Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Pol theta), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Pol theta plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Pol theta modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Pol theta in different genomic loci; one ortholog is homologous to the Pol theta C-terminal polymerase domain, and the other is homologous to the Pol theta helicase domain, called Pol theta-polymerase and Pol theta-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol theta-helicase from the nuclear extract. Orc1/Cdc6 and Pol theta-helicase directly interacted, and Pol theta-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Pol theta-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol theta-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Pol theta), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Pol theta plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Pol theta modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Pol theta in different genomic loci; one ortholog is homologous to the Pol theta C-terminal polymerase domain, and the other is homologous to the Pol theta helicase domain, called Pol theta-polymerase and Pol theta-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol theta-helicase from the nuclear extract. Orc1/Cdc6 and Pol theta-helicase directly interacted, and Pol theta-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Pol theta-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol theta-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Author summary

Atlas of Schistosoma mansoni long non-coding RNAs and their expression correlation to protein-coding genes

Long non-coding RNAs (lncRNAs) have been widely discovered in several organisms with the help of high-throughput RNA sequencing. LncRNAs are over 200 nt-long transcripts that do not have protein-coding (PC) potential, having been reported in model organisms to act mainly on the overall control of PC gene expression. Little is known about the functionality of lncRNAs in evolutionarily ancient non-model metazoan organisms, like Schistosoma mansoni, the parasite that causes schistosomiasis, one of the most prevalent infectious-parasitic diseases worldwide. In a recent transcriptomics effort, we identified thousands of S. mansoni lncRNAs predicted to be functional along the course of parasite development. Here, we present an online catalog of each of the S. mansoni lncRNAs whose expression is correlated to PC genes along the parasite lifecycle, which can be conveniently browsed and downloaded through a new web resource http://verjolab.usp.br. We also provide access now to navigation on the co-expression networks disclosed in our previous publication, where we correlated mRNAs and lncRNAs transcriptional patterns across five life-cycle stages/forms, pinpointing biological processes where lncRNAs might act upon.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Author summary

Atlas of Schistosoma mansoni long non-coding RNAs and their expression correlation to protein-coding genes

Long non-coding RNAs (lncRNAs) have been widely discovered in several organisms with the help of high-throughput RNA sequencing. LncRNAs are over 200 nt-long transcripts that do not have protein-coding (PC) potential, having been reported in model organisms to act mainly on the overall control of PC gene expression. Little is known about the functionality of lncRNAs in evolutionarily ancient non-model metazoan organisms, like Schistosoma mansoni, the parasite that causes schistosomiasis, one of the most prevalent infectious-parasitic diseases worldwide. In a recent transcriptomics effort, we identified thousands of S. mansoni lncRNAs predicted to be functional along the course of parasite development. Here, we present an online catalog of each of the S. mansoni lncRNAs whose expression is correlated to PC genes along the parasite lifecycle, which can be conveniently browsed and downloaded through a new web resource http://verjolab.usp.br. We also provide access now to navigation on the co-expression networks disclosed in our previous publication, where we correlated mRNAs and lncRNAs transcriptional patterns across five life-cycle stages/forms, pinpointing biological processes where lncRNAs might act upon.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi / Novo alelo do gene A2 descrito em Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90 percent similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

A leishmaniose visceral (LV) é uma zoonose amplamente disseminada, causada no Brasil pela Leishmania (Leishmania) infantum chagasi. Flebotomíneos vetores adquirem o agente etiológico, alimentando-se do sangue de animais contaminados, como cachorros ou animais selvagens. A doença é endêmica no Brasil, e focos de epidemia são relatados em cidades densamente povoadas por todo o país. Muitas manifestações clínicas relacionadas à infecção por Leishmania estão ligadas à relação parasito-hospedeiro, e vários possíveis fatores de virulência dos parasitas, que causam a LV, são alvos de estudo, tais como os genes A2. O gene A2 foi isolado pela primeira vez em 1994 e, em seguida, em 2005, três novos alelos foram descritos em Leishmania (Leishmania) infantum. No presente estudo, um fragmento do gene A2 de uma população clonal de L.(L.) infantum chagasi foi amplificado por PCR e sua sequência de nucleotídeos determinada. O fragmento mostrou 90 por cento de similaridade com alelos do gene A2 de Leishmania (Leishmania) donovani e de L. (L.) infantum, descritos na literatura. Entretanto, a tradução da sequência de nucleotídeos mostra diferenças na sequência de aminoácidos da proteína, que podem ser essenciais em determinar a variabilidade do gene A2 em espécies do complexo L. (L.) donovani e representa uma ferramenta adicional na compreenssão do papel dessa família de genes na virulência e imunidade da leishmaniose visceral. O conhecimento dessa variação é importante para o desenvolvimento de testes diagnósticos mais precisos e ferramentas mais eficazes no controle da doença.