title: Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD).
abstract: 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.
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK. isabel.mauricio@lshtm.ac.uk
authors.forename: Isabel L
authors.initials: IL
authors.lastname: Mauricio
authors.forename: Matthew
authors.initials: M
authors.lastname: Yeo
authors.forename: Mehdi
authors.initials: M
authors.lastname: Baghaei
authors.forename: Daniela
authors.initials: D
authors.lastname: Doto
authors.forename: Francine
authors.initials: F
authors.lastname: Pratlong
authors.forename: Eva
authors.initials: E
authors.lastname: Zemanova
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Dedet
authors.forename: Julius
authors.initials: J
authors.lastname: Lukes
authors.forename: Michael A
authors.initials: MA
authors.lastname: Miles
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D005838:Genotype; D006239:Haplotypes; D007527:Isoenzymes / Q000235:genetics; D007893:Leishmania donovani / Q000145:classification* / Q000201:enzymology / Q000235:genetics; D018314:Leishmania infantum / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D010274:Parasitology / Q000379:methods; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D007527:Isoenzymes; D015800:Protozoan Proteins