Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.

Longitudinal analysis of antigen specific response in individuals with Schistosoma mansoni infection in an endemic area of Minas Gerais, Brazil.

BACKGROUND: Immunoepidemiologic studies have shown a relationship between IgE and IgG4 antibodies with age and with resistance and susceptibility to infection. It is believed that the IgE and IgG4 responses to soluble egg antigen (SEA) can be used for serological analysis of infection and post-treatment status. This study aimed to evaluate the association between Schistosoma mansoni infection and anti-SEA IgG4 and IgE reactivities, and determine whether these reactivities could be used as biomarkers of infection. METHODS: Between 2001 and 2009, a longitudinal study was performed in which parasitologic and blood specimens and socioeconomic and water-contact information were collected from 127 individuals. All patients positive for S. mansoni infection were treated. RESULTS: Schistosomiasis prevalence and the geometric mean of the egg count in 2001 were 59% and 61.05, respectively, decreasing to 26.8% and 8.78 in 2009. IgG4 anti-SEA reactivity in infected individuals was significantly higher than that in uninfected individuals at all time points. Analysis of receiver-operating characteristic (ROC) area showed that the IgG4 anti-SEA antibodies were able to predict infection by S. mansoni at each time point. CONCLUSION: IgG4 anti-SEA reactivity can be used as a biomarker for immune monitoring of the presence of infection with S. mansoni in endemic areas.

Identification of a new Schistosoma mansoni SMYB1 partner: putative roles in RNA metabolism.

SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.

Effect of human TGF-ß on the gene expression profile of Schistosoma mansoni adult worms.

Schistosoma mansoni is responsible for schistosomiasis, a parasitic disease that affects 200 million people worldwide. Molecular mechanisms of host-parasite interaction are complex and involve a crosstalk between host signals and parasite receptors. TGF-ß signaling pathway has been shown to play an important role in S. mansoni development and embryogenesis. In particular human (h) TGF-ß has been shown to bind to a S. mansoni receptor, transduce a signal that regulates the expression of a schistosome target gene. Here we describe 381 parasite genes whose expression levels are affected by in vitro treatment with hTGF-ß. Among these differentially expressed genes we highlight genes related to morphology, development and cell cycle that could be players of cytokine effects on the parasite. We confirm by qPCR the expression changes detected with microarrays for 5 out of 7 selected genes. We also highlight a set of non-coding RNAs transcribed from the same loci of protein-coding genes that are differentially expressed upon hTGF-ß treatment. These datasets offer potential targets to be explored in order to understand the molecular mechanisms behind the possible role of hTGF-ß effects on parasite biology.

Schistosoma mansoni infection in a rural area of the Jequitinhonha Valley, Minas Gerais, Brazil: analysis of exposure risk.

This study examines the relative contribution of age-specific total IgE levels, eosinophils and water contact behavior to the prevalence and intensity (geometric mean egg counts) of Schistosoma mansoni infection in the poor rural population of Virgem das Graças in northern Minas Gerais State. In bivariate analysis, age was strongly correlated with both prevalence and intensity of infection, while eosinophil levels with prevalence only (p<0.0001); IgE levels and 5 demographic and socioeconomic variables were moderately correlated with prevalence (p<0.05), as were number of persons per room and TBM (total body minutes) with egg counts. In multivariate analysis, after controlling for demographic and socioeconomic factors, only total IgE levels were significantly correlated with both prevalence (p=0.248, 95% CI=1.01-1.11) and intensity (p=0.0217, 95% CI=0.01-0.14) of infection and eosinophil levels with prevalence (p=0.0005, 95% CI=1.07-1.24). Although any causal relationship cannot be confirmed by a cross-sectional study, we demonstrated an associated decrease in prevalence and intensity of S. mansoni infection with increased IgE levels.

A unique nuclear receptor direct repeat 17 (DR17) is present within the upstream region of Schistosoma mansoni female-specific p14 gene.

The eggs produced by sexually mature female Schistosma mansoni are responsible for the pathogenesis of the disease. The eggshell precursor gene p14 is expressed only in the vitelline cells of sexually mature female worms in response to a yet unidentified male stimulus. Herein, we report the identification of a novel nuclear receptor response element in the upstream region of the p14 gene. This element contains the canonical hexameric DNA core motif, 5'-PuGGTCA, composed of an atypically spaced direct repeat (DR17). Schistosome nuclear receptors SmRXR1 and SmNR1 specifically bound to the p14-DR17 element as a heterodimer. SmRXR1, but not SmNR1, bound to the motif as a monomer. Introduction of mutations in the TCA core sequence completely abolished the binding by SmRXR1/SmNR1 heterodimer. This finding supports our hypothesis that the expression of Schistosoma mansonip14 gene is regulated through the nuclear receptor signaling pathway.

Schistosoma mansoni: Heterologous complementation of a yeast null mutant by SmRbx, a protein similar to a RING box protein involved in ubiquitination.

The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.

Socioeconomic determinants of schistosomiasis in a poor rural area in Brazil.

The objective of this paper is to identify and quantify socioeconomic determinants of Schistosoma mansoni infection in the rural area of Virgem das Graças in Minas Gerais State of Brazil. A cross-sectional study was carried out to examine the prevalence and intensity of schistosomiasis in relation to socioeconomic characteristics of the households. Log-binomial regression analysis was used to examine the data on both the household and individual levels, analyzing the prevalence ratios for the association of schistosomiasis and socioeconomic variables related to the head of the household. Multiple comparisons through mixed effect modeling were used to examine the relationship between intensity of infection (geometric mean egg counts) and different levels of socioeconomic variables, respectively. In the univariate analysis, place of residence, number of persons per room, and lack of motorized transport were associated with schistosomiasis at the household level and age and unsafe water contact at the individual level. Age, unsafe water contact, number of persons per room, household possessions and lack of education of head of household remained significant predictors of schistosomiasis in the multivariable analysis. Only age was significantly associated with intensity of infection of individuals. It is concluded that widespread poverty, the rural environment, and weak socioeconomic differentiation that result in intense contact with infective water appear to minimize the protective effect of piped water supply and other socioeconomic parameters on schistosomiasis found in other studies. The potential role of socioeconomic development in conjunction with schistosomiasis control is described and areas for further studies are identified.

The spatial distribution of Schistosoma mansoni infection before and after chemotherapy in the Jequitinhonha Valley in Brazil

Schistosomiasis prevalence and egg counts remained low one year after chemotherapy in most households in a hyperendemic rural area in northern Minas Gerais but several distinct spatial patterns could be observed in relation to IgE levels and to a lesser extent to exposure risk (TBM) and type of water supply. An inverse relationship between pre-treatment household prevalence and egg counts on the one hand and post-treatment IgE levels on the other were noted in two of the five communities. Low exposure risk was associated with the low pre-treatment infection rates in the central village but did not contribute to the decline of infection rates after chemotherapy in the study area, as indicated by the significant increase in water contact during the posttreatment period (p < 0.0001). Distance between households and the streams and socioeconomic factors were also unimportant in predicting the spatial distribution of infection. These results are consistent with the production and antiparasitic effect of high levels of IgE in Schistosoma mansoni infection.

The spatial distribution of Schistosoma mansoni infection before and after chemotherapy in the Jequitinhonha Valley in Brazil.

Schistosomiasis prevalence and egg counts remained low one year after chemotherapy in most households in a hyperendemic rural area in northern Minas Gerais but several distinct spatial patterns could be observed in relation to IgE levels and to a lesser extent to exposure risk (TBM) and type of water supply. An inverse relationship between pre-treatment household prevalence and egg counts on the one hand and post-treatment IgE levels on the other were noted in two of the five communities. Low exposure risk was associated with the low pre-treatment infection rates in the central village but did not contribute to the decline of infection rates after chemotherapy in the study area, as indicated by the significant increase in water contact during the posttreatment period (p < 0.0001). Distance between households and the streams and socioeconomic factors were also unimportant in predicting the spatial distribution of infection. These results are consistent with the production and antiparasitic effect of high levels of IgE in Schistosoma mansoni infection.

Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni.

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

Human schistosomiasis mansoni: intensity of infection differentially affects the production of interleukin-10, interferon-gamma and interleukin-13 by soluble egg antigen or adult worm antigen stimulated cultures.

The effect of the intensity of infection (eggs per gram faeces, epg) on the production of interferon-gamma (INF-gamma), interleukin (IL)-10 and IL-13 by peripheral blood mononuclear cells (PBMCs) from individuals living in a Schistosoma mansoni-endemic area was evaluated. In vitro stimulation of PBMCs with soluble egg antigen (SEA) resulted in significantly higher secretion levels of IFN-gamma in egg-negative individuals compared with those with an intensity of infection of more than 100 epg. In contrast, the egg-positive group produced significantly higher amounts of IL-10. Levels of IL-13 did not differ significantly between egg-positive and egg-negative groups. These findings suggest that IL-10 is an important cytokine in the control of the T helper cell (Th) type 1 responses during human S. mansoni infection, shifting the immune response from Th0 in egg-negative individuals from an endemic area to a Th2 polarization in chronic infected individuals.

Exposure to Schistosoma mansoni infection in a rural area in Brazil. Part III: household aggregation of water-contact behaviour.

Much research points to the importance of the household in the study of water-borne diseases such as schistosomiasis. An important aspect of the household is the clustering of domestic activities associated with water collection, storage and usage. Such activities can result in the sharing of water-contact sites and water-contact behaviour, which expose household members to similar risks of infection. In previous studies, we determined that shared residence accounted for 28% of the variance in Schistosoma faecal egg excretion rates. We now quantify the effect of shared residence on the variation in water-related health behaviours. We found that shared residence accounted for 30% of the variation in total water contacts per week. It also accounted for a large proportion of the variation in individual water-contact behaviour: e.g. agricultural contacts (63%), washing limbs (56%) or bathing (41%). These results implicate the household as an important composite measure of the complex relationships between socioeconomic, environmental and behavioural factors that influence water-contact behaviour and, therefore, the transmission of schistosomiasis. Our results also support a focus on the household in the implementation of schistosomiasis prevention and control efforts.

High levels of IgG4 to Schistosoma mansoni egg antigens in individuals with periportal fibrosis.

Specific IgG4 and IgE responses to adult worm antigen and soluble egg antigen (SEA) were examined in 267 individuals from an area in which schistosomiasis mansoni is endemic. Based on information obtained from clinical and sonographic examinations of this sample, the individuals were divided in three groups: 1) 204 individuals without periportal fibrosis, and liver and spleen enlargements; 2) 41 individuals without periportal fibrosis, but presenting with organopathy, with or without organomegaly; and 3) 22 individuals with periportal fibrosis, regardless of their status as having hepatomegaly and/or splenomegaly. Levels of IgG4 to SEA were significantly higher in sera from patients with fibrosis compared with the patients from the other two groups. We also found significantly higher levels of IgG4 against SEA in egg-negative patients with fibrosis compared with egg-negative patients from the other two groups. This report demonstrates a specific humoral response in patients presenting with initial fibrosis, a form of schistosomiasis transient between intestinal and severe hepatosplenic.