Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of similar to 1.7 kb located in 2 chromosomal bands of 600-720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes. (c) 2010 Elsevier Ireland Ltd. All rights reserved.Brazilian agency CNPqConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CDCH-UCV in VenezuelaCDCH-UCV in Venezuel

Alignment of predicted amino acid sequences from entire cruzipain of <i>T. cruzi</i> (TcI, TcII, TcIII, TcVI and Tcbat) and homologues from <i>T. cruzi</i>-like (<i>T. c. marinkellei</i> and <i>T. dionisii</i>), <i>T. rangeli</i> and <i>T. b. brucei</i>.

<p>Pre, pro, catalytic domain and C-terminal extension amino acid sequences of cruzipain genes from <i>T. cruzi</i> Sylvio X10.6 and G (TcI), TCC1994 (Tcbat), Y and Esmeraldo cl3 (TcII), M6241 cl6 (TcIII), CL Brener (TcVI) Non-Esmeraldo-like (TcIII) and Esmeraldo-like (TcII) haplotypes and homologues from <i>T. c. marinkellei</i> (344), <i>T. dionisii</i> (211), <i>T. rangeli</i> (LDG and AM80) and <i>T. b. brucei</i> (TREU 927). The CATL family signatures of pro-domain motifs ERFININ (ERFN) and GNFD (GTFD) are indicated in bold and underlined, the subsites S1, S2 and S2′ are in bold, and the conserved Trp181 are indicated by (*).The glutamine [Q] of the oxyanion hole, cysteine [C], histidine [H] and asparagine [N] of catalytic triad in the catalytic domain, and 8 cysteines in the C-terminal extension are indicated by arrow heads.</p

Synteny of a locus containing cruzipain genes in <i>T. cruzi</i>, <i>T. dionisii</i> and <i>T. b. brucei</i>.

<p>Segments from the chromosome 6 of <i>T. cruzi</i> CL Brener non-Esmeraldo-like and Esmeraldo-like haplotypes, corresponding to TcIII and TcII, respectively, showing 3 to 4 cruzipain gene copies (entire or partial) flanked by orthologous genes marked with different colors according to the legend. Data from the draft assembly of <i>T. cruzi</i> G, M6241 cl6 and <i>T. dionisii</i> allowed to place one, three or two cruzipain gene copies, respectively, within the same syntenic region (figure do not reflect their actual position on chromosomes). Syntenic region from the chromosome 6 of <i>T. b. brucei</i> comprising 11 copies in tandem of brucipain genes was included in the alignment. The shades of vertical gray bars indicate the variable degrees of divergence between sequences according to the legend. The accession codes of all contigs/scaffolds and GenBank accession numbers (in bold) are presented below the corresponding sequences.</p

Polymorphism and network analyses of catalytic domain sequences of cruzipain genes from <i>T. cruzi</i> isolates of TcI-VI and Tcbat.

<p>(A) Polymorphic nucleotide sites on catalytic domains of cruzipain encoding genes; (B) Network based on polymorphic nucleotides constructed with the Neighbour-Net algorithm excluding all conserved sites and with Uncorrected p-distance. The numbers in nodes correspond to bootstrap values from 100 replicates. CLBrener1-5 are sequences from TriTrypDB: Tc00.1047053509429.320, Tc00.1047053507537.20, Tc00.1047053507603.270, Tc00.1047053507603.260 and Tc00.1047053507537.10; *GenBank accession numbers of all sequences included in these analyses are listed on Table1. Major types of sequences from <i>T. cruzi</i> isolates of different DTUs are indicated by different colors according to the legend.</p

Network genealogies of predicted amino acid sequences from all domains of genes encoding cruzipain in <i>T. cruzi</i> and homologues in other trypanosome species.

<p>Networks produced using the Neighbour-Net algorithm in SplitsTree v4.11.3, excluding all conserved sites and with Uncorrected p-distance. Networks were produced using entire sequences (A), pre- and pro-domains (B) or restricted to catalytic domains (C) of cruzipain encoding genes from the different trypanosomes are indicated by different symbols and colors according to the legend. Numbers in nodes correspond to support values estimated by performing 100 bootstrap replicates using the same parameter optimized for network inferences.</p