Genetic profiling of the isoprenoid and sterol biosynthesis pathway genes of Trypanosoma cruzi.

In Trypanosoma cruzi the isoprenoid and sterol biosynthesis pathways are validated targets for chemotherapeutic intervention. In this work we present a study of the genetic diversity observed in genes from these pathways. Using a number of bioinformatic strategies, we first identified genes that were missing and/or were truncated in the T. cruzi genome. Based on this analysis we obtained the complete sequence of the ortholog of the yeast ERG26 gene and identified a non-orthologous homolog of the yeast ERG25 gene (sterol methyl oxidase, SMO), and we propose that the orthologs of ERG25 have been lost in trypanosomes (but not in Leishmanias). Next, starting from a set of 16 T. cruzi strains representative of all extant evolutionary lineages, we amplified and sequenced ∼ 24 Kbp from 22 genes, identifying a total of 975 SNPs or fixed differences, of which 28% represent non-synonymous changes. We observed genes with a density of substitutions ranging from those close to the average (∼ 2.5/100 bp) to some showing a high number of changes (11.4/100 bp, for the putative lathosterol oxidase gene). All the genes of the pathway are under apparent purifying selection, but genes coding for the sterol C14-demethylase, the HMG-CoA synthase, and the HMG-CoA reductase have the lowest density of missense SNPs in the panel. Other genes (TcPMK, TcSMO-like) have a relatively high density of non-synonymous SNPs (2.5 and 1.9 every 100 bp, respectively). However, none of the non-synonymous changes identified affect a catalytic or ligand binding site residue. A comparative analysis of the corresponding genes from African trypanosomes and Leishmania shows similar levels of apparent selection for each gene. This information will be essential for future drug development studies focused on this pathway.

A genomic scale map of genetic diversity in Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi, the causal agent of Chagas Disease, affects more than 16 million people in Latin America. The clinical outcome of the disease results from a complex interplay between environmental factors and the genetic background of both the human host and the parasite. However, knowledge of the genetic diversity of the parasite, is currently limited to a number of highly studied loci. The availability of a number of genomes from different evolutionary lineages of T. cruzi provides an unprecedented opportunity to look at the genetic diversity of the parasite at a genomic scale. RESULTS: Using a bioinformatic strategy, we have clustered T. cruzi sequence data available in the public domain and obtained multiple sequence alignments in which one or two alleles from the reference CL-Brener were included. These data covers 4 major evolutionary lineages (DTUs): TcI, TcII, TcIII, and the hybrid TcVI. Using these set of alignments we have identified 288,957 high quality single nucleotide polymorphisms and 1,480 indels. In a reduced re-sequencing study we were able to validate ~ 97% of high-quality SNPs identified in 47 loci. Analysis of how these changes affect encoded protein products showed a 0.77 ratio of synonymous to non-synonymous changes in the T. cruzi genome. We observed 113 changes that introduce or remove a stop codon, some causing significant functional changes, and a number of tri-allelic and tetra-allelic SNPs that could be exploited in strain typing assays. Based on an analysis of the observed nucleotide diversity we show that the T. cruzi genome contains a core set of genes that are under apparent purifying selection. Interestingly, orthologs of known druggable targets show statistically significant lower nucleotide diversity values. CONCLUSIONS: This study provides the first look at the genetic diversity of T. cruzi at a genomic scale. The analysis covers an estimated ~ 60% of the genetic diversity present in the population, providing an essential resource for future studies on the development of new drugs and diagnostics, for Chagas Disease. These data is available through the TcSNP database (http://snps.tcruzi.org).

A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.

BACKGROUND: Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci. METHODOLOGY/PRINCIPAL FINDINGS: We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI-TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites. CONCLUSIONS/SIGNIFICANCE: Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.