Exploiting genetic polymorphisms in metabolic enzymes for rapid screening of Leishmania infantum genotypes

Abstract

BackgroundLeishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing.ResultsPartial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples.ConclusionsA HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3143-7) contains supplementary material, which is available to authorized users.

Application of next generation sequencing (NGS) for descriptive analysis of 30 genomes of Leishmania infantum isolates in Middle-North Brazil

“…Also, several genes associated with drug resistance development were shown to contain non-synonymous SNPs or nonsense mutations 34 . Additionally mutations in exonic regions have been demonstrated to help differentiate isolates from a population and knowing about their impact on different Leishmania isolates's pathogenicity 31,37 . Finally, the presence of non-synonymous SNPs in genes located in conserved regions, important for parasite metabolism 31 and associated with increased parasite load 30 , classify these genes as candidates to detect differences in virulence from isolates and species.…”

“…Synonymous variants do not alter the primary structure of polypeptide, but may have negative effects on the stability and structure of mRNA and proteins, and may contribute to the complexity of the infectious diseases, being interesting targets for the identification of genetic factors associated with virulence 35 . For example, in L. infantum , nine silent SNPs were detected in the malic enzyme gene (class of enzymes that catalyze the reduction of NADP + and needed to maintain intracellular redox homeostasis of the parasite), one of which distinguishes strains from the same zymodemo 36 , 37 . Also, several genes associated with drug resistance development were shown to contain non-synonymous SNPs or nonsense mutations 34 .…”

Genetic tools discriminate strains of Leishmania infantum isolated from humans and dogs in Sicily, Italy

“…Therefore, during the past years, different molecular approaches were developed to characterize isolates and to identify intra-specie polymorphism. In particular, the combined molecular tools included polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), high-resolution melt (HRM)-based assay [14], sequencing of protein coding (i.e., gp63, hsp70, cpb) or noncoding regions (i.e., internal transcribed spacer-1, repetitive DNA sequences) [15]. Currently, microsatellites-based identification seems to be the best way to identify the circulating strains, although the introduction of the whole genome sequencing represents a revolutionary step and will lead to massive improvements.…”

Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis

“…The qPCR-ML, qPCR-ama and qPCR-ITS1 amplicons were analyzed by HRM protocol on a Rotor-Gene 6000 instrument as described previously [ 24 ] with few modifications. Briefly, HRM was carried out over the range from 80 to 90 °C (qPCR-ML, qPCR-ama) or 75 to 85 °C (qPCR-ITS1), rising at 0.1 °C/s and waiting for 2 s at each temperature.…”