Aspectos parasitológicos, imunológicos e moleculares da resposta dependente do receptor do tipo Toll 9 na infecção experimental por cepas de diferentes linhagens de Trypanosoma cruzi

Abstract

Toll-like receptors (TLR) are important components of the innate immune system, responsible for the recognition of Pathogen Associated Molecular Patterns (PAMPs). TLR9 was initially identified as responsible for the recognition of unmethylated CpG motifs derived from genomic DNA of bacteria and viruses. More recently, it was demonstrated that the genomic DNA of trypanosomatids, such as Trypanosoma cruzi, exhibits a TLR9-dependent immunostimulatory activity and that this receptor is essential in the recognition of the parasite and host resistance in experimental infections. Previous studies by our group have identified immunostimulatory CpG motifs in the genome of T. cruzi CL Brener strain. These sequences are not randomly distributed in the genome, but instead are enriched in regions containing gene clusters encoding surface proteins and other T. cruzi specific sequences. These regions are highly polymorphic in the two haplotypes of the hybrid CL Brener strain, which led us to speculate that the abundance of these CpG motifs could vary in the genome of different T. cruzi lineages, and if so, contribute to a differential activation of TLR9. To test this hypothesis, in this work, we studied the role of TLR9 in infections caused by T. cruzi strains that belong to different evolutionary lineages: Colombiana (TcI), Y (TcII) and CL Brener (TcVI). These strains were selected for this study because they display a distinct pattern of parasitaemia and virulence in experimental infections. Experimental infections of wild type and TLR9-/- mice with Colombiana, Y and CL Brener strains revealed a lower importance of TLR9 in controlling parasitemia and mortality in infections with Colombiana, when compared with infections with CL Brener and Y strains. Immunostimulatory assays using dendritic cells and macrophages from C57BL/6 and TLR9 -/- incubated with equal amounts of genomic DNA of different strains revealed no difference in the production of nitric oxide and IL-12 among the strains. However, assays using dendritic cells incubated with the same number of live trypomastigotes of the three strains of T. cruzi revealed a lower ratio of the TNF- and IL-12 production by dendritic cells from C57BL/6 and TLR9-/- mice for Colombiana compared with those values for the other strains. These results support the hypothesis that Colombiana strain has lower abundance of CpG motifs in its genome compared with CL Brener and Y strains, and suggest possible differences in the size of the genomes of these strains. This hypothesis was confirmed by sequencing the genomes of Y and Colombiana strains and by performing comparative analysis with the genome of CL Brener. CL Brener and Y strains have a higher abundance of CpG motifs that have high immunostimulatory activity, whereas the genome of Colombiana is enriched with CpG motifs with lower immunostimulatory properties. Comparative analyzes including the genomes of Sylvio (TcI), Arequipa (TcI), Esmeraldo (TcII) and Tulahuén (TcVI) revealed that this profile is conserved within T. cruzi phylogenetic lineages. Our results reveal that infection caused by distinct T. cruzi strains promotes a differential activation of the TLR9 receptor, leading to variable production of proinflammatory cytokines that are necessary for parasite clearance. The content of CpG motifs in the genome of different T. cruzi strains is therefore an important factor affecting virulence, pathogenesis and parasite adaptation to the vertebrate host.