Trafs (TNF receptor associated factors) de Schistosoma mansoni: caracterização molecular e filogenética para o entendimento de seus papéis no desenvolvimento parasitário e no parasitismo
Data
2024
Autores
Bertevello, Claudio Romero 
Orientadores
Santos, Katia Cristina Oliveira Pereira
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Caracterização in silico e análise filogenética dos genes homólogos dos TRAFs de S. mansoni e de outros helmintos utilizando os dados públicos de genoma e transcriptoma. Métodos: O contexto genômico e as marcas de H3K4me3 das TRAFs de S. mansoni foram analisados por um genome browser. Os níveis de expressão dos transcritos de SmTRAF1 e SmTRAF2 foram obtidos em publicações e bancos que continham dados de expressão nos estágios de desenvolvimento, em infecções unissexual e bissexual e expressão espacial de sequenciamento single-cell de vermes adultos. Buscas por homólogos de TRAF nos outros helmintos foram realizadas por BLAST e por HMMER nos genomas depositados no Wormbase Parasite. A análise dos domínios conservados e regiões “coiled-coil” foram feitas utilizando os programas SMART, CDD e MarCoil, respectivamente. O alinhamento global dos domínios MATH para a filogenia foi realizado por MUSCLE, e a análise filogenética foi computada por Inferência Bayesiana utilizando sequências referências dos TRAFs de mamíferos, aves e invertebrados. Resultados: Marcas de H3K4me3 para TRAF1 (Smp_336340) revelaram picos em miracídio (isoforma 1), adulto e cercaria (isoformas 2 e 3), indicando que o início da predição gênica está correto. A isoforma 1 de SmTRAF1 apresentou expressão em cercaria, e menor expressão em esquistossômulos. A isoforma 2 é mais expressa em miracídios e cercaria, e a isoforma 3 foi expressa em miracídio e machos. SmTRAF1 é mais expresso em machos e fêmeas de infecção unissexual (imaturos), com o mesmo padrão nas gônadas destes parasitas. A expressão espacial de SmTRAF1 é detectada na maioria dos tipos celulares, com destaque em células progenitoras de neoblastos, neoblastos e neurônios. O TRAF 2, que não é encontrado na montagem atual do genoma, possui expressão somente em cercaria. A busca por homólogos de TRAF em outros helmintos resultou na identificação de 45 genes preditos em 31 espécies de platelmintos e 70 genes preditos em 57 espécies de nematoides (79,49% e 43,85% das espécies depositadas no Wormbase parasite, respectivamente). As análises filogenéticas dos domínios MATH indicaram que os TRAFs dos helmintos (inclusive TRAF1 de S. mansoni) se agruparam majoritariamente com os TRAF4 das sequências referências. A exceção foi SmTRAF2 se agrupou junto às sequencias referências de TRAF2 e alguns homólogos de M. lignano e S. mediterranea (platelmintos de vida livre) se agruparam no mesmo ramo que TRAF3. Conclusões: As isoformas de SmTRAF1 (agora denominada SmTRAF4) se expressam diferencialmente ao longo dos estágios de desenvolvimento e do status de maturação sexual e SmTRAF2 é expresso exclusivamente em cercarias, indicando envolvimento em processos biológicos e vias de sinalização distintos. A maioria das espécies de platelmintos depositadas no Wormbase Parasite possuem homólogos de TRAF, opostamente ao observado em nematoides. Estes homólogos são essencialmente ortólogos de TRAF4, o TRAF evolutivamente mais ancestral que classicamente interage com receptores de NGF.
Objective: In silico characterization and phylogenetic analysis of TRAF homologous genes from S. mansoni and other helminths using public genome and transcriptome data. Methods: The genomic context and H3K4me3 marks of S. mansoni TRAFs were analyzed using a genome browser. The expression levels of SmTRAF1 and SmTRAF2 transcripts were obtained from publications and databases that contained expression data on developmental stages, unisexual and bisexual infections, and spatial expression from single-cell sequencing of adult worms. Searches for TRAF homologs in other helminths were conducted using BLAST and HMMER in the genomes deposited in Wormbase Parasite. The analysis of conserved domains and “coiled-coil” regions was performed using the SMART, CDD, and MarCoil programs, respectively. Global alignment of MATH domains for phylogenetic analysis was carried out using MUSCLE, and phylogenetic analysis was computed using Bayesian Inference with reference sequences from TRAFs of mammals, birds, and invertebrates. Results: H3K4me3 marks for SmTRAF1 (Smp_336340) revealed peaks in miracidia (isoform 1), adults, and cercariae (isoforms 2 and 3), indicating that the start of the gene prediction is accurate. SmTRAF1 isoform 1 showed expression in cercariae, with lower expression in schistosomula. Isoform 2 was most expressed in miracidia and cercariae, while isoform 3 was expressed in miracidia and males. SmTRAF1 is expressed more in males and females of unisexual infections (immature), with a similar pattern observed in the gonads of these parasites. The spatial expression of SmTRAF1 is detected in most cell types, especially in progenitor cells of neoblasts, neoblasts, and neurons. SmTRAF2, which is not found in the current genome assembly, is expressed only in cercariae. The search for TRAF homologs in other helminths identified 45 predicted genes in 31 flatworm species and 70 predicted genes in 57 nematode species (79.49% and 43.85% of the species deposited in Wormbase Parasite, respectively). Phylogenetic analyses of the MATH domains indicated that helminth TRAFs (including TRAF1 from S. mansoni) mostly grouped with TRAF4 from reference sequences. The exception was SmTRAF2, which grouped next to the TRAF2 reference sequences, with some homologs from M. lignano and S. mediterranea (free-living flatworms) sharing the same branch as TRAF3. Conclusions: SmTRAF1 isoforms (now referred to as SmTRAF4) are differentially expressed across developmental stages and sexual maturation status, while SmTRAF2 is expressed exclusively in cercariae, indicating involvement in distinct biological processes and signaling pathways. Contrary to observations in nematodes, most flatworm species deposited in Wormbase Parasite possess TRAF homologs, which are primarily orthologs of TRAF4, the evolutionarily most ancestral TRAF that classically interacts with NGF receptors.
Objective: In silico characterization and phylogenetic analysis of TRAF homologous genes from S. mansoni and other helminths using public genome and transcriptome data. Methods: The genomic context and H3K4me3 marks of S. mansoni TRAFs were analyzed using a genome browser. The expression levels of SmTRAF1 and SmTRAF2 transcripts were obtained from publications and databases that contained expression data on developmental stages, unisexual and bisexual infections, and spatial expression from single-cell sequencing of adult worms. Searches for TRAF homologs in other helminths were conducted using BLAST and HMMER in the genomes deposited in Wormbase Parasite. The analysis of conserved domains and “coiled-coil” regions was performed using the SMART, CDD, and MarCoil programs, respectively. Global alignment of MATH domains for phylogenetic analysis was carried out using MUSCLE, and phylogenetic analysis was computed using Bayesian Inference with reference sequences from TRAFs of mammals, birds, and invertebrates. Results: H3K4me3 marks for SmTRAF1 (Smp_336340) revealed peaks in miracidia (isoform 1), adults, and cercariae (isoforms 2 and 3), indicating that the start of the gene prediction is accurate. SmTRAF1 isoform 1 showed expression in cercariae, with lower expression in schistosomula. Isoform 2 was most expressed in miracidia and cercariae, while isoform 3 was expressed in miracidia and males. SmTRAF1 is expressed more in males and females of unisexual infections (immature), with a similar pattern observed in the gonads of these parasites. The spatial expression of SmTRAF1 is detected in most cell types, especially in progenitor cells of neoblasts, neoblasts, and neurons. SmTRAF2, which is not found in the current genome assembly, is expressed only in cercariae. The search for TRAF homologs in other helminths identified 45 predicted genes in 31 flatworm species and 70 predicted genes in 57 nematode species (79.49% and 43.85% of the species deposited in Wormbase Parasite, respectively). Phylogenetic analyses of the MATH domains indicated that helminth TRAFs (including TRAF1 from S. mansoni) mostly grouped with TRAF4 from reference sequences. The exception was SmTRAF2, which grouped next to the TRAF2 reference sequences, with some homologs from M. lignano and S. mediterranea (free-living flatworms) sharing the same branch as TRAF3. Conclusions: SmTRAF1 isoforms (now referred to as SmTRAF4) are differentially expressed across developmental stages and sexual maturation status, while SmTRAF2 is expressed exclusively in cercariae, indicating involvement in distinct biological processes and signaling pathways. Contrary to observations in nematodes, most flatworm species deposited in Wormbase Parasite possess TRAF homologs, which are primarily orthologs of TRAF4, the evolutionarily most ancestral TRAF that classically interacts with NGF receptors.
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Citação
BERTEVELLO, Claudio Romero. Trafs (TNF receptor associated factors) de Schistosoma mansoni: caracterização molecular e filogenética para o entendimento de seus papéis no desenvolvimento parasitário e no parasitismo. 2024. 184 f. Dissertação (Mestrado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2024.