Identification and characterization of microRNAs in Biomphalaria tenagophila and comparative analysis of their expression in Schistosoma mansoni-resistant and -susceptible snail populations.

BACKGROUND: Schistosomiasis is a neglected tropical disease caused by Schistosoma and affects over 240 million people worldwide. One of the most prominent causative agents is Schistosoma mansoni, which develops inside the intermediate host. Biomphalaria tenagophila is the second most important vector of schistosomiasis in Brazil and the Taim population is completely resistant to infection by S. mansoni. OBJECTIVE: This study aims to identify and characterize B. tenagophila microRNAs (miRNAs) and evaluate their differential expression in S. mansoni-susceptible and -resistant populations of B. tenagophila. METHODS: Two populations of B. tenagophila snails, susceptible and resistant to S. mansoni infection, were used to investigate the small RNA response of these snails after being infected with the parasite. Small RNA sequencing and quantitative real-time PCR were employed to identify and validate differentially expressed miRNAs. Bioinformatics analysis were performed to identify miRNA precursors and mature and evaluate their differential expression. FINDINGS: The study predicted 173 mature miRNAs and 123 precursors. Among them were six Lophotrochozoa-specific miRNAs, three mollusk-specific miRNAs, and six pre-miRNAs in a cluster. The small RNA sequencing and RT-PCR of B. tenagophila samples allowed assessing the expression patterns of miRNAs. MAIN CONCLUSIONS: The results obtained may support future studies in Biomphalaria spp., generating a global impact on disease control.

Assessment of the accuracy of 11 different diagnostic tests for the detection of Schistosomiasis mansoni in individuals from a Brazilian area of low endemicity using latent class analysis.

Background: Schistosomiasis is a parasitic disease associated with poverty. It is estimated that 7.1 million people are infected with Schistosoma mansoni in Latin America, with 95% of them living in Brazil. Accurate diagnosis and timely treatment are important measures to control and eliminate schistosomiasis, but diagnostic improvements are needed to detect infections, especially in areas of low endemicity. Methodology: This research aimed to evaluate the performance of 11 diagnostic tests using latent class analysis (LCA). A cross-sectional survey was undertaken in a low endemicity area of the municipality of Malacacheta, Minas Gerais, Brazil. Feces, urine, and blood samples were collected from 400 residents older than 6 years of age, who had not been treated with praziquantel in the 12 months previous to the collection of their samples. The collected samples were examined using parasitological (Helm Test® kit Kato-Katz), nucleic acid amplification tests -NAATs (PCR, qPCR and LAMP on urine; PCR-ELISA, qPCR and LAMP on stool), and immunological (POC-CCA, the commercial anti-Schistosoma mansoni IgG ELISA kit from Euroimmun, and two in-house ELISA assays using either the recombinant antigen PPE or the synthetic peptide Smp150390.1) tests. Results: The positivity rate of the 11 tests evaluated ranged from 5% (qPCR on urine) to 40.8% (commercial ELISA kit). The estimated prevalence of schistosomiasis was 12% (95% CI: 9-15%) according to the LCA. Among all tests assessed, the commercial ELISA kit had the highest estimated sensitivity (100%), while the Kato-Katz had the highest estimated specificity (99%). Based on the accuracy measures observed, we proposed three 2-step diagnostic approaches for the active search of infected people in endemic settings. The approaches proposed consist of combinations of commercial ELISA kit and NAATs tests performed on stool. All the approaches had higher sensitivity and specificity than the mean values observed for the 11 tests (70.4 and 89.5%, respectively). Conclusion: We showed that it is possible to achieve high specificity and sensitivity rates with lower costs by combining serological and NAATs tests, which would assist in the decision-making process for appropriate allocation of public funding aiming to achieve the WHO target of eliminating schistosomiasis as a public health problem by 2030.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor.

BACKGROUND: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. METHODS: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. RESULTS: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). CONCLUSIONS: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors.

The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.

Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host.

Sm16 is an immunomodulatory protein that seems to play a key role in the suppression of the cutaneous inflammatory response during Schistosoma mansoni penetration of the skin of definitive hosts. Therefore, Sm16 represents a potential target for protective immune responses induced by vaccination. In this work, we generated the recombinant protein rSm16 and produced polyclonal antibodies against this protein to evaluate its expression during different parasite life-cycle stages and its location on the surface of the parasite. In addition, we analyzed the immune responses elicited by immunization with rSm16 using two different vaccine formulations, as well as its ability to induce protection in Balb/c mice. In order to explore the biological function of Sm16 during the course of experimental infection, RNA interference was also employed. Our results demonstrated that Sm16 is expressed in cercaria and schistosomula and is located in the schistosomula surface. Despite humoral and cellular immune responses triggered by vaccination using rSm16 associated with either Freund's or alum adjuvants, immunized mice presented no reduction in either parasite burden or parasite egg laying. Knockdown of Sm16 gene expression in schistosomula resulted in decreased parasite size in vitro but had no effect on parasite survival or egg production in vivo. Thus, our findings demonstrate that although the vaccine formulations used in this study succeeded in activating immune responses, these failed to promote parasite elimination. Finally, we have shown that Sm16 is not vital for parasite survival in the definitive host and hence may not represent a suitable target for vaccine development.

The Schistosoma mansoni cyclophilin A epitope 107-121 induces a protective immune response against schistosomiasis.

Great efforts have been made to identify promising antigens and vaccine formulations against schistosomiasis. Among the previously described Schistosoma vaccine candidates, cyclophilins comprise an interesting antigen that could be used for vaccine formulations. Cyclophilin A is the target for the cyclosporine A, a drug with schistosomicide activity, and its orthologue from Schistosoma japonicum induces a protective immune response in mice. Although Schistosoma mansoni cyclophilin A also represents a promising target for anti-schistosome vaccines, its potential to induce protection has not been evaluated. In this study, we characterized the cyclophilin A (SmCyp), initially described as Smp17.7, analyzed its allergenic potential using in vitro functional assays, and evaluated its ability to induce protection in mice when administered as an antigen using different vaccine formulations and strategies. Results indicated that SmCyp could be successfully expressed by mammalian cells and bacteria. The recombinant protein did not promote IgE-reporter system activation in vitro, demonstrating its probable safety for use in vaccine formulations. T and B-cell epitopes were predicted in the SmCyp sequence, with two of them located within the active isomerase site. The most immunogenic antigen, SmCyp (107-121), was then used for immunization protocols. Immunization with the SmCyp gene or protein failed to reduce parasite burden but induced an immune response that modulated the granuloma area. In contrast, immunization with the synthetic peptide SmCyp (107-121) significantly reduced worm burden (48-50%) in comparison to control group, but did not regulate liver pathology. Moreover, the protection observed in mice immunized with the synthetic peptide was associated with the significant production of antibodies against the SmCyp (107-121) epitope. Therefore, in this study, we identified an epitope within the SmCyp sequence that induces a protective immune response against the parasite, thus representing a promising antigen that could be used for vaccine formulation against schistosomiasis.

Tackling Hypotheticals in Helminth Genomes.

Advancements in genome sequencing have led to the rapid accumulation of uncharacterized 'hypothetical proteins' in the public databases. Here we provide a community perspective and some best-practice approaches for the accurate functional annotation of uncharacterized genomic sequences.

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/ß HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.

Impact of mitomycin C on the mRNA expression signatures of immunological biomarkers in eosinophilic nasal polyposis.

BACKGROUND: The topical application of mitomycin C has been evaluated as a complementary therapy for eosinophilic nasal polyposis (ENP). However, the mechanism underlying the additional benefits of mitomycin C for the control of eosinophilic inflammation and prevention of posttherapeutic relapse remains to be elucidated. In this work, the aim was to characterize the gene expression profile by quantitative real-time polymerase chain reaction (qPCR) of proinflammatory and regulatory biomarkers that are typically associated with ENP and to assess the impact of the topical application of mitomycin C on the nasal mucosal tissue immunologic milieu after ENP surgery. METHODS: We have selected 20 patients with ENP that were recommended to undergo surgical intervention. Normal mucosal tissue was obtained from healthy nasal mucosa from six patients with absence of eosinophilic infiltration. To test the effect of mitomycin C, one side of the maxillary sinus mucosa was selected for topical application of this drug and the other received no further treatment and acted as the control. The genes interleukin-4 (IL-4), IL-5, IL-10, IL-13, chemokine (C-C motif) ligand 5 (CCL5), CCL24, colony-stimulating factor 2 (CSF2), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-alpha), and beta actin (ACTB) were selected for gene expression analysis by qPCR. RESULTS: The data showed higher expression of proinflammatory biomarkers and lower levels of regulatory TGFB1 transcripts in ENP mucosal tissue. Surgery with topical application of mitomycin C induced a prominent transcriptional down-regulation of the immunologic biomarkers, CCL24, TNF-alpha, CSF2, and IL-5, in ENP mucosal tissue. Additionally, this treatment restored the levels of chemokines and cytokines to those observed in the nasal mucosal tissue of control subjects, except for TGFB1, which remained below the reference pattern. Moreover, CSF2 was identified as a putative biomarker with significant predictive value for complementary prophylactic purposes after surgery in ENP patients. CONCLUSION: After the characterization of the expression signatures of immunologic biomarkers in ENP, we observed that the topical use of mitomycin C is important for the reestablishment of the immunologic microenvironment of a normal expression profile of biomarkers involved in ENP mucosal tissue.

Impact of mitomycin C on the mRNA expression signatures of immunological biomarkers in eosinophilic nasal polyposis.

BACKGROUND: The topical application of mitomycin C has been evaluated as a complementary therapy for eosinophilic nasal polyposis (ENP). However, the mechanism underlying the additional benefits of mitomycin C for the control of eosinophilic inflammation and prevention of posttherapeutic relapse remains to be elucidated. In this work, the aim was to characterize the gene expression profile by quantitative real-time polymerase chain reaction (qPCR) of proinflammatory and regulatory biomarkers that are typically associated with ENP and to assess the impact of the topical application of mitomycin C on the nasal mucosal tissue immunologic milieu after ENP surgery. METHODS: We have selected 20 patients with ENP that were recommended to undergo surgical intervention. Normal mucosal tissue was obtained from healthy nasal mucosa from six patients with absence of eosinophilic infiltration. To test the effect of mitomycin C, one side of the maxillary sinus mucosa was selected for topical application of this drug and the other received no further treatment and acted as the control. The genes interleukin-4 (IL-4), IL-5, IL-10, IL-13, chemokine (C-C motif) ligand 5 (CCL5), CCL24, colony-stimulating factor 2 (CSF2), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-alpha), and beta actin (ACTB) were selected for gene expression analysis by qPCR. RESULTS: The data showed higher expression of proinflammatory biomarkers and lower levels of regulatory TGFB1 transcripts in ENP mucosal tissue. Surgery with topical application of mitomycin C induced a prominent transcriptional down-regulation of the immunologic biomarkers, CCL24, TNF-alpha, CSF2, and IL-5, in ENP mucosal tissue. Additionally, this treatment restored the levels of chemokines and cytokines to those observed in the nasal mucosal tissue of control subjects, except for TGFB1, which remained below the reference pattern. Moreover, CSF2 was identified as a putative biomarker with significant predictive value for complementary prophylactic purposes after surgery in ENP patients. CONCLUSION: After the characterization of the expression signatures of immunologic biomarkers in ENP, we observed that the topical use of mitomycin C is important for the reestablishment of the immunologic microenvironment of a normal expression profile of biomarkers involved in ENP mucosal tissue.

Phenotypic screen of early-developing larvae of the blood fluke, schistosoma mansoni, using RNA interference.

RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1alpha), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced ( approximately 1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.