Molecular characterization of lipoamide dehydrogenase gene in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Lipoamide dehydrogenase (LipDH) is a flavin-containing disulfide oxidoreductase from the same group of thioredoxin reductase, glutathione reductase and trypanothione reductase. This enzyme is found in the mitochondria of all aerobic organisms where it takes part in at least three important multienzyme complexes from the citric acid cycle. In this study, we performed a phylogenetic analysis comparing the amino acid sequence of the LipDH from Trypanosoma cruzi (TcLipDH) with the LipDH from other organisms. Subsequently, the copy number of the TcLipDH gene, the mRNA and protein levels, and the enzymatic activity of the LipDH were determined in populations and strains of T. cruzi that were either resistant or susceptible to benznidazole (BZ). In silico analysis showed the presence of two TcLipDH alleles in the T. cruzi genome. It also showed that TcLipDH protein has less than 55% of identity in comparison to the human LipDH, but the active site is conserved in both of them. Southern blot results suggest that the TcLipDH is a single copy gene in the genome of the T. cruzi samples analyzed. Northern blot assays showed one transcript of 2.4 kb in all T. cruzi populations. Northern blot and Real Time RT-PCR data revealed that the TcLipDH mRNA levels were 2-fold more expressed in the BZ-resistant T. cruzi population (17LER) than in its susceptible pair (17WTS). Western blot results revealed that the TcLipDH protein level is 2-fold higher in 17LER sample in comparison to 17WTS sample. In addition, LipDH activity was higher in the 17LER population than in the 17WTS. Sequencing analysis revealed that the amino acid sequences of the TcLipDH from 17WTS and 17LER populations are identical. Our findings show that one of the mechanisms associated with in vitro-induced BZ resistance to T. cruzi correlates with upregulation of LipDH enzyme.

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species.

Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb(III)) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3- to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb(III) susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb(III) exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb(III)-sodium nitrate or Sb(III)-potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb(III) alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb(III) and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb(III) susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb(III).

Ubiquitin-specific proteases are differentially expressed throughout the Schistosoma mansoni life cycle.

BACKGROUND: The ubiquitination process can be reversed by deubiquitinating enzymes (DUBs). These proteases are involved in ubiquitin processing, in the recovery of modified ubiquitin trapped in inactive forms, and in the recycling of ubiquitin monomers from polyubiquitinated chains. The diversity of DUB functions is illustrated by their number and variety of their catalytic domains with specific 3D architectures. DUBs can be divided into five subclasses: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs or UBPs), ovarian tumour proteases (OTUs), Machado-Joseph disease proteases (MJDs) and JAB1/MPN/Mov34 metalloenzymes (JAMMs). METHODS: Considering the role that the ubiquitin-proteasome system has been shown to play during the development of Schistosoma mansoni, our main goal was to identify and characterize SmUSPs. Here, we showed the identification of putative ubiquitin-specific proteases using bioinformatic approaches. We also evaluated the gene expression profile of representative USP family members using qRT-PCR. RESULTS: We reported 17 USP family members in S. mansoni that present a conservation of UCH domains. Furthermore, the putative SmUSP transcripts analysed were detected in all investigated stages, showing distinct expression during S. mansoni development. The SmUSPs exhibiting high expression profiles were SmUSP7, SmUSP8, SmUSP9x and SmUSP24. CONCLUSION: S. mansoni USPs showed changes in expression levels for different life cycle stages indicating their involvement in cellular processes required for S. mansoni development. These data will serve as a basis for future functional studies of USPs in this parasite.

Characterisation of major vault protein during the life cycle of the human parasite Schistosoma mansoni.

Vaults are ribonucleoproteins (13 MDa) highly conserved among lower and higher eukaryotes. Their association produces a complex composed of three proteins named Major Vault Protein (MVP), vault (PolyADP-ribose) polymerase (VPARP) and Telomerase-associated protein (TEP1), plus a small untranslated RNA. The exact function of this complex is unknown, although the biological role of vaults has been associated with multidrug resistance phenotypes and signal transduction pathways. Genomic analysis showed that model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, do not possess genes encoding vaults. However, we have found that vault-related genes are present in the Schistosoma mansoni genome. These observations raised questions on the involvement of vaults in mechanisms of adaptation of the parasite in its mammalian host. Therefore, molecular characterisation of the putative Major Vault Protein performed using bioinformatics tools showed that this vault component is highly conserved in S. mansoni. The MVP expression level was quantified by qRT-PCR using total RNA from susceptible (LE) and resistant (LE-PZQ) adult worm lineages, cercariae and mechanically transformed schistosomula (MTS) cultured for 3.5, 24, 48 and 72 h in vitro. Our results suggest a stage-specific expression in all developmental stages analysed. Western blotting has shown up-regulation of SmMVP in the MTS-3.5, 72 h and resistant adult worms, and similar levels in all other stages. Furthermore, SmMVP was found differentially expressed in adult males and females from the susceptible lineage. Further studies should clarify whether SmMVP is somehow linked to drug resistance in S. mansoni.

Praziquantel treatment decreases Schistosoma mansoni genetic diversity in experimental infections.

BACKGROUND: Schistosomiasis has a considerable impact on public health in many tropical and subtropical areas. In the new world, schistosomiasis is caused by the digenetic trematode Schistosoma mansoni. Chemotherapy is the main measure for controlling schistosomiasis, and the current drug of choice for treatment is praziquantel (PZQ). Although PZQ is efficient and safe, its repetitive large-scale use in endemic areas may lead to the selection of resistant strains. Isolates less susceptible to PZQ have been found in the field and selected for in the laboratory. The impact of selecting strains with a decreased susceptibility phenotype on disease dynamics and parasite population genetics is not fully understood. This study addresses the impact of PZQ pressure on the genetics of a laboratory population by analyzing frequency variations of polymorphic genetic markers. METHODOLOGY: Infected mice were treated with increasing PZQ doses until the highest dose of 3 × 300 mg/Kg was reached. The effect of PZQ treatment on the parasite population was assessed using five polymorphic microsatellite markers. Parasitological and genetic data were compared with those of the untreated control. After six parasite generations submitted to treatment, it was possible to obtain a S. mansoni population with decreased susceptibility to PZQ. In our experiments we also observed that female worms were more susceptible to PZQ than male worms. CONCLUSIONS: The selective pressure exerted by PZQ led to decreased genetic variability in S. mansoni and increased endogamy. The understanding of how S. mansoni populations respond to successive drug pressure has important implications on the appearance and maintenance of a PZQ resistance phenotype in endemic regions.

NEDD8 conjugation in Schistosoma mansoni: genome analysis and expression profiles.

NEDD8 is an ubiquitin-like molecule that covalently binds to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to bind to p53 and p73, as well as all Cullin family proteins, which are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes. Here, we focused on a genomic analysis of the genes involved in the NEDD8 conjugation pathway in Schistosoma mansoni. The results revealed seven genes related to NEDD8 conjugation that are conserved in Schistosoma japonicum, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We performed quantitative RT-PCR (qRT-PCR), which showed differential profiles for Smnedd8, Smapp1, Smuba3, Smube2f, Smdcn1, Smrbx and Smsenp8 throughout the life cycle of S. mansoni. Upregulation was observed in 3-day-old schistosomula and adult worms for all analysed genes. We also analysed the transcription levels of Cullin family members Smp63 and Smp73, and observed upregulation in early schistosomula, while cercariae and adult worms showed expression levels similar to one another. Taken together, these results suggest that the NEDDylation/DeNEDDylation pathway controls important cellular regulators during worm development from cercariae to schistosomula and, finally, to adult.

Molecular characterization of SUMO E2 conjugation enzyme: differential expression profile in Schistosoma mansoni.

SUMO-dependent post-translational modification is implicated in a variety of cellular functions including gene expression regulation, nuclear sub-localization, and signal transduction. Conjugation of SUMO to other proteins occurs in a similar process to ubiquitination, which involves three classes of enzymes: an E1 activating, an E2 conjugating, and an E3 target-specific ligase. Ubc9 is the unique SUMO E2 enzyme known to conjugate SUMO to target substrates. Here, we present the molecular characterization of this enzyme and demonstrate its expression profile during the S. mansoni life cycle. We have used bioinformatic approaches to identify the SUMO-conjugating enzyme, the SmUbc9-like protein, in the Schistosoma mansoni databases. Quantitative RT-PCR was employed to measure the transcript levels of SUMO E2 in cercariae, adult worms, and in vitro cultivated schistosomula. Furthermore, recombinant SmUbc9 was expressed using the Gateway system, and antibodies raised in rats were used to measure SmUbc9 protein levels in S. mansoni stages by Western blotting. Our data revealed upregulation of the SmUbc9 transcript in early schistosomula followed by a marked differential gene expression in the other analyzed stages. The protein levels were maintained fairly constant suggesting a post-transcriptional regulation of the SmUbc9 mRNA. Our results show for the first time that S. mansoni employs a functional SUMO E2 enzyme, for the conjugation of the SUMO proteins to its target substrates.

Preliminary analysis of miRNA pathway in Schistosoma mansoni.

RNA silencing refers to a series of nuclear and cytoplasmatic processes involved in the post-transcriptional regulation of gene expression or post-transcriptional gene silencing (PTGS), either by sequence-specific mRNA degradation or by translational arrest. The best characterized small RNAs are microRNAs (miRNAs), which predominantly perform gene silencing through post-transcriptional mechanisms. In this work we used bioinformatic approaches to identify the parasitic trematode Schistosoma mansoni sequences that are similar to enzymes involved in the post-transcriptional gene silencing mediated by miRNA pathway. We used amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases identifying a total of 13 putative proteins in the parasite. In addition, the transcript levels of SmDicer1 and SmAgo2/3/4 were identified by qRT-PCR using cercariae, adult worms, eggs and in vitro cultivated schistosomula. Our results showed that the SmDicer1 and SmAgo2/3/4 are differentially expressed during schistosomula development, suggesting that the miRNA pathway is regulated at the transcript level and therefore may control gene expression during the life cycle of S. mansoni.

Hepatitis C and hepatitis B virus infection in different hemodialysis units in Belo Horizonte, Minas Gerais, Brazil.

The prevalence, virological and epidemiological aspects of the hepatitis C virus (HCV) and the hepatitis B virus (HBV) infections vary among hemodialysis patients in different countries. Aiming at analyzing these aspects of HCV and HBV infections in hemodialysis patients in Belo Horizonte, MG, Brazil, we studied three hemodialysis units including 434 patients. Serology was used to detect anti-HCV and HBsAg. Reverse trancriptase nested polymerase chain reaction (RT-nested-PCR) of the 5'-noncoding region was used to detect circulating HCV RNA and restriction fragment length polymorphism analysis for genotyping. Seroprevalence varied from 26.5% to 11.1% for hepatitis C and from 5.9% to 0% for hepatitis B. Risk factors observed for HBV and/or HCV infections were the number of patients per dialysis unit, duration of treatment, number of clinics attended, number of blood units transfused, and lower level scholarity. Alanine aminotransferase levels were altered with a higher frequency in HBV or HCV seropositive patients. Half of ten patients, negative for anti-HCV, had detectable viremia by RT-nested-PCR, indicating that this technique should be used to confirm infections in this group of patients. The HCV genotype 1 was the most frequently observed, followed by the genotype 2, but no correlation was detected between genotype and clinical or epidemiological data.

Hepatitis C and hepatitis B virus infection in different hemodialysis units in Belo Horizonte, Minas Gerais, Brazil

The prevalence, virological and epidemilogical aspects of the hepatitis C virus (HCV) and the hepatitis B virus (HBV) infections vary among hemodialysis patients in different countries. Aiming at analyzing these aspects of HCV and HBV infections in hemodialysis patients in Belo Horizonte, MG, Brazil, we studied three hemodialysis units including 434 patients. Serology was used to detect anti-HCV and HBsAg. Reverse trancriptase nested polymerase chain reaction (RT-nested-PCR) of the 5'-noncoding region was used to detect circulating HCV RNA and restriction fragment length polymorphism analysis for genotyping. Seroprevalence varied from 26.5 percent to 11.1 percent for hepatitis C and from 5.9 percent to 0 percent for hepatitis B. Risk factors observed for HBV and/or HCV infections were the number of patients per dialysis unit, duration of treatment, number of clinics attended, number of blood units transfused, and lower level scholarity. Alanine aminotransferase levels were altered with a higher frequency in HBV or HCV seropositive patients. Half of ten patients, negative for anti-HCV, had detectable viremia by RT-nested-PCR, indicating that this technique should be used to confirm infections in this group of patients. The HCV genotype 1 was the most frequently observed, followed by the genotype 2, but no correlation was detected between genotype and clinical or epidemiological data