RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania)

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982

Characterization of Trypanosoma rangeli strains isolated in Central and South America : an overview

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.

Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Resumen del taller sobre el uso de la reacción en cadena de la polimerasa (PCR) para distinguir entre Trypanosoma cruzi y Trypanosoma rangeli / Summary of the workshop: use of the polymerase chain reaction (PCR) to differentiate Trypanosoma cruzi from Trypanosoma rangeli

Este taller se realizó con el propósito de transferir la tecnología de la reacción en cadena de la polimerasa (PCR) para la detección de T. cruzi y T. rangeli a laboratorios en Colombia involucrados en el diagnóstico y estudios epidemiológicos de la enfermedad de Chagas. Para demostración de la técnica se utilizaron muestras clínicas y epidemiológicas de áreas endémicas colombianas. En los ensayos se emplearon muestras de tripanosomas provenientes de diferentes medios de cultivo para evaluar el posible efecto de los componentes de estos medios sobre la sensibilidad de PCR. Se hizo extracción de ADN utilizando los métodos de ebullición, lisis hipotónica y geneclean. El ADN se amplificó utilizando oligonucleótidos sintéticos, correspondientes a una secuencia conservada de 22 nucleótidos dentro de un gen mini-exón. Los dos organismos fueron distinguidos por las movilidades electroforéticas de sus respectivos productos de amplificación, confirmando su identidad con sondas intergénicas específicas de especie, marcadas con digoxigenina dUTP. La hibridación se visualizó con la reacción de color del NBT. De un total de 28 muestras analizadas, se lograron 17 identificaciones que coincidieron con la clasificación original. De cinco muestras desconocidas, tres fueron identificadas como T. rangeli y dos como infecciones mixtas. Se presentaron resultados ambiguos en dos muestras, ocasionados por contaminación en PCR. Solamente dos muestras no se pudieron identificar mediante PCR por problemas en la extracción del ADN de la muestra. Teniendo en cuenta estos resultados preliminares se abre la posibilidad de utilizar esta técnica como una herramienta útil o método adicional a las técnicas de rutina para detectar y diagnósticar la enfermedad de Chagas en Colombia