Database of Trypanosoma cruzi repeated genes: 20 000 additional gene variants

<p>Abstract</p> <p>Background</p> <p>Repeats are present in all genomes, and often have important functions. However, in large genome sequencing projects, many repetitive regions remain uncharacterized. The genome of the protozoan parasite <it>Trypanosoma cruzi </it>consists of more than 50% repeats. These repeats include surface molecule genes, and several other gene families. In the <it>T. cruzi </it>genome sequencing project, it was clear that not all copies of repetitive genes were present in the assembly, due to collapse of nearly identical repeats. However, at the time of publication of the <it>T. cruzi </it>genome, it was not clear to what extent this had occurred.</p> <p>Results</p> <p>We have developed a pipeline to estimate the genomic repeat content, where shotgun reads are aligned to the genomic sequence and the gene copy number is estimated using the average shotgun coverage. This method was applied to the genome of <it>T. cruzi </it>and copy numbers of all protein coding sequences and pseudogenes were estimated. The 22 640 results were stored in a database available online. 18% of all protein coding sequences and pseudogenes were estimated to exist in 14 or more copies in the <it>T. cruzi </it>CL Brener genome. The average coverage of the annotated protein coding sequences and pseudogenes indicate a total gene copy number, including allelic gene variants, of over 40 000.</p> <p>Conclusion</p> <p>Our results indicate that the number of protein coding sequences and pseudogenes in the <it>T. cruzi </it>genome may be twice the previous estimate. We have constructed a database of the <it>T. cruzi </it>gene repeat data that is available as a resource to the community. The main purpose of the database is to enable biologists interested in repeated, unfinished regions to closely examine and resolve these regions themselves using all available shotgun data, instead of having to rely on annotated consensus sequences that often are erroneous and possibly misleading. Five repetitive genes were studied in more detail, in order to illustrate how the database can be used to analyze and extract information about gene repeats with different characteristics in <it>Trypanosoma cruzi</it>.</p

A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi

Fil: Ferella, Marcela. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Montalvetti, Andrea. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Rohloff, Peter. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Miranda, Kildare. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Fang, Jianmin. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Reina, Silvia. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Kawamukai, Makoto. University Matsue. Faculty of Life and Environmental Science. Department of Applied Bioscience and Biotechnology; Japón.Fil: Bua, Jacqueline. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Nilsson, Daniel. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Pravia, Carlos. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Katzin, Alejandro. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Casera, María B. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Áslund, Lena. Uppsala University. Department of Genetics and Pathology; Suecia.Fil: Andersson, Björn. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Docampo, Roberto. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Bontempi, Esteban. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén"; Argentina.We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ∼ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target

Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

There is an urgent need to substitute the highly toxic arsenic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with Trypanosoma brucei. We exploited the inability of trypanosomes to engage in de novo purine synthesis as a therapeutic target. Cordycepin was selected from a trypanocidal screen of a 2200-compound library. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice inoculated with the human pathogenic subspecies T. brucei rhodesiense or T. brucei gambiense even after parasites had penetrated into the brain. Successful treatment was achieved by intraperitoneal, oral or subcutaneous administration of the compounds. Treatment with the doublet also diminished infection-induced cerebral inflammation. Cordycepin induced programmed cell death of the parasites. Although parasites grown in vitro with low doses of cordycepin gradually developed resistance, the resistant parasites lost virulence and showed no cross-resistance to trypanocidal drugs in clinical use. Our data strongly support testing cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT

The Short Non-Coding Transcriptome of the Protozoan Parasite Trypanosoma cruzi

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16–61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95–98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3′ end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes

Detection and characterization of novel proteins in Trypanosoma cruzi

Trypanosoma cruzi is a flagellated protozoan parasite. It infects a wide range of mammals, including humans. Human T. cruzi infections are endemic to South and Central America. The parasite is transmitted to humans mainly through an insect from the Triatominae subfamily, which feeds from mammals and defecates at the site of the wound, which allows the parasites in the faeces to infect the damaged cells at the bite area. The disease is named Chagas disease in honour of Carlos Chagas who first detected the parasites in the insect, and in this way determined that the vector of the disease was the triatomine bug. There is no cure for Chagas disease and if it is left untreated it can be lethal in the initial stage of the infection, especially for children. If the affected patients develop the chronic form of the disease, there is a high risk of organ deterioration due to the long term presence of the parasites. Only two drugs are used at present to treat Chagas disease, Nifurtimox and Benznidazole. These drugs were developed in 1960s-70s and they can cause severe side effects. Although preventive and control measures have been effective to reduce transmission, there are still 15,000 deaths per year and over 20 million people are at risk of contracting the disease. Chagas disease is one of the socalled neglected tropical diseases, for which there is little interest from pharmaceutical companies to develop new drugs. In response to the critical need for new safe and effective drugs, much research has been performed by many groups in the field, in order to expand the knowledge on T. cruzi biology and to study different enzymes and metabolic pathways that differ from humans. My aim in this thesis was to detect novel proteins in T. cruzi and characterize them by means of: assessing their localization, determining their enzymatic activity, infering putative identity, if unknown by homology comparisons, and determine if they were really expressed in the parasite. In paper I, we detected a polyprenyl synthase in the T. cruzi EST database. We have expressed and characterized this protein. The enzymes of the polyprenyl synthase family are involved in the synthesis of isoprenoids, which are essential for cell function. The identified protein was a solanesyl diphosphate synthase (TcSPPS) that had all the conserved motifs of the family, presented polyprenyl synthase activity and synthesized the maximum chain isoprenoid, solanesyl diphosphate. Long chain isoprenoids are used in ubiquinone biosynthesis. This was shown by the ability of TcSPPS to complement an E. coli strain deficient for ubiquinone production. By using immunofluorescence microscopy, immunogold electron microscopy and cell fractionation we localized TcSPPS to the glycosomes, a peroxisome-like organelle of T. cruzi. In paper II, we report the localization of a short polyprenyl synthase, farnesyl diphosphate synthase (FPPS) in both T. cruzi and Trypanosoma brucei, a closely related trypanosome to T. cruzi that causes sleeping sickness in Africa. Short chain polyprenyl synthases produce short isoprenoids that are utilized to for example modify signalling proteins that utilize the isoprenoid arm to attach to membranes and receptors. As we found the TcSPPS in the glycosomes, we wanted to determine if the entire part of the isoprenoid pathway where these two enzymes take part, was compartmentalized to the glycosomes or not. We found that this was not the case. Both TcFPPS and TbFPPS are present in the cytoplasm. In paper III, we partially characterized a third polyprenyl synthase of T. cruzi, TcPPS. We detected this protein by western blots analysis in the three stages of the parasite and in the cytoplasm of epimastigotes in T. cruzi. It is an unusual protein, due to the 747 amino acid sequence and a molecular weight of 85 kDa, compared to the usual size for the family, which is around 40 kDa. Another particular feature was the presence of a domain of unknown function, DUF2006, which is unrelated to the conserved polyprenyl synthase conserved motifs. We hypothesized that this enzyme could be a GGPPS. The recombinant TcPPS had polyprenyl activity, but the preferred substrate was GPP instead of FPP, the preferred substrates of GGPPSs. In paper IV, we used a mass spectrometry based proteomic approach to detect novel proteins in an organelle enriched sample from T. cruzi epimastigotes. The organellar proteins were separated by 2DGE and 1DGE and subsequently subjected to LC-MS/MS. The results from mass spectrometry were used to search against the T. cruzi translated genome. The search rendered 396 protein identifications. For 173 of them, this was the first expression data reported in T. cruzi. Furthermore, the proteins in the sample belonged to several organellar compartments and the level of cytoplasmic and highly abundant surface proteins was much reduced. We located five novel proteins to the acidocalcisome, mitochondrion, ER and cytoplasmic vesicles, through immunofluorescence microscopy of epitopetagged over-expressed clones. In summary, this work has contributed to the detection and characterization of several novel proteins in T. cruzi, and has answered various questions and has generated new hypotheses to be tested

Proteomics in Trypanosoma cruzi--localization of novel proteins to various organelles

The completion of the genome sequence of Trypanosoma cruzi has been followed by several studies of protein expression, with the long-term aim to obtain a complete picture of the parasite proteome. We report a proteomic analysis of an organellar cell fraction from T. cruzi CL Brener epimastigotes. A total of 396 proteins were identified by LC-MS/MS. Of these, 138 were annotated as hypothetical in the genome databases and the rest could be assigned to several metabolic and biosynthetic pathways, transport, and structural functions. Comparative analysis with a whole cell proteome study resulted in the validation of the expression of 173 additional proteins. Of these, 38 proteins previously reported in other stages were not found in the only large-scale study of the total epimastigote stage proteome. A selected set of identified proteins was analyzed further to investigate gene copy number, sequence variation, transmembrane domains, and targeting signals. The genes were cloned and the proteins expressed with a c-myc epitope tag in T. cruzi epimastigotes. Immunofluorescence microscopy revealed the localization of these proteins in different cellular compartments such as ER, acidocalcisome, mitochondrion, and putative cytoplasmic transport or delivery vesicles. The results demonstrate that the use of enriched subcellular fractions allows the detection of T. cruzi proteins that are undetected by whole cell proteomic methods

Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq

Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most ofthe genome was transcribed in trophozoites grown in vitro, but at vastly different levels.RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology

Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq

Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most ofthe genome was transcribed in trophozoites grown in vitro, but at vastly different levels.RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated DuringGiardia-Host Cell Interactions

Giardia intestinaliscolonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used anin vitromodel of the parasite's interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) inGiardia, which might relate to the establishment of infection and disease induction.Giardiatrophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles duringGiardia-host cell interactions

Cloning of the F fusion protein gene from human respiratory syncytial virus in Escherichia coli

El Virus Respiratorio Sincicial Humano (HRSV) es la principal causa de infección respiratoria aguda en niños pequeños. Su proteína de fusión F es responsable de la penetración del virus en células hospedadoras. F es antigénicamente conservada en los 2 subtipos virales (A y B) de HVRS e induce “in vivo” una respuesta de anticuerpos neutralizantes capaz de limitar la replicación viral...Fil: Palazon, Eliana Gisel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaFil: Ferella, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; ArgentinaFil: Gonzalez, F.. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; ArgentinaFil: Videla, Cristina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Vintiñi, Elisa Ofelia. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; ArgentinaFil: Zamora, A.. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaFil: Dus Santos, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Medina, Marcela Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaXXXV Jornadas Científicas de la Asociación de Biología de TucumánTafí del ValleArgentinaAsociación de Biología de Tucumá