RESUMO
Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (SmSEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in SmSEPT10, showing binding to phosphatidylinositol phosphates. SmSEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by SmSEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in SmSEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as SmSEPT10.
Assuntos
Schistosoma mansoni , Septinas , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Septinas/metabolismo , Septinas/química , Septinas/genética , Animais , Ligação Proteica , Domínios Proteicos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Lipídeos/químicaRESUMO
Biomphalaria glabrata is a freshwater snail and the obligatory intermediate host of Schistosoma mansoni parasite, the etiologic agent of intestinal Schistosomiasis, in South America and Caribbean. Interestingly in such host-parasite interactions, compatibility varies between populations, strains or individuals. This observed compatibility polymorphism is based on a complex molecular-matching-phenotype, the molecular bases of which have been investigated in numerous studies, notably by comparing between different strains or geographical isolates or clonal selected snail lines. Herein we propose to decipher the constitutive molecular support of this interaction in selected non-clonal resistant and susceptible snail strain originating from the same natural population from Brazil and thus having the same genetic background. Thanks to a global RNAseq transcriptomic approach on whole snail, we identified a total of 328 differentially expressed genes between resistant and susceptible phenotypes among which 129 were up-regulated and 199 down-regulated. Metabolomic studies were used to corroborate the RNAseq results. The activation of immune genes and specific metabolic pathways in resistant snails might provide them with the capacity to better respond to parasite infection.
Assuntos
Biomphalaria , Interações Hospedeiro-Parasita , Metabolômica , Fenótipo , Schistosoma mansoni , Transcriptoma , Biomphalaria/parasitologia , Biomphalaria/genética , Animais , Schistosoma mansoni/genética , Interações Hospedeiro-Parasita/genética , Brasil , Perfilação da Expressão Gênica , Esquistossomose mansoni/parasitologiaRESUMO
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.
Assuntos
Biomphalaria , MicroRNAs , Humanos , Animais , Biomphalaria/genética , MicroRNAs/genética , Schistosoma mansoni/genética , Brasil , Biologia ComputacionalRESUMO
Schistosoma mansoni is a parasitic flatworm that causes a human disease called schistosomiasis, or bilharzia. At the genomic level, S. mansoni is AT-rich, but has some compositional heterogeneity. Indeed, some regions of its genome are GC-rich, mainly in the regions located near the extreme ends of the chromosomes. Recently, we showed that, despite the strong bias towards A/T ending codons, highly expressed genes tend to use GC-rich codons. Here, we address the following question: are highly expressed sequences biased in their amino acid frequencies? Our analyses show that these sequences in S. mansoni, as in species ranging from bacteria to human, are strongly biased in nucleotide composition. Highly expressed genes tend to use GC-rich codons (in the first and second codon positions), which code the energetically cheapest amino acids. Therefore, we conclude that amino acid usage, at least in highly expressed genes, is strongly shaped by natural selection to avoid energetically expensive residues. Whether this is an adaptation to the parasitic way of life of S. mansoni, is unclear since the same pattern occurs in free-living species.
Assuntos
Platelmintos , Animais , Humanos , Platelmintos/genética , Schistosoma mansoni/genética , Aminoácidos/genética , Códon , BactériasRESUMO
Introduction: The genus Biomphalaria in Brazil includes 11 species and one subspecies, three of which are intermediate hosts of Schistosoma mansoni. Due to the recent evolution of this group, some species are difficult to identify based on morphological characters, making the use of genetic markers necessary for species identification. This study aimed to evaluate the use of partial sequences of the cytochrome c oxidase I (coi) gene for the identification of Biomphalaria species using phylogenetic reconstruction and species delimitation algorithms. The study tested the use of DNA barcoding technique for species delimitation within the genus. Methods: DNA barcoding was performed by sequencing a partial region of the coi gene from specimens, and the sequences were analyzed using phylogenetic reconstruction and algorithms to delimit Operational Taxonomic Units (OTUs). Results: The study found that the use of the coi gene in the reconstruction of the phylogeny of the genus might be an alternative for understanding the evolution and dispersion of species. However, this marker alone is not enough to solve complex taxonomic problems within the genus. A total of 223 sequences were analyzed, 102 of which could be separated using the barcode gap, enabling the correct identification of seven taxa. Discussion: The study demonstrated that accurate mollusk identification is necessary for effective schistosomiasis control. The DNA barcoding methodology was found to be promising for accurate mollusk identification, which is crucial for concentrating schistosomiasis control efforts in places where it is needed.
Assuntos
Biomphalaria , Animais , Biomphalaria/genética , Filogenia , Código de Barras de DNA Taxonômico/métodos , DNA , Schistosoma mansoni/genéticaRESUMO
The trematode parasite Schistosoma mansoni causes schistosomiasis, which affects over 200 million people worldwide. Schistosomes are dioecious, with egg laying depending on the females' obligatory pairing with males. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential that have been involved in other species with reproduction, stem cell maintenance, and drug resistance. In S. mansoni, we recently showed that the knockdown of one lncRNA affects the pairing status of these parasites. Here, we re-analyzed public RNA-Seq data from paired and unpaired adult male and female worms and their gonads, obtained from mixed-sex or single-sex cercariae infections, and found thousands of differentially expressed pairing-dependent lncRNAs among the 23 biological samples that were compared. The expression levels of selected lncRNAs were validated by RT-qPCR using an in vitro unpairing model. In addition, the in vitro silencing of three selected lncRNAs showed that knockdown of these pairing-dependent lncRNAs reduced cell proliferation in adult worms and their gonads, and are essential for female vitellaria maintenance, reproduction, and/or egg development. Remarkably, in vivo silencing of each of the three selected lncRNAs significantly reduced worm burden in infected mice by 26 to 35%. Whole mount in situ hybridization experiments showed that these pairing-dependent lncRNAs are expressed in reproductive tissues. These results show that lncRNAs are key components intervening in S. mansoni adult worm homeostasis, which affects pairing status and survival in the mammalian host, thus presenting great potential as new therapeutic target candidates.
Assuntos
Parasitos , RNA Longo não Codificante , Esquistossomose mansoni , Masculino , Feminino , Animais , Camundongos , Schistosoma mansoni/genética , RNA Longo não Codificante/genética , Fertilidade/genética , Reprodução , Parasitos/genética , Esquistossomose mansoni/parasitologia , MamíferosRESUMO
The laboratory diagnosis of intestinal schistosomiasis, carried out by detecting parasite eggs in feces, has low sensitivity when applied to individuals with low parasitic load. Serological tests can be more sensitive for the diagnosis of the disease. Therefore, the objective of this work was to develop and evaluate an ELISA-based immunoenzymatic assay, using a Schistosoma mansoni multiepitope antigen (ELISA IgG anti-SmME). For this, the amino acid sequences of S. mansoni cathepsin B and asparaginyl endopeptidase were submitted to the prediction of B cell epitopes and, together with peptide sequences obtained from earlier works, were used in the construction of a minigene. The multiepitope protein was expressed in Escherichia coli and the performance of the ELISA IgG anti-SmME for schistosomiasis was evaluated using serum samples from 107 individuals either egg positive or negative. In addition, 11 samples from individuals with other helminth infections were included. The ELISA IgG anti-SmME showed a sensitivity of 81.1% and a specificity of 46.1%. Further analysis revealed a 77.2% sensitivity in diagnosis of individuals with egg counts of ≤12 epg (eggs per gram feces) and 87.5% for individuals with 1399 epg. It is worth mentioning that, to our knowledge, this was the first study using a multiepitope recombinant antigen in an ELISA for diagnosis of intestinal schistosomiasis, which demonstrated promising results in the diagnosis of individuals with low parasitic loads.
Assuntos
Esquistossomose mansoni , Animais , Humanos , Esquistossomose mansoni/diagnóstico , Schistosoma mansoni/genética , Antígenos de Helmintos , Sensibilidade e Especificidade , Contagem de Ovos de Parasitas , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/parasitologia , Anticorpos Anti-Helmínticos , Imunoglobulina GRESUMO
Wild rodent species are naturally infected by Schistosoma mansoni; however, the genetic characterization of the parasite, its parasitological features, and its role in human schistosomiasis are poorly understood. In this study, we isolated and characterized Schistosoma from naturally infected Holochilus sciureus, called HS strain, collected from a schistosomiasis endemic region in Maranhão State, Brazil. To isolate the parasite, miracidia obtained from the livers of H. sciureus were used to infect Biomphalaria glabrata of sympatric (called SB) and allopatric (called BH) strains, and the produced cercariae were subcutaneously inoculated into hamsters and/or BALB/c mice. Parasitological kinetics in experimentally infected hosts were evaluated, and the tRNACys-12S (referred to as 16S herein) and cox 1 regions of mtDNA from isolated worms were amplified and sequenced. Only miracidia obtained from infected mice, but not from hamsters, were capable of infecting B. glabrata, allowing maintenance of the isolated parasite. Cox1 and 16S mtDNA sequences showed 100% similarity with S. mansoni, and phylogenetic analysis showed that the HS strain of S. mansoni forms an assemblage with isolates from America and Kenya, confirming the conspecificity. Experimental infection of B. glabrata SB with S. mansoni HS resulted in two peaks of cercariae shedding at 45 and 70 days post-infection (dpi) and caused higher mortality than in B. glabrata BH. The worm recovery rate in mice was approximately 13%, and the peak of egg elimination occurred at the 10th week post-infection. Therefore, S. mansoni obtained from H. sciureus was successfully isolated, genetically characterized, and maintained in mice, allowing further study of this schistosome strain.
Assuntos
Biomphalaria , Esquistossomose mansoni , Trematódeos , Animais , Humanos , Camundongos , Schistosoma mansoni/genética , Esquistossomose mansoni/parasitologia , Arvicolinae , Roedores/parasitologia , Brasil , Filogenia , Biomphalaria/parasitologia , Sigmodontinae , CercáriasRESUMO
Schistosomiasis is a life-threatening infectious disease categorized by the World Health Organization as a public health issue. New molecular diagnostic alternatives for intestinal schistosomiasis caused by Schistosoma mansoni, such as the loop-mediated isothermal amplification (LAMP), a fast and simple amplification technique, have been proposed for control of this NTD in low-endemicity locations. A LAMP assay was performed to detect the internal transcribed spacer 1 ribosomal gene of S. mansoni (SmITS1-LAMP) in 322 DNA extracted from stool samples from schistosomiasis endemic area in Brazil. Kato-Katz analysis of human stool samples was used as the gold standard test, detecting 144 positive samples. SmITS1-LAMP detection limit achieved a maximum analytical sensitivity of 10 fg/µL using S. mansoni genomic DNA, subsequently detecting 17/144 (11.8%) positive samples. SmITS1-LAMP sensitivity and specificity were 12% (95%CI: 7%-18%) and 93% (95%CI: 89%-96%), respectively. Positive predictive value (PPV) and negative predictive value (NPV) were 59% (95%CI: 39% - 76%); and 57% (95%CI: 51% - 62%), respectively. Most cases involved men (61.8%), predominantly young adults (20-39 years old) in cases diagnosed by Kato-Katz and adults (40-59 years old) in cases diagnosed by LAMP. The low number of eggs per gram of stool (1-99 EPG) was the most frequently identified by both Kato-Katz and LAMP. Further studies are needed to evaluate the applicability of SmMIT-LAMP on Schistosoma mansoni diagnosis and surveillance of schistosome infections.
Assuntos
Esquistossomose mansoni , Esquistossomose , Masculino , Adulto Jovem , Animais , Humanos , Adulto , Pessoa de Meia-Idade , Brasil/epidemiologia , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/epidemiologia , Schistosoma mansoni/genética , Fezes , Sensibilidade e Especificidade , PrevalênciaRESUMO
Schistosomiasis is a tropical neglected disease commonly associated with rural areas; however, urban schistosomiasis has been reported worldwide, and increasing urbanization is one of the most important demographic shifts of the 20th and now 21st centuries. The pattern of urbanization is not uniform so that within the same city the rates and sources of population increase vary. Here, we report on the parasite composition in one neighbourhood in the metropolitan area of Salvador, Bahia, Brazil. Using epidemiological data and population genetics, we find evidence for local transmission and maintenance of Schistosoma mansoni infection within an urban population and little contribution from rural-urban migration. Our findings provide direction for local mitigation strategies and to assist the public living in this neighbourhood to interrupt the local transmission cycle.
Assuntos
Esquistossomose mansoni , Esquistossomose , Animais , Schistosoma mansoni/genética , Brasil/epidemiologia , Esquistossomose/epidemiologia , Esquistossomose/prevenção & controle , Esquistossomose/veterinária , Esquistossomose mansoni/epidemiologia , Esquistossomose mansoni/prevenção & controle , Esquistossomose mansoni/veterinária , População UrbanaRESUMO
Patterns of diversity in pathogen genomes provide a window into the spatiotemporal spread of disease. In this study, we tested the hypothesis that Schistosoma mansoni parasites form genetic clusters that coincide with the communities of their human hosts. We also looked for genetic clustering of parasites at the sub-community level. Our data consists of 14 microsatellite DNA markers, typed from pooled DNA samples from [Formula: see text] infected individuals living in three Brazilian communities. We found a one-to-one correspondence between genetic clusters found by K-means cluster analysis and communities when [Formula: see text]. These clusters are also easily identified in a neighbor-joining tree and principal coordinates plots. K-means analysis with [Formula: see text] also reveals genetic clusters of parasites at the sub-community level. These sub-clusters also appear on the neighbor-joining tree and principal coordinates plots. A surprising finding is a genetic relationship between subgroups in widely separated human communities. This connection suggests the existence of common transmission sites that have wide influence. In summary, the genetic structure of S. mansoni in Brazil juxtaposes local isolation that is occasionally broken by long-range migration. Permanent eradication of schistosomes will require both local efforts and the identification of regional infection reservoirs.
Assuntos
Genética Populacional , Schistosoma mansoni/genética , Esquistossomose mansoni/parasitologia , Animais , Brasil , Análise por Conglomerados , Interações Hospedeiro-Parasita/genética , Humanos , Repetições de Microssatélites , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/transmissãoRESUMO
Long non-coding RNAs (lncRNAs) emerged in the past 20 years due to massive amounts of scientific data regarding transcriptomic analyses. They have been implicated in a plethora of cellular processes in higher eukaryotes. However, little is known about lncRNA possible involvement in parasitic diseases, with most studies only detecting their presence in parasites of human medical importance. Here, we review the progress on lncRNA studies and their functions in protozoans and helminths. In addition, we show an example of knockdown of one lncRNA in Schistosoma mansoni, SmLINC156349, which led to in vitro parasite adhesion, motility, and pairing impairment, with a 20% decrease in parasite viability and 33% reduction in female oviposition. Other observed phenotypes were a decrease in the proliferation rate of both male and female worms and their gonads, and reduced female lipid and vitelline droplets that are markers for well-developed vitellaria. Impairment of female worms' vitellaria in SmLINC156349-silenced worms led to egg development deficiency. All those results demonstrate the great potential of the tools and methods to characterize lncRNAs as potential new therapeutic targets. Further, we discuss the challenges and limitations of current methods for studying lncRNAs in parasites and possible solutions to overcome them, and we highlight the future directions of this exciting field.
Assuntos
Helmintos , Parasitos , Doenças Parasitárias , RNA Longo não Codificante , Animais , Feminino , Perfilação da Expressão Gênica , Helmintos/genética , Masculino , Parasitos/genética , RNA Longo não Codificante/genética , Schistosoma mansoni/genéticaRESUMO
Schistosoma mansoni is a trematode flatworm that parasitizes humans and produces a disease called bilharzia. At the genomic level, it is characterized by a low genomic GC content and an "isochore-like" structure, where GC-richest regions, mainly placed at the extremes of the chromosomes, are interspersed with low GC-regions. Furthermore, the GC-richest regions are at the same time the gene-richest, and where the most heavily expressed genes are placed. Taking these features into account, we decided to reanalyze the codon usage of this flatworm. Our results show that a) when all genes are considered together, the strong mutational bias towards A + T leads to a predominance of A/T-ending codons, b) a multivariate analysis discriminates between highly and lowly expressed genes, c) the sequences expressed at highest levels display a significant increase in G/C-ending codons, d) when comparing the molecular distances with a closely related species the synonymous distance in highly expressed genes is significantly lower than in lowly expressed sequences. Therefore, we conclude that despite previous results, which were performed with a small sample of genes, codon usage in S. mansoni is the result of two forces that operate in opposite directions: while mutational bias leads to a predominance of A/T codons, translational selection, working at the level of speed, increment G/C ending triplets.
Assuntos
Uso do Códon , Platelmintos , Animais , Composição de Bases , Códon , Platelmintos/genética , Schistosoma mansoni/genéticaRESUMO
BACKGROUND: Schistosomiasis a neglected tropical disease endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. METHODS: Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. RESULTS: Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. CONCLUSIONS: Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques.
Assuntos
Biomphalaria/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Schistosoma mansoni/genética , Animais , Brasil , Doenças Endêmicas , Esquistossomose mansoni/epidemiologia , Especificidade da EspécieRESUMO
Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real/normas , Schistosoma mansoni/genética , Animais , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Estágios do Ciclo de Vida/genética , Masculino , Fases de Leitura Aberta/genética , Padrões de Referência , Transcriptoma/genéticaRESUMO
Freshwater snails of the genus Biomphalaria serve as intermediate hosts for the digenetic trematode Schistosoma mansoni, the etiological agent for the most widespread form of intestinal schistosomiasis. As neuropeptide signaling in host snails can be altered by trematode infection, a neural transcriptomics approach was undertaken to identify peptide precursors in Biomphalaria glabrata, the major intermediate host for S. mansoni in the Western Hemisphere. Three transcripts that encode peptides belonging to the FMRF-NH2 -related peptide (FaRP) family were identified in B. glabrata. One transcript encoded a precursor polypeptide (Bgl-FaRP1; 292 amino acids) that included eight copies of the tetrapeptide FMRF-NH2 and single copies of FIRF-NH2 , FLRF-NH2 , and pQFYRI-NH2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH2 and 1 copy of SKPYMRF-NH2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH2 peptide. The three precursors shared a common signal peptide, suggesting a genomic organization described previously in gastropods. Immunohistochemical studies were performed on the nervous systems of B. glabrata and B. alexandrina, a major intermediate host for S. mansoni in Egypt. FMRF-NH2 -like immunoreactive (FMRF-NH2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH2 -li neurons. This study supports the participation of FMRF-NH2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria.
Assuntos
FMRFamida/genética , Neuropeptídeos/genética , Esquistossomose mansoni/genética , Transcriptoma/genética , Sequência de Aminoácidos , Animais , Biomphalaria , FMRFamida/análise , FMRFamida/metabolismo , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Imagem Óptica/métodos , Schistosoma mansoni/genética , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/metabolismoRESUMO
Acute schistosomiasis (AS) manifests with a broad spectrum of clinical features in pediatric populations. Diagnosis may be difficult in the absence of detectable numbers of eggs. As a result, new approaches may be required to achieve an accurate diagnosis. Optimal praziquantel (PZQ) treatment regimen for young children is debatable. Also, the post-treatment response is still poorly evaluated due to the lack of reliable markers. A group of 6 children (a toddler and 5 pre-school children) and one pre-adolescent were investigated for AS clinical manifestations and followed-up for two years after treatment. Ova detection was performed by Kato-Katz (KK) and presence of Schistosoma mansoni DNA was assessed by real-time PCR (rt-PCR) in stool samples. IgG and IgE anti-Schistosoma levels and urinary antigen were detected by ELISA and point-of-care circulating cathodic antigen (POC-CCA) testing in serum and urine, respectively. AS clinical symptoms were present in 5/7 (71.4%) of the infected children, and hypereosinophilia was detected in all of them. Ova detection and serology were positive in only 3/7 (44.9%) and 4/7 (57.1%), respectively. However, real-time PCR (rt-PCR) showed the presence of Schistosoma DNA in 6/7 (85.7%) of the cases, and urinary antigen was detected in all infected children. The long-term follow-up after treatment with three doses of PZQ (80mg/kg/dose), showed high cure rates (CR) as demonstrated by the DNA-based assay as well as reduced levels of side effects. CR based on urinary antigen detection ranged from 28.6 to 100%, being the highest CR due to double testing the 2-year post-treatment samples. The results suggest that high dose and repeated treatment with PZQ might be effective for AS in young children. Also, new laboratory markers should be considered to diagnosis and monitor the drug response.
Assuntos
Anti-Helmínticos/uso terapêutico , Parasitologia , Praziquantel/uso terapêutico , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/tratamento farmacológico , Adolescente , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/urina , Biomarcadores/sangue , Biomarcadores/urina , Pré-Escolar , DNA de Helmintos/genética , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Glicoproteínas/urina , Proteínas de Helminto/urina , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Lactente , Masculino , Contagem de Ovos de Parasitas , Testes Imediatos , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/parasitologia , Testes Sorológicos , Resultado do TratamentoRESUMO
BACKGROUND: Schistosomiasis is a disease caused by Schistosoma. Due to its complex life cycle, evolutionary position and sexual dimorphism, schistosomes have several mechanisms of gene regulation. MicroRNAs (miRNAs) are short endogenous RNAs that regulate gene expression at the post-transcriptional level by targeting mRNA transcripts. OBJECTIVES: Here, we tested 12 miRNAs and identified their putative targets using a computational approach. METHODS: We performed the expression profiles of a set of miRNAs and their putative targets during the parasite's life cycle by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). FINDINGS: Our results showed differential expression patterns of the mature miRNAs sma-miR-250; sma-miR-92a; sma-miR-new_4-3p; sma-miR-new_4-5p; sma-miR-new_5-5p; sma-miR-new_12-5p; sma-miR-new_13-3p and sma-miR-new_13-5p. Interestingly, many of the putative target genes are linked to oxidative phosphorylation and are up-regulated in adult-worms, which led us to suggest that miRNAs might play important roles in the post-transcriptional regulation of genes related to energetic metabolism inversion during parasite development. It is noteworthy that the expression of sma-miR-new_13-3p exhibited a negative correlation on SmNADH:ubiquinone oxidoreductase complex I. MAIN CONCLUSIONS: Our analysis revealed putative miRNA genes related to important biological processes, such as transforming growth factor beta (TGF-ß) signaling, proteasome regulation, glucose and lipid metabolism, immune system evasion and transcriptional regulation.
Assuntos
MicroRNAs , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Estágios do Ciclo de Vida/genética , MicroRNAs/genética , Schistosoma mansoni/genética , Transdução de SinaisRESUMO
The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.
Assuntos
Leucina/efeitos dos fármacos , Leucina/genética , Mutação Puntual , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Animais , Artrite , Leucina/química , Leucina/metabolismo , Elastase de Leucócito/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Neutrófilos , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato , Receptor 4 Toll-Like/genética , TranscriptomaRESUMO
Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target-based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2-((4-fluorophenyl)amino)-3-hydroxynaphthalene-1,4-dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure-guided design of SmDHODH inhibitors. DATABASE: The structural data are available in Protein Data Bank (PDB) database under the accession code number 6UY4.