Differential expression profiles of miRNAs and their putative targets in Schistosoma mansoni during its life cycle

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-β) signaling, proteasome regulation, glucose and lipid metabolism, immune system evasion and transcriptional regulation.

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.

Tegumentary leishmaniasis in the State of Amazonas: what have we learned and what do we need?

This study evaluated the occurrence of American tegumentary leishmaniasis (ATL) in the State of Amazonas, Brazil, in the last 30 years with emphasis on the last 10 years (2001 to 2010). The disease was predominantly observed in males (76.2%), in the 21- to 30-year-old age group (26.6%) and in extractive workers (43.7%); 3.3% of the cases were the mucosal form. The endemic channel shows the disease seasonality, with a predominance of cases at the beginning and end of each year. The number of cases by municipality in the period of 2001-2010 shows the maintenance of the endemic in the localities where the highest numbers of cases have always been registered, namely, Manaus, Rio Preto da Eva, Itacoatiara and Presidente Figueiredo. The comparison of data from 2001 to 2005 and from 2006 to 2010 showed the emergence of this disease in municipalities that had been previously unaffected. In the last years, there has been a significant increase in the activities of control, diagnosis and treatment of leishmaniasis in the State of Amazonas. In conclusion, the historical series of ATL analyzed in this study suggests that the transmission foci remain and are even expanding, though without continuous transmission in the intra- or peridomicile settings. Moreover, the disease will persist in the Amazon while the factors associated with infection acquisition relative to forest exploitation continue to have economic appeal. There is a real expectation of wide variations in disease incidence that can be influenced by climate and economic aspects.

Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10 percent IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71 percent at 48 h vs 14.5 percent), even after two weeks (47 percent vs 1.9 percent). Also, the percentage of extracellular stages augmented (25.3 percent vs 1.1 percent at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis

Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba

The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD) profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC) reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed (au)