RESUMO
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
Interactions between the environment, parasites, vectors, and/or intermediate hosts are complex and involve several factors that define the success or failure of an infection. Among these interactions that can affect infections by a parasite, it is possible to highlight the genetic and epigenetic mechanisms in hosts and parasites. The interaction between genetics, epigenetics, infection, and the host's internal and external environment is decisive and dictates the outcome of a parasitic infection and the resistance, susceptibility, and transmission of this parasite. Epigenetic changes become important mediators in the regulation of gene expression, allowing the evasion of the parasite to immune host barriers, its transmission to new hosts, and the end of its development cycle. Epigenetics is a new frontier in the understanding of the interaction mechanisms between parasite and host that, along with information from the gene regions associated with complex phenotypic variations, the Quantitative Trait Loci, brings new possibilities to investigate more modern and efficient approaches to the treatment, control, and eradication of parasitic diseases. In this brief review, a general overview of the use of epigenetic information and mapping of Quantitative Trait Loci was summarized, both in genes of parasites and hosts, for understanding the mechanisms of resistance and/or susceptibility in parasitic relationships; also, the main search platforms were quantitatively compared, aiming to facilitate access data produced over a period of twenty years.
Assuntos
Epigênese Genética , Interações Hospedeiro-Parasita/genética , Doenças Parasitárias/genética , Locos de Características Quantitativas , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Resistência à Doença , Predisposição Genética para Doença , Humanos , Doenças Parasitárias/parasitologiaRESUMO
Schistosoma mansoni causes schistosomiasis, which affects 240 million people, and 700 million people are living at risk of infection. Epigenetic mechanisms are important for transcriptional control and are well-known conserved transcriptional co-regulators in evolution, already described in mammal, yeast, protozoa and S. mansoni, responsible for heterochromatization and gene silence mechanisms through the formation of complexes of transcriptional repression in chromatin. Previous results from another group have shown that HP1 (SmCBX) proteins form chromatin complexes with SmMDB2/3 proteins and regulate stem cells and oviposition in parasite adult worms. In addition, results from other groups have shown that cercariae are transcriptionally silent and epigenetic mechanisms are involved in the regulation of gene expression in this stage. In this work, our aim was to give insights into SmHP1 and proteins involved in transcriptional regulation in the cercariae stage. Using monoclonal anti-HP1 antibody for Western blotting, immunoprecipitation, and mass spectrometry, we preliminarily determined nuclear proteins that putatively interact with HP1 to form complexes to regulate gene expression, heterochromatin formation, and translational complexes in the cercariae stage. So far, our data is to give some insights into nuclear interactors in S. mansoni cercariae. SIGNIFICANCE: The significance of this original paper is the evidence for Heterochromatin Protein (HP1), interaction with nuclear proteins in the cercariae stage. Schistosoma mansoni cercariae are the infective stage of the human beings in endemic areas of schistosomiasis, a neglected disease, most prevalent in Brazil and Africa. While cercariae are waiting for a host, it does not feed, gene expression is silent and protein synthesis is stopped. These biochemical mechanisms are recovered when cercariae find a human host, but all proteins and mechanisms are not still elucidated. Until now, literature shows that these phenomena are regulated by epigenetics mechanisms, dependent of histone posttranslational modifications. But we have few pieces of evidence about the other proteins that participates in these processes and which are the co-regulators of expression.
Assuntos
Cercárias , Schistosoma mansoni , Animais , Brasil , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Feminino , HumanosRESUMO
The proteasome is the key player in the cellular protein degradation machinery and is pivotal for protein homeostasis and Schistosoma mansoni (S. mansoni) survival. Our group study provides insights into proteasome inhibitors and reveals that selective schistosomiasis agents represent an interesting branch of proteasome research linked to the development of new drugs for this neglected disease. Here, we explored the phenotypic response of S. mansoni to b-AP15, a bis-benzylidine piperidone that inhibits 26S proteasome deubiquitinases (DUBs), ubiquitin-specific protease 14 (USP14), and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5). b-AP15 induces a modest decrease in egg production in vitro and reduces viability, leading to the death of parasite couples. This inhibitor also induces a twofold increase in the accumulation of polyubiquitinated proteins in S. mansoni adult worms and causes tegument changes such as disintegration, wrinkling, and bubble formation, both throughout the length of the parasite and in the oral sucker. b-AP15 alters the cell organelles of adult S. mansoni worms, and we specifically observed mitochondrial alterations, which are suggestive of proteotoxic stress leading to autophagy. Taken together, these results indicate that the deubiquitinase function of the proteasome is essential for the parasite and support the hypothesis that the proteasome constitutes an interesting drug target for the treatment of schistosomiasis.
Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Oviposição/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Feminino , Proteínas de Helminto/metabolismo , Piperidonas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Schistosoma mansoni/metabolismo , Schistosoma mansoni/fisiologia , Ubiquitinação/efeitos dos fármacosRESUMO
Mansonic schistosomiasis is a neglected disease transmitted by Biomphalaria spp. snails. Understanding what happens inside the intermediate host is important to develop more efficient ways of reducing schistosomiasis prevalence. Our purpose was to characterize metabolic and immunological changes in Biomphalaria glabrata 24 h after exposure to Schistosoma mansoni. For this purpose, proteins were extracted from snails' whole tissue with Tris-Urea buffer and digested with tripsin. Mass spectrometry was performed and analyzed with MaxQuant and Perseus software. Also, the hemolymph of five snails 24 h post exposure was collected, and the numbers of hemocytes, levels of urea, uric acid, nitric oxide, calcium, glycogen and alanine and aspartate aminotransferases activities were assessed. Snails were also dissected for measurement of glycogen content in the cephalopodal region and gonoda-digestive gland complex. Globin domain proteins were found to be up-regulated; also the number of circulating hemocytes was significantly higher after 24 h of exposure to the parasite. NO levels were higher 24 h post exposure. Several proteins associated with energy metabolism were found to be up-regulated. Glycogen analysis showed a significant decrease in the gonad-digestive gland complex glycogen content. We found several proteins which seem to be associated with the host immune response, most of which were up-regulated, however some were down-regulated, which may represent an important clue in understanding B. glabrata - S. mansoni compatibility.
Assuntos
Biomphalaria/imunologia , Biomphalaria/parasitologia , Interações Hospedeiro-Parasita , Fatores Imunológicos/análise , Metaboloma , Proteoma/análise , Schistosoma mansoni/crescimento & desenvolvimento , Animais , Biologia Computacional , Imunidade Inata , Espectrometria de MassasRESUMO
BACKGROUND: Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE: The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS: The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS: 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS: Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.
Assuntos
Biomphalaria/genética , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética , Animais , Biomphalaria/enzimologia , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Filogenia , Valores de Referência , Transcriptoma , UbiquitinaçãoRESUMO
BACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.
Assuntos
Humanos , Ubiquitina/análise , Complexo de Endopeptidases do Proteassoma , Estudo de Associação Genômica Ampla/métodos , Biomphalaria/parasitologiaRESUMO
In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos CD36/imunologia , Eritrócitos/parasitologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Infecções Assintomáticas , Células CHO , Adesão Celular/genética , Adesão Celular/imunologia , Cricetulus , Eritrócitos/imunologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Malária Falciparum/parasitologia , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms. .
Assuntos
Animais , Humanos , Anticorpos Antiprotozoários/imunologia , /imunologia , Eritrócitos/parasitologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Infecções Assintomáticas , Células CHO , Cricetulus , Adesão Celular/genética , Adesão Celular/imunologia , Eritrócitos/imunologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Malária Falciparum/parasitologia , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Small ubiquitin-like modifier (SUMO) conjugation of proteins occurs through a concert action of enzymes using a similar ubiquitination mechanism. After a C-terminal peptide is cleaved from the SUMO precursor by a protease to reveal a di-glycine motif, SUMO is activated by an E1 enzyme (Aos1/Uba2) and conjugated to target proteins by the sole E2 enzyme (Ubc9) guided to the appropriate substrates by the SUMO E3 ligase. Previous reports from our group showed that Schistosoma mansoni has two paralogs of SUMO: one E2 conjugation Ubc9 and two SUMO-specific proteases (SENPs). The differential gene expression profile observed for SUMO pathway genes throughout the S. mansoni life cycle attests for the distinct patterns of SUMO conjugates observed during parasite development particularly during the cercariae to schistosomula transition. To continue this investigation, we here analysed the repertoire of SUMO E3 ligases and their expression profiles during cercariae/schistosomula transition. In silico analysis through S. mansoni databases showed two conserved SUMO E3 ligases: protein inhibitor of activated STAT (PIAS) and Ran-binding protein 2 (RanBP2). Furthermore, expression levels of the SUMO E3 ligases were measured by qRT-PCR using total RNA from cercariae, adult worms and mechanically transformed schistosomula. Our data showed an up-regulation of expression in lung schistosomula and adult worm stages. In conclusion, the differential expression of SmPIAS and SmRanBP2 during schistosomula development was similar to the expression levels of all genes related to SUMO conjugation, thereby suggesting that the control of protein activity, localisation or stability during cercariae to schistosomula transition is SUMO-dependent.