CA88, a nuclear repetitive DNA sequence identified in Schistosoma mansoni, aids in the genotyping of nine Schistosoma species of medical and veterinary importance

CA88 is the first long nuclear repetitive DNA sequence identified in the blood fluke, Schistosoma mansoni. The assembled S. mansoni sequence, which contains the CA88 repeat, has 8,887 nucleotides and at least three repeat units of approximately 360 bp. In addition, CA88 also possesses an internal CA microsatellite, identified as SmBr18. Both PCR and BLAST analysis have been used to analyse and confirm the CA88 sequence in other S. mansoni sequences in the public database. PCR-acquired nuclear repetitive DNA sequence profiles from nine Schistosoma species were used to classify this organism into four genotypes. Included among the nine species analysed were five sequences of both African and Asian lineages that are known to infect humans. Within these genotypes, three of them refer to recognised species groups. A panel of four microsatellite loci, including SmBr18 and three previously published loci, has been used to characterise the nine Schistosoma species. Each species has been identified and classified based on its CA88 DNA fingerprint profile. Furthermore, microsatellite sequences and intra-specific variation have also been observed within the nine Schistosoma species sequences. Taken together, these results support the use of these markers in studying the population dynamics of Schistosoma isolates from endemic areas and also provide new methods for investigating the relationships between different populations of parasites. In addition, these data also indicate that Schistosoma magrebowiei is not a sister taxon to Schistosoma mattheei, prompting a new designation to a basal clade.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Imunologia e ParasitologiaInstituto de Pesquisas René Rachou-FiocruzThe Natural History Museum Department of ZoologySanta Casa de Belo Horizonte Programa de Pós-Graduação e PesquisaUniversidade Federal de Ouro Preto Escola de Farmácia Laboratório de Pesquisas ClínicasUNIFESP, EPM, Imunologia e ParasitologiaSciEL

Identification and characterization of Tc1/mariner-like DNA transposons in genomes of the pathogenic fungi of the Paracoccidioides species complex

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>(Eukaryota, Fungi, Ascomycota) is a thermodimorphic fungus, the etiological agent of paracoccidioidomycosis, the most important systemic mycoses in Latin America. Three isolates corresponding to distinct phylogenetic lineages of the <it>Paracoccidioides </it>species complex had their genomes sequenced. In this study the identification and characterization of class II transposable elements in the genomes of these fungi was carried out.</p> <p>Results</p> <p>A genomic survey for DNA transposons in the sequence assemblies of <it>Paracoccidioides</it>, a genus recently proposed to encompass species <it>P. brasiliensis </it>(harboring phylogenetic lineages S1, PS2, PS3) and <it>P. lutzii </it>(<it>Pb01-like </it>isolates), has been completed. Eight new <it>Tc1/mariner </it>families, referred to as Trem (<b>Tr</b>ansposable <b>e</b>lement <b>m</b>ariner), labeled A through H were identified. Elements from each family have 65-80% sequence similarity with other <it>Tc1/mariner </it>elements. They are flanked by 2-bp TA target site duplications and different termini. Encoded DDD-transposases, some of which have complete ORFs, indicated that they could be functionally active. The distribution of Trem elements varied between the genomic sequences characterized as belonging to <it>P. brasiliensis </it>(S1 and PS2) and <it>P. lutzii</it>. TremC and H elements would have been present in a hypothetical ancestor common to <it>P. brasiliensis </it>and <it>P. lutzii</it>, while TremA, B and F elements were either acquired by <it>P. brasiliensis </it>or lost by <it>P. lutzii </it>after speciation. Although TremD and TremE share about 70% similarity, they are specific to <it>P. brasiliensis </it>and <it>P. lutzii</it>, respectively. This suggests that these elements could either have been present in a hypothetical common ancestor and have evolved divergently after the split between <it>P. brasiliensis </it>and <it>P. Lutzii</it>, or have been independently acquired by horizontal transfer.</p> <p>Conclusions</p> <p>New families of <it>Tc1/mariner </it>DNA transposons in the genomic assemblies of the <it>Paracoccidioides </it>species complex are described. Families were distinguished based on significant BLAST identities between transposases and/or TIRs. The expansion of Trem in a putative ancestor common to the species <it>P. brasiliensis </it>and <it>P. lutzii </it>would have given origin to TremC and TremH, while other elements could have been acquired or lost after speciation had occurred. The results may contribute to our understanding of the organization and architecture of genomes in the genus <it>Paracoccidioides</it>.</p

Eukaryotic Protein Kinases (ePKs) of the Helminth Parasite Schistosoma mansoni

<p>Abstract</p> <p>Background</p> <p>Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The <it>Schistosoma mansoni </it>genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the <it>S. mansoni </it>predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets.</p> <p>Results</p> <p>We have identified 252 ePKs, which corresponds to 1.9% of the <it>S. mansoni </it>predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that <it>S. mansoni </it>has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in <it>S. mansoni </it>or belong to an expanded family in this parasite. Only 16 <it>S. mansoni </it>ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite.</p> <p>Conclusions</p> <p>Our approach has improved the functional annotation of 40% of <it>S. mansoni </it>ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of <it>S. mansoni </it>in response to diverse environments during the parasite development, vector interaction, and host infection.</p

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Efficacy of seed extract of Bixa orellana against monogenean gill parasites and physiological aspects of Colossoma macropomum after bath treatment

This study evaluated the use of therapeutic baths with acetone extract of Bixa orellana seeds on the hematological, biochemical and hormonal parameters and plasma cortisol levels of tambaqui (Colossoma macropomum) parasitized by the monogenean Anacanthorus spathulatus. The extract showed in vitro anthelmintic activity against the parasites, and the fish tolerated the concentrations used in the toxicity test. Based on these results, an in vivo test was performed. A total of 180 juveniles of tambaqui were divided into six treatment groups in triplicate: group 1: basal (fish non-treated and non-parasitized), group 2: exposed to acetone 0.2% and parasitized fish, group 3: control (non-treated and parasitized fish), group 4: parasitized fish treated with 125 μg·mL-1 of annatto extract for 2 h bath for two consecutive days, group 5: parasitized fish treated with 250 μg·mL-1 of annatto extract for 2 h bath for two consecutive days, group 6: parasitized fish treated with 125 μg·mL-1 of extract for a single bath for 12 h. After the last bath, parasitological, hematological, biochemical and hormonal analysis were performed. Annatto extract showed 100% efficacy in all concentrations and times of bath evaluated. Hemoglobin concentration and hematocrit percentage were higher in treated fish with 250 μg·mL-1 2 h and 125 μg·mL-1 12 h than that observed in the non-treated fish groups. Glucose was significantly higher in annatto-treated fish and cortisol was significantly higher in acetone group fish compared to other groups. Significant decrease in thrombocyte number was observed in fish after bath with acetone 0.2% compared to basal group, 125 μg·mL-1 2 h and 125 μg·mL-1 12 h, as well as decreased number of circulating lymphocytes in fish after bath with acetone 0.2% and 125 μg·mL-1 12 h in relation to non-treated fish (control). On the other hand, significant increase in WBC count was found in fish treated with 125 μg·mL-1 12 h in relation to basal and acetone groups. This is the first report on the use of seeds of B. orellana against monogenean parasites of fish. In vitro study model used with gills in Petri dishes and their in situ observation was successful and could be a useful tool for testing substances to treat fish parasites. Annatto extract bath is an efficacious alternative for treating monogeneans. However, more studies must be carried out for better understanding of the mechanism of anthelmintic activity, isolation of bioactive substances and toxicological evaluation before testing in farming conditions. Statement of relevance: Chemotherapies have been mostly used to treat fish parasites. However, they present consequences to both to environment and human health. Alternatives have been studied to improve the fish health status and control fish parasites. Phytotherapy shows several advantages in controlling parasites and improving the fish health status. This study shows by the first time the use of B. orellana in controlling monogenean parasites and its effects on the hematological, biochemical and hormonal parameters. © 2016 Elsevier B.V

Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of <it>Trypanosoma cruzi</it>

Abstract Background Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS). Methods In the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms. Results In silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6 Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level. Conclusion The BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.</p

Hematology and biochemistry of Colossoma macropomum co-infected with Aeromonas hydrophila and monogenean Anacanthorus spathulatus after treatment with seed extract of Bixa orellana

This study evaluated the use in vitro and in vivo of Bixa orellana seed extracts on the hematological and biochemical parameters of Colossoma macropomum co-infected with bacterium and monogenean, Anacanthorus spathulatus. The extract presented antimicrobial and antiparasitic activity in vitro against the pathogens. Fish supported the toxicity test and in vivo assay used 180 fish distributed in six treatments in triplicate: non-parasitized fish non-injected with A. hydrophila; non-parasitized fish non-injected exposed to acetone 0.2%; parasitized fish injected non-treated with the extract; parasitized fish injected treated with 125 μg mL−1 of extract in 2 h bath for two consecutive days; parasitized fish injected treated with 250 μg mL−1 of extract in 2 h bath for two consecutive days; parasitized fish injected treated with 125 μg mL−1 of extract for 12 h. After last bath, the fish were examined. Acetonic extract showed minimal inhibitory concentration of 125 μg mL−1 against A. hydrophila in the in vitro test and 100% efficacy against monogenean with therapeutic baths in relation to control non-treated in the in vivo test. Aeromonas hydrophila infection did not cause mortality. The gross pathology analysis showed ascites and hemorrhagic liver, kidney and spleen. Increased hemoglobin concentration, mean corpuscular hemoglobin concentration and total number of erythrocytes was followed by a decrease in hematocrit percentage in non-treated fish compared to basal group. Lymphocytes from fish in the control group were higher than in the other groups. Glucose was higher in treated fish than that found in those of basal and control. The results demonstrated that B. orellana bath was an effective alternative to treat fish diseases. © 2018 Elsevier B.V