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

T. cruzi-Like and Other Trypanosome Species. PLoS ONE 7(6): e38385. doi:10.1371/journal.pone.0038385 Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi , T. cruzi -Like and Other Trypanosome Species Luciana Lima 0 Paola A. Ortiz 0 Fla via Maia da Silva 0 Joa o Marcelo P. Alves 0 Myrna G. Serrano 0 Alane P. Cortez 0 Silvia C. Alfieri 0 Gregory A. Buck 0 Marta M. G. Teixeira 0 Mauricio Martins Rodrigues, Federal University of Sao Paulo, Brazil 0 1 Departamento de Parasitologia, ICB, Universidade de Sa o Paulo, Sa o Paulo, Sa o Paulo, Brasil, 2 Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University , Richmond , Virginia, United States of America, 3 Faculdade de Farma cia, Universidade Federal de Goia s , Goiania, Goia s , Brasil 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. - Funding: This research was funded by grants from Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Cientfico e Tecnolo gico) to MMGT and from the NSF (USA) Grant: Assembling the Tree of Life: Phylum Euglenozoa (GAB, PI). PAO is doctorate fellow sponsored by CNPq-PROTAX (Programa Nacional de Taxonomia do CNPq). LL, APC and FMS are postdoctoral fellows sponsored by CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nvel Superior - Brasil). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Cathepsin L-like (CATL) are cysteine proteases that play important roles in cell invasion, growth, differentiation, immunity, immune-modulation, virulence, pathogenicity and survival of pathogenic protozoans. Different isoforms of CATL are encoded by a large gene family and perform distinct roles in the interactions of the trypanosomes with vertebrate hosts and vectors, differing in stage, cellular localization and expression level during the life cycle. This functional and structural diversification may have contributed to the adaptation of different trypanosome species to their different life cycles, vertebrate hosts and vectors [14]. Trypanosoma cruzi is the type species of the subgenus Schizotrypanum and a complex of genetically heterogeneous isolates distributed in 6 intraspecific subdivisions denominated discrete typing units (DTUs), TcI-TcVI [5,6], and one new genotype (Tcbat) identified in Brazilian bats [7]. Closest relatives of T. cruzi are the bat-restricted T. c. marinkellei followed by T. dionisii, which are referred as T. cruzi-like due to morphologically indistinguishable blood and culture forms [811]. Development within mammalian cells in vitro is a feature shared by all species of the subgenus Schizotrypanum, while in vivo only T. cruzi has been proven to infect mammals other than chiropterans [7,9,10,1214]. Bats infected by T. cruzi-like species show nests of amastigotes in cardiac, skeletal and stomach muscle cells likewise T. cruzi in a range of hosts including man. Recent studies demonstrated that T. dionisii and T. cruzi invade mammalian cells through a common mechanism involving lysosome mobilization to the site of parasite entry [15,16]. Previous studies showed that T. cruzi and T. dionisii share similar molecules with important roles in host-parasite interactions such as phospholipids and cysteine proteases [17,18] as well as epitopes associated to autoimmunity in Chagas disease [19]. Nevertheless, T. dionisii differ from T. cruzi in surface glycoproteins involved in host-cell interactions [16]. Besides morphology and in vitro and in vivo behavior, T. cruzi-like species share cellular, biochemical and immunological features with T. cruzi and, hence, can be valuable as non-infective to humans models for studies of T. cruzi and as targets for trials of drugs, vaccines and diagnosis [10,20]. Similar to T. cruzi, all T. cruzi-like isolates differentiate from epimastigotes to infective metacyclic trypomastigotes in cultures and in the vector gut; bats are infected by licking vector feces contaminated with trypanosomes on their fur and/or by ingesting the infected vectors themselves [8,9]. Differently from T. cruzi that can be transmitted by several genera of triatomine bugs, transmission of T. c. marinkellei seems to involve only triatomines of the genus Cavernicola, while cimicids are vectors of T. dionisii [8,9]. In addition to T. cruzi, T. rangeli was reported infecting humans and non-human primates, chiropterans and mammals of other orders and are both transmitted by triatomines [21,22]. The mechanisms underpinning vertebrate and vector specificities of these trypanosomes are unknown. Previous studies demonstrated that two main CATL enzymes are expressed by T. cruzi, the major isoform (.75 copies) addressed in the present study and designated as cruzipain, is the archetype of a large multigene family organized in tandem repeats expressed in all life cycle stag (...truncated)