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 parasite...

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Bibliographic Details
Published in:International journal for parasitology 2006-06, Vol.36 (7), p.757-769
Main Authors: Mauricio, Isabel L., Yeo, Matthew, Baghaei, Mehdi, Doto, Daniela, Pratlong, Francine, Zemanova, Eva, Dedet, Jean-Pierre, Lukes, Julius, Miles, Michael A.
Format: Article
Language:English
Subjects:
6-Phosphogluconate dehydrogenase
Alleles
Animals
Aspartate aminotransferase
Base Sequence
Biological and medical sciences
DNA, Protozoan - genetics
Fundamental and applied biological sciences. Psychology
Genotype
Glucose-6-phosphate isomerase
Haplotypes
Isoenzymes - genetics
Leishmania donovani
Leishmania donovani - classification
Leishmania donovani - enzymology
Leishmania donovani - genetics
Leishmania infantum
Leishmania infantum - classification
Leishmania infantum - genetics
Life cycle. Host-agent relationship. Pathogenesis
Molecular Sequence Data
Multilocus sequence typing
Nucleoside hydrolases
Parasitology - methods
Phylogeny
Polymerase Chain Reaction - methods
Protozoa
Protozoan Proteins - genetics
Sequence Analysis, DNA
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Summary: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.
ISSN:0020-7519
1879-0135
DOI:10.1016/j.ijpara.2006.03.006