miRNAs responsive to S. mansoni miracidia exposure, five were significantly reduced in their abundance, and correspondingly, these five miRNAs were determined to putatively target six genes with significantly elevated expression and that have been previously associated with immune responses in other animal species, including humans. In conclusion, this study demonstrates the central importance of a functional miRNA pathway in snails, which potentially forms a critical component of the immune response of snails to parasite exposure. Further, the data reported in this study provide additional evidence of the complexity of the molecular response of B. glabrata to S. mansoni infection: a molecular response that could be targeted in the future to overcome parasite infection and, in turn, human schistosomiasis.

Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis.

BACKGROUND: Schistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis. METHODS: We developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda. FINDINGS: The S. japonicum SHERLOCK assay achieved 93-100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples. INTERPRETATION: These schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases. FUNDING: Australian Infectious Diseases Research Centre seed grant (2022) and National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria.

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-gamma and lymphotoxin alpha, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4(+) and CD8(+) T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4(+) T cell responses by helminth coinfection amplified CD4(+) T cell-mediated parasite sequestration, whereas vaccination could generate CD4(+) T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

Identification of Putative Neuropeptides That Alter the Behaviour of Schistosoma mansoni Cercariae.

Elucidating the infectivity of Schistosoma mansoni, one of the main etiological agents of human schistosomiasis, requires an improved understanding of the behavioural mechanisms of cercariae, the non-feeding mammalian infective stage. This study investigated the presence and effect of cercariae-derived putative neuropeptides on cercarial behaviour when applied externally. Cercariae were peptidomically analysed and 11 neuropeptide precursor proteins, all of which were specific to the Schistosoma genus and most of which highly expressed in the cercarial stage, were identified in cercariae for the first time. Protein-protein interaction analysis predicted the interaction of various neuropeptide precursors (e.g., Sm-npp-30, Sm-npp-33, Sm-npp-35) with cercarial structural proteins (e.g., myosin heavy chain and titin). In total, nine putative neuropeptides, selected based on their high hydrophobicity and small size (~1 kilodalton), were tested on cercariae (3 mg/mL) in acute exposure (1 min) and prolonged exposure (360 min) behavioural bioassays. The peptides AAYMDLPW-NH2, NRKIDQSFYSYY-NH2, FLLALPSP-OH, and NYLWDTRL-NH2 stimulated acute increases in cercarial spinning, stopping, and directional change during active states. However, only NRKIDQSFYSYY-NH2 caused the same behavioural changes at a lower concentration (0.1 mg/mL). After prolonged exposure, AAYMDLPW-NH2 and NYLWDTRL-NH2 caused increasing passive behaviour and NRKIDQSFYSYY-NH2 caused increasing body-first and head-pulling movements. These findings characterise behaviour-altering novel putative neuropeptides, which may inform future biocontrol innovations to prevent human schistosomiasis.

Analysis of rhodopsin G protein-coupled receptor orthologs reveals semiochemical peptides for parasite (Schistosoma mansoni) and host (Biomphalaria glabrata) interplay.

Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections. A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia. Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.

Identification of Schistosoma mansoni miracidia attractant candidates in infected Biomphalaria glabrata using behaviour-guided comparative proteomics.

Schistosomiasis, caused by infection with Schistosoma digenetic trematodes, is one of the deadliest neglected tropical diseases in the world. The Schistosoma lifecycle involves the miracidial infection of an intermediate freshwater snail host, such as Biomphalaria glabrata. Dispersing snail host-derived Schistosoma miracidia attractants has been considered a method of minimising intermediate host infections and, by extension, human schistosomiasis. The attractiveness of B. glabrata to miracidia is known to be reduced following infection; however, the relationship between duration of infection and attractiveness is unclear. Excretory-secretory proteins (ESPs) most abundant in attractive snail conditioned water (SCW) are key candidates to function as miracidia attractants. This study analysed SCW from B. glabrata that were naïve (uninfected) and at different time-points post-miracidia exposure (PME; 16h, 1-week, 2-weeks and 3-weeks PME) to identify candidate ESPs mediating Schistosoma mansoni miracidia behaviour change, including aggregation and chemoklinokinesis behaviour (random motion, including slowdown and increased turning rate and magnitude). Miracidia behaviour change was only observed post-addition of naïve and 3W-PME SCW, with other treatments inducing significantly weaker behaviour changes. Therefore, ESPs were considered attractant candidates if they were shared between naïve and 3W-PME SCW (or exclusive to the former), contained a predicted N-terminal signal peptide and displayed low identity (<50%) to known proteins outside of the Biomphalaria genus. Using these criteria, a total of 6 ESP attractant candidates were identified, including acetylcholine binding protein-like proteins and uncharacterised proteins. Tissue-specific RNA-seq analysis of the genes encoding these 6 ESPs indicated relatively high gene expression within various B. glabrata tissues, including the foot, mantle and kidney. Acetylcholine binding protein-like proteins were highly promising due to their high abundance in naïve and 3W-PME SCW, high specificity to B. glabrata and high expression in the ovotestis, from which attractants have been previously identified. In summary, this study used proteomics, guided by behavioural assays, to identify miracidia attractant candidates that should be further investigated as potential biocontrols to disrupt miracidia infection and minimise schistosomiasis.

Comparative study of excretory-secretory proteins released by Schistosoma mansoni-resistant, susceptible and naïve Biomphalaria glabrata.

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.

The insulin receptor is a transmission blocking veterinary vaccine target for zoonotic Schistosoma japonicum.

Insulin receptors have been previously identified in Schistosoma japonicum that can bind human insulin. We used the purified recombined protein of the ligand domain of S.japonicum insulin receptor 2 (SjLD2) in three independent murine vaccine/challenge trials. Compared with controls, vaccination of mice with SjLD2 resulted in a significant reduction in faecal eggs, the stunting of adult worms and a reduction in liver granuloma density in all three trials. Furthermore, in the final trial, in which mature intestinal eggs were also quantified, there was a reduction in their number. These results suggest that development of a vaccine based on rSjLD2 for preventing transmission of zoonotic schistosomiasis is feasible.