Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD)

Multilocus enzyme electrophoresis is the gold standard for identification of Leishmania species and strains. Drawbacks include: only amino acid polymorphisms affecting electrophoretic mobility are detected; distinct allozymes can have coincident mobilities; few characters are available; and parasites must be cultured in bulk. So far, thousands of Leishmania strains have been phenotyped by multilocus enzyme electrophoresis. Here, we sequence enzyme-coding genes to provide a PCR-based higher resolution equivalent of multilocus enzyme electrophoresis, particularly for Leishmania infantum. Of 15 enzymes used for multilocus enzyme electrophoresis (MON typing) we have sequenced aspartate aminotransferase, glucose-6-phosphate isomerase, nucleoside hydrolase 1, nucleoside hydrolase 2 and 6-phosphogluconate dehydrogenase. Heterozygous alleles were common, with multiple heterozygous sites within a single locus for several of the genes. Haplotypes were resolved by allele-specific PCR and allele-specific sequencing. Heterozygous haplotypes conformed to the haplotypes of putative parents. One strain appeared to be hybrid across two genetic groups of the Leishmania donovani complex. In most cases, a single amino acid polymorphism was responsible for change in enzyme mobility. Some indistinguishable phenotypes were produced by distinct genotypes. Silent genetic polymorphisms provided enhanced discrimination over multilocus enzyme electrophoresis, for example, by subdividing the zymodeme MON-1. The PCR-based genotyping that we describe could be applied directly to clinical samples or to small volume cultures and in a multilocus sequence typing format. Furthermore, it can be used to detect recombination indirectly and for population genetics studies.     

Leishmania major and Leishmania tropica: II. Effect of an immunomodulator, S(2) complex on the enzymes of the parasites

S(2) complex has been reported to have a direct antileishmanial effect. The possibility that the direct antileishmanial effect may be due to inhibition of key enzymes involved in glucose metabolism and/ or enzymes associated with virulence was investigated. Cell pellets were prepared from cultures of both axenic amastigotes and promastigotes of Leishmania major (MHOM/IQ/93/MRC6) and L. tropica (MHOM/IQ/93/MRC2). S(2) complex, at various concentrations, was added to the enzyme extracts prepared from the pellets. Results show that in the Embden-Meyerhof pathway, both hexokinase and glucose-phosphate isomerase but not fructophosphokinase were dose dependently inhibited. In the hexose-monophosphate shunt both glucose-6-phosphate dehydrogenase and ribose-5-phosphate isomerase were dose dependently inhibited. Malic dehydrogenase and malic enzyme from the citric-acid cycle were both dose dependently inhibited but succinate dehydrogenase from the same pathway was not inhibited. Both enzymes associated with virulence (protease and acid phosphatase), showed activation rather than inhibition at higher doses of S(2) complex. Thus, the direct antileishmanial effect of S(2) complex may result, partially or entirely, from the inhibition of enzymes that are necessary for the parasites' carbohydrate metabolism.     

The genus Allochromatium (Chromatiales Chromatiaceae) revisited: a study on its intragenic structure based on multilocus sequence analysis (MLSA) and DNA-DNA hybridization (DDH)

In this study, the taxonomic status of anoxygenic photosynthetic bacteria belonging to the genus Allochromatium is revisited. The inter- and intraspecies relationship of seven Allochromatium strains, including a set of well described type strains, were examined by DNA-DNA hybridization (DDH) and multilocus sequence analysis (MLSA) using segments of seven protein-coding genes. The re-sequencing of the 16S rRNA, the internal transcriber spacer (ITS), multi-gene analysis and DDH comparison indicated that both type strains Allochromatium vinosum DSM 180^T and Allochromatium minutissimum DSM 1376^T are closely related to each other forming an independent cluster together with the strains A. vinosum DSM 183 and DSM 1686. The internal comparison of members of this A. vinosum phylogroup showed values of DDH relatedness above 80% and concatenated sequence similarities (4744 bp) above 98%. In contrast, the MLSA scheme has identified A. vinosum strain BH-2 as a separate lineage. Strain BH-2 was first classified as a member of the species A. vinosum based on DDH comparison. However, this strain showed the lowest similarity values of the 16S rRNA gene and concatenated sequences, as well as amino acid identity (AAI) when compared to other Allochromatium strains, suggesting that strain BH-2 may represent a new species.     

Leishmania donovani complex (Kinetoplastida, Trypanosomatidae): Comparison of deoxyribonucleic acid based techniques for typing of isolates from Ethiopia

In Ethiopia, visceral leishmaniasis (VL) is an increasing public health concern. Recently, a new outbreak of VL claimed the lives of hundreds of Ethiopians. Mapping its distribution and the identification of the causative Leishmania species is important for proper use of resources and for control planning. The choice of appropriate typing technique is the key for determining the infecting species. Here we compared three deoxyribonucleic acid (DNA) based markers. We used, for the first time, cpbE and cpbF (cpbE/F) PCR-RFLP and demonstrated that it clearly differentiates Leishmania donovani from Leishmania infantum. The cpbE/F PCR-RFLP gave identical banding pattern for all L. donovani strains irrespective of their geographic origin. With the K26 (primers) PCR-RFLP, the L. donovani strains gave a banding pattern different from L. infantum and showed variation with geographic origin. The Ethiopian isolates typed as L. donovani by the PCR-RFLP of the cpbE/F (gene) and K26 (primers) showed two types of patterns with the T2/B4 (primers) PCR-RFLP; one group with L. infantum-like and the other L. donovani-like pattern. Phylogenetic analysis using cpbE/F sequences showed variation with geographic origin of strains and the African strains of L. donovani are more distantly related to L. infantum. Moreover, the Ethiopian isolates were seen to be closely related to the Sudanese, Kenyan and Indian strains. Thus, we recommend the use of more than one marker to study the population genetics of L. donovani complex.