Marcel Marín

Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist.

The gene encoding the metacyclogenesis-associated transcript Mat-1 is conserved in the genus Leishmania and shows a tendency to form dimers upon protein expression.

Abstract The Leishmania infantum Mat-1 gene – recently described in L. major as a highly stage-specific, metacyclogenesis-associated transcript – has been cloned. The 420-bp Mat-1 coding region is conserved with respect to the L. major gene (82% sequence homology). Analysis of the predicted amino-acid sequence reveals structural motifs showing homology with the class of leucine-zipper transcription factors. Southern-blot hybridization analysis suggests that Mat-1 is a low-copy-number gene, probably consisting of two gene copies. The recombinant Mat-1 protein expressed in fusion with the Escherichia coli maltose-binding protein shows a tendency to form dimers in the presence of the leucine-rich C-terminal domain. Bacteria expressing the Mat-1 open reading frame are highly growth-attenuated and tend to delete or modify the insert, which suggests that expression of Mat-1 is toxic for the bacteria.