Juan Fernando Alzate

Apoptotic markers in protozoan parasites

The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.

Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity.

Leishmania infantum expresses a mitochondrial nuclease homologous to EndoG that migrates to the nucleus in response to an apoptotic stimulus

It is increasingly accepted that single-celled organisms, such as Leishmania parasites, are able to undergo a cell death process that resembles apoptosis in metazoans and is induced by a variety of stimuli. However, the molecular mechanisms that participate and regulate this death process are still very poorly described, and very few of the participating molecules have been identified. Because DNA degradation is probably the most frequently characterized event during programmed cell death in Leishmania parasites, we have focused on identifying a candidate nuclease responsible for this effect during the cell death process. The results presented herein demonstrate that Leishmania infantum promastigotes express a nuclease similar to the endonuclease G of higher eukaryotes which, according to its predicted structure, belongs to the beta beta alpha metal superfamily of nucleases. Its cation dependence resembles that of the EndoGs present in other organisms and, similarly to them, it is inhibited by moderate concentrations of K+ or Na+. L. infantum EndoG contains a signal peptide that causes its translocation to the mitochondrion where it is maintained under normal growth conditions. However, under the pressure of a death stimulus such as edelfosine treatment, L. infantum EndoG is released from the single mitochondrion and translocates to the nucleus, where it is thought to participate in the process of DNA degradation that is associated with programmed cell death. Our results also demonstrate that overexpression of the nuclease in edelfosine-treated promastigotes causes a significant increase in the percentage of TUNEL-positive parasites.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Edelfosine Induces an Apoptotic Process in Leishmania infantum That Is Regulated by the Ectopic Expression of Bcl-XL and Hrk

ABSTRACT The alkyl-lysophospholipids edelfosine and miltefosine induce apoptosis in Leishmania infantum promastigotes. The finding that edelfosine-induced cell death can be regulated by the ectopic expression of the antiapoptotic and proapoptotic members of the Bcl-2 family of proteins Bcl-XL and Hrk suggests that this process is similar to apoptosis in eukaryotic cells.

The method used to sample ulcers influences the diagnosis of cutaneous leishmaniasis

Before beginning treatment for cutaneous leishmaniasis, parasitological confirmation of the disease is required. The most commonly used diagnostic procedures are microscopy and culture of samples taken from the active edge of the lesion. In this study, we compared the sensitivity of previous diagnostic procedures with the polymerase chain reaction (PCR), using smears taken from the edge of the lesion and its centre. The sensitivity was greater with smears taken from the centre of the lesion, both for microscopical examination (85%) and for PCR (81%), compared to those obtained from the edge of the lesion (69% and 58% respectively). When PCR was carried out on biopsy material from the edge of the lesion the sensitivity was 63%.

Revisiting the reference genomes of human pathogenic Cryptosporidium species: reannotation of C. parvum Iowa and a new C. hominis reference

Cryptosporidium parvum and C. hominis are the most relevant species of this genus for human health. Both cause a self-limiting diarrhea in immunocompetent individuals, but cause potentially life-threatening disease in the immunocompromised. Despite the importance of these pathogens, only one reference genome of each has been analyzed and published. These two reference genomes were sequenced using automated capillary sequencing; as of yet, no next generation sequencing technology has been applied to improve their assemblies and annotations. For C. hominis, the main challenge that prevents a larger number of genomes to be sequenced is its resistance to axenic culture. In the present study, we employed next generation technology to analyse the genomic DNA and RNA to generate a new reference genome sequence of a C. hominis strain isolated directly from human stool and a new genome annotation of the C. parvum Iowa reference genome.

Probing the Dimerization Interface of Leishmania infantum Trypanothione Reductase with Site‐Directed Mutagenesis and Short Peptides

Binding at the interface: We tested the inhibitory activity of a set of peptide sequences derived from an α-helix of the dimeric trypanothione reductase from Leishmania infantum. Replacement of a glutamic acid residue with a lysine promoted monomer dissociation and enzyme inhibition.

Mitochondrial genome sequence of the potato powdery scab pathogen Spongospora subterranea

Abstract Spongospora subterranea is a soil-borne obligate parasite responsible for potato powdery scab disease. S. subterranea is a member of the order Plasmodiophorida, a protist taxa that is related to Cercozoa and Foraminifera but the fine details of these relationships remain unresolved. Currently there is only one available complete mtDNA sequence of a cercozoan, Bigelowiella natans. In this work, the mitochondrial sequence of a S. subterranea isolate infecting an Andean variety of S. tuberosum ssp. andigena (Diacol-Capiro) is presented. The mtDNA codes for 16 proteins of the respiratory chain, 11 ribosomal proteins, 3 ribosomal RNAs, 24 tRNAs, a RNA processing RNaseP, a RNA-directed polymerase, and two proteins of unknown function. This is the first report of a mtDNA genome sequence from a plasmodiophorid and will be useful in clarifying the phylogenetic relationship of this group to other members in the supergroup Rhizaria once more mtDNA sequences are available.

Complete mitochondrial genome of the Andean morphotype of Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae)

Abstract The South America fruit fly Anastrepha fraterculus s.l. is an important pest of fruits in Latin America and it is really a complex with at least eight cryptic species. In this work, we report the complete mitochondrial genome for the Andean morphotype of A. fraterculus. The mitochondrial genome is 16,739 nucleotides in size; includes 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. Phylogenetic analysis was performed using all the protein-coding genes with other 19 species from Tephritidae.