The role of natural selection in shaping genetic variation in a promising Chagas disease drug target: Trypanosoma cruzi trans-sialidase

Abstract

Rational drug design creates innovative therapeutics based on knowledge of the biological target to provide more effective and responsible therapeutics. Chagas disease, endemic throughout Latin America, is caused by Trypanosoma cruzi, a protozoan parasite. Current therapeutics are problematic with widespread calls for new approaches. Researchers are using rational drug design for Chagas disease and one target receiving considerable attention is the T. cruzi trans-sialidase protein (TcTS). In T. cruzi, trans-sialidase catalyzes the transfer of sialic acid from a mammalian host to coat the parasite surface membrane and avoid immuno-detection. However, the role of TcTS in pathology variance among and within genetic variants of the parasite is not well understood despite numerous studies. Previous studies reported the crystalline structure of TcTS and the TS protein structure in other trypanosomes where the enzyme is often inactive. However, no study has examined the role of natural selection in genetic variation in TcTS. To understand the role of natural selection in TcTS DNA sequence and protein variation, we examined a 471 bp portion of the TcTS gene from 48 T. cruzi samples isolated from insect vectors. Because there may be multiple parasite genotypes infecting one insect and there are multiple copies of TcTS per parasite genome, all 48 sequences had multiple polymorphic bases. To resolve these polymorphisms, we examined cloned sequences from two insect vectors. The data are analyzed to understand the role of natural selection in shaping genetic variation in TcTS and interpreted in light of the possible role of TcTS as a drug target. The analysis highlights negative or purifying selection on three amino acids previously shown to be important in TcTS transfer activity. One amino acid in particular, Tyr342, is a strong candidate for a drug target because it is under negative selection and amino acid substitutions inactivate TcTS transfer activity. AUTHOR SUMMARY: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted to humans and other mammals primarily by Triatomine insects. Being endemic in many South and Central American countries and affecting millions of people the need for new more effective and safe therapies is evident. Here, we examine genetic variation and natural selection on DNA (471 bp) and amino acid (157 aa) sequence data of the T. cruzi trans-sialdiase (TcTS) protein, often suggested as a candidate for rational drug design. In our surveyed region of the protein there were five amino acid residues that have been shown to be integral to the function of TcTS. We found that three were under strong negative selection making them ideal candidates for drug design; however, one was under balancing selection and should be avoided as a drug target. Our study provides new information into identifying potential targets for a new Chagas drug.

Keywords: Chagas disease; Genetic variation; Natural selection; Rational drug design; Trans-sialidase; Triatoma dimidiata; Triatoma nitida; Trypanosoma cruzi.

Fig. 1

Experimental design. (A) DNA was extracted from the abdomens of infected insect vectors. (B) There could be one (top panel) or multiple (lower panel) parasite genotypes per insect indicated by numbers 1 and 2. (C) Within each parasite nucleus there can be 1–>20 copies of the TcTS group 1 gene indicated by letters a-i. Based on the analysis of published genomes, most of these are unique haplotypes, but some haplotypes occur multiple times in a genome or in multiple parasite genotypes. (D) PCRp sequence with heterozygous peaks. (E) Sequences from clones with heterozygous peaks resolved.

Fig. 2

Best Maximum Likelihood tree reconstruction for the trans-sialidase (TS) protein family constructed from T. cruzi TS samples and GenBank reference samples for each representative group for the TS protein family. Boostrap values (0–100) are indicated at branch nodes.

Fig. 3

Summary of results showing DNA and amino acid sequence variation, and results of the analysis of Selecton test for natural selection based on analysis of the 10 clone sequences. Rows (identified with ALL CAPITAL LETTERS) indicate: SELECTION, the Selecton scores represented by color (see key on Figure); POSITION, the amino acid position in the TcTS protein (our sequences cover amino acids 275-431); AA, the amino acid for each of the 10 clones; and DNA, the corresponding nucleotides for each amino acid. The blue line shows the Selecton score for each amino acid with the red horizontal line indicating neutral evolution or no selection. The blue histograms show the conservation of each amino acid site (1 = no variation, 0 = all sites unique).

Fig. 4

Comparison of selecton values and type of selection detected for analysis of clonal sequences, PCRp sequences, and multiple genomes.

Fig. 5

Three-dimension projections of Trypanosoma cruzi trans-sialidase (TcTS) molecule with amino acids in the sequenced region shaded to indicate Selecton scores.