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Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD + , while Lmex_IDH-90 solely used NADP + as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent K m value towards isocitrate (2.0 μM vs 43 μM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD + and NADP + , respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in two cell compartments of Leishmania mexicana promastigotes. These activities could be attributed to two different isoenzymes, one residing in glycosomes, the other in the cytosol. We have cloned and sequenced the genes for both isoenzymes. The glycosomal enzyme is encoded by two tandemly linked genes of identical sequence and contains features frequently found in glycosomal enzymes: the presence of peptide insertions, a small carboxy-terminal extension with a potential glycosomal targeting signal (-SKM) and an excess of positively charged residues (net charge +7). Only one open reading frame was detected for the cytosolic enzyme. The amino acid sequences of the two proteins are only 55% identical. We discuss some evolutionary aspects of the observed organization of the GAPDH genes in the Trypanosomatidae and the role of the two isoenzymes in the metabolism of these organisms. The possibility to develop GAPDH-specific inhibitors that will be effective against the enzyme of various parasitic members of this family is explored.

In spite of the wide use of electrokaryotypes for Leishmania identification, the number, ploidy and associated functions of the chromosomal bands still remain controversial topics. In the present work, we studied these problems in the pathogenic organism Leishmania mexicana using the clamped homogeneous electric field electrophoresis (CHEF) technique, which allows the separation of uniform chromosomal bands in one run. We arrived at the following general conclusions: (i) a comparative densitometric study using haploid Saccharomyces cerevisiae cells as standard reveals that although L. mexicana is an aneuploid organism, its larger bands are diploid; (ii) a total of 18 chromosomal bands ranging from 3.2 to 0.245 Mbp were resolved. These molecules summed to 1.34 X 10(8) bp, a value within the range of the Leishmania genome; (iii) in hybridisation experiments using different housekeeping gene probes, the majority hybridised with chromosomal band 17 or 18 of L. mexicana, with additional locations for some genes; (iv) the presence of the ubiquitous leishmanial (CA/GT)n sequence in the DNA probes could lead to erroneous gene localisation.

A recombinant DNA ribosomal gene spacer of Leishmania braziliensis Y was used as probe to test different Leishmania species. Based on the similarity of their restriction patterns, three groups were distinguished with respect to international Leishmania references: first a group with a similar restriction pattern to L. braziliensis Y and the reference organism L. mexicana garnhami JAP78; a second group with restriction patterns similar to the reference organism L. mexicana mexicana M379; and finally a group where all the restriction patterns were related to the reference organism L. braziliensis braziliensis M2903. These results support the existence of L. garnhami as an independent Leishmania species; they confirm previous studies on L. mexicana and L. braziliensis and open the way for the more exact diagnosis of New World Leishmaniasis.

The DNA karyotypes of three species and several subspecies of New World Leishmania were found to be distinct. The karyotypes were more similar among closely related isolates than among more distantly related groups. Two classes of chromosomal DNA differences were detected among stocks; +/- 50 kb size differences among DNAs, some of which were shown to contain homologous sequences, and DNAs having no obvious corresponding chromosomal DNA in other isolates. A total of 14-24 chromosomal DNA bands were resolved, depending on the isolate, but densitometric analyses suggest that these isolates contain 26-33 distinct DNA molecules. These molecules total about 2.5 X 10(7) bp, a substantial fraction of the genomic DNA. The chromosomal DNA locations of gene sequences homologous to alpha- and beta-tubulin, ribosomal RNA, thymidylate synthetase-dihydrofolate reductase, and the H-region sequence were determined. The homologous sequences were located on chromosomal DNAs of similar, but not identical sizes among different stocks. We also found species- and some subspecies-specific beta-tubulin chromosomal loci. We conclude that the DNA karyotype is useful for stock identification, taxonomy, and gene localization in Leishmania. Its potential for identifying the species and subspecies in natural infections appears less useful unless applied in conjunction with specific hybridization probes.