Estudo do papel de trans-sialidases de Trypanosoma cruzi durante a interação parasito-hospedeiro

Abstract

Trypanosoma cruzi, the causative agent of Chaga’s Disease, presents multiple multigenic families such as mucins, trans-sialidases and MASP. Trans-sialidases (TS) represent the largest multigenic family in the T. cruzi genome with over 1400 gene copies, in which around 16 sequences encode enzymatically active TS (aTS), classified as group I-TS. T. cruzi is not able to produce sialic acid de novo. aTS are responsible for transferring sialic acid from host glycoconjugates to mucins present on the parasite surface, working as a mechanism of molecular mimicry and subverting its recognition by the host. T. cruzi does not have functional RNAi machinery which has limited reverse genetics studies on this pathogen. Using CRISPR/Cas9 technology, we generated for the first-time knockout parasites for members of the multigenic TS family that encodes aTS (aTS-KØ), which display undetectable levels of TS activity (Burle-Caldas et al, 2022). We evaluated the role of aTS expression, and its catalytic activity, in the host-parasite relationship during the passage of T. cruzi in two hosts: invertebrate (triatomines) and vertebrate (mammals) by using aTS-depleted parasites (aTS-KØ). Here, we noticed for the first time that T. cruzi does not require TS activity to complete its life cycle in Rhodinius prolixus. Conversely, aTS acitivity is a necessary step for its maintenance in mammals in vivo and in vitro. Although the initial stages of the intracellular development of T. cruzi are not affected (invasion and multiplication), we observed that final stages of the cell cycle are dependent of aTS (differentiation and egress). These findings endorsed that this is not a cell-specific phenomenon and also confirmed previous published data by our group. In vitro infection of epithelial cells with aTS-KØ has significantly lower inflammatory cytokines mRNA transcript levels such as IL-1-β, IL-1-α, IL-6 and CXCL8 compared to wild-type parasites. The differential cytokine induction may be related to a persistent MEK/ERK1/2 protein kinase activation axis during aTS-KØ infection, suggesting an indirect ERK1/2 regulatory role of the host cell anti-inflammatory response. Furthermore, we showed, that the activation of anti-apoptotic pathways by T. cruzi in epithelial cells is independent of aTS. Taken together, previous data and the data presented here suggest that aTS mutants represent a highly attenuated strain that could be used as a vaccine strategy, in addition to allowing complex studies of the role of aTS as virulence factors.