Aspectos genéticos de parasitos e hospedeiros envolvidos na patogênese da doença de chagas

Abstract

Chagas disease, which is caused by Trypanosoma cruzi, afflicts more than 18 million people in the American continent. Both a complex populational structure and a extensive intraespecific variability are observed in this parasite, but a great number of genetic andbiochemical markers support the taxon subdivision into at least two major lineages: T. cruzi I - associated to the wild transmission cycles; and the T. cruzi II - associated to the domestic transmission cycles. The human disease shows variable presentation in the chronic phase, withlow parasitism levels and undeterminable clinical routes, ranges from completely asymptomatic to massive manifestations. The disease results from complex interactions between parasites andhosts, but the reasons for the different patterns of clinical manifestations remain elusive. To clarify these questions it is necessary to understand better both the parasites and hosts genetic factors and how they interact during the pathogenesis. In order to investigate the structure and evolution of Trypanosoma cruzi populations we profiled 75 strains of the parasite with five nuclear microsatellite loci, 24S RNA genes, andsequence polymorphisms in the mitochondrial cytochrome oxidase subunit II, cytochrome B and NADH dehydrogenase subunit 1 genes. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-clusterA), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH).The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24S rDNA and microsatellite profiling data were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes thatwere clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose three ancestral lineages that we call, respectively, T. cruzi I, T. cruzi II and T. cruzi III. At least twohybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial donor (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi III and the mitochondrial recipient was T. cruzi II. By using a new molecular tool resulting of the combination of heminested PCR amplification and real time PCR we obtained the sensitiveness and resolution of typing the T. cruzi 24S directly from chronically infected tissues. In twenty-seven hearts, esophagus and colon samples of twenty-five patients from Minas Gerais and Goiás states we detected only the rDNA allele type 1, which correlates well to the T. cruzi II 7 lineage. These data confirm previous evidences of T. cruzi II being responsible for the disease, at least in that geographic region. Furthermore, we genotyped the parasites in the blood stream and in brain biopsy from a Chagasic patient with AIDS. Analyses of rDNA 24S and mini-exon genes and of microsatellite and LSSP/PCR profiles indicate a bigger clonal diversity at brain than at the blood stream and that the two populations were genetically diverse, since in the blood we found the T. cruzi II lineage while a hybrid population was found in the brain. With the aim of investigating further the T. cruzi differential distribution into host tissues, we used the murine model of thedisease, where there is evidence of a correlation between tissue tropism and MHC haplotypes (denominated H-2 in mice). In order to study this we used both in vivo and ex vivo procedures. First we infected mice congenic for the H-2 region [C57BLK/sJ (H-2d), C57BL/6 (H-2b), BALB/c (H-2d)and BALB/B10 (H-2b)] with an artificial mixture of two parasites [JG (T. cruzi II) and Col1.7G2 (T. cruzi I)]. For all experiments, the prevalence of a strain in detriment to the other into cardiac tissue is correlated to the H-2 haplotype, were the Col1.7G2 strain colonization is improved by the H-2bhaplotype, regardless of the genetic background. The results presented in this work reinforce the notion that the genetic variation in T. cruziand its host influence the course of the Chagas disease.