Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.

Evidence of hybridization, mitochondrial introgression and biparental inheritance of the kDNA minicircles in Trypanosoma cruzi I

“…There are six major T. cruzi lineages (named TcI to TcVI) [26,27]. Particularly, the natural hybrids TcV and TcVI appear to be heterozygous in most loci which is unexpected under the parasexual model [28,29] but fits the hypothesis of a reductive division prior to cell fusion. A recent study based on sequencing of 45 TcI genomes from a restricted geographical area showed that both mechanisms (sexual and parasexual) may occur in natural populations [20].…”

Parasite Genomics Protocols

“…It is important to appreciate in these analyses that many aspects of genes are correlated [ 52 ], so further analysis will be required to determine which aspect(s) of the gene causes the correlation [ 53 , 54 ]. A balanced analysis should take into account that statistically signifi cant p -values can be obtained with large data sets, even when the strength of the effect is very weak (i.e., high p -value, but low correlation coeffi cient rho ) .…”

“…MLST data offer minimal subjectivity in analysis and are transferable and electronically portable, allowing for interlaboratory comparisons without the exchange of reference isolates. Our research group, along with others [ 51 ], is presently expanding this panel of loci with the aim of formalizing an MLST scheme that can be used for high resolution genetic diversity studies [ 52 ] .…”

New insights intoTrypanosoma cruzievolution and genotyping based on system-wide protein expression profiles (PhyloQuant)

“…Other factors influencing disease outcome include mixed infections, infection routes, host genetics and a range of eco-geographical factors (30,38). Different molecular techniques have been used to study the genetic diversity of T. cruzi such as kinetoplast DNA minicircle (39), RAPDs (40) and more recently by polymorphism of the spliced leader intergenic region (SL-IR) (41)(42)(43) and multilocus sequence typing (44)(45)(46). In 1999, a consensus nomenclature was proposed, placing T. cruzi strains into two major groups; T. cruzi I and T. cruzi II (47).…”