Date of Award
Doctor of Philosophy
Igor C. Almeida
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected infectious disease that is becoming a world health concern. This obligate intracellular parasite employs a diversity of molecules and strategies to successfully invade a wide variety of mammalian cells and modulate host immune responses, which are essential features for completion of its life cycle in the host. The major plasma membrane antigens of T. cruzi infective trypomastigote forms are glycosylphosphatidylinositol (tGPI)-anchored mucin-like glycoproteins. Although previous studies demonstrated that the proinflammatory responses induced by tGPIs are mediated by Toll-like receptor (TLR) 2, the involvement of other TLRs and coreceptors has not been investigated yet. The focus of the first part of this dissertation was to investigate the molecular events involved in the interaction between T. cruzi and host cells and particularly, the upstream molecules implicated in tGPIs recognition by the innate immune system and the influence of the tGPI structural features on the biological activity and receptor/coreceptor requirement. To overcome the limitations related to heterogeneity and quantity of native GPI anchors, chemically synthesized tGPIs (stGPIs) were used. The stGPIs were preferentially recognized by the TLR2 and TLR6 heterodimer while co-expression of CD14 and CD36 accessory molecules induced a significant enhancement in cellular responses as assessed by NF-kappa B activation and IL-8 production. Further insights into the influence of the tGPI structural features on the biological activity and receptor/coreceptor requirement were also revealed. Intriguingly, in contrast to the well established role of TLR2 as one of the primary sensor of the innate immune defense against pathogens, activation of host cell TLR2 signaling by trypomastigote-released, GPI-containing vesicles was shown to facilitate T. cruzi invasion. This unanticipated role of TLR2 in host cell invasion by T. cruzi was further investigated. Clearly, TLR2-specific ligands significantly increased parasite infection in distinct cell types. In line with these findings, silencing of TLR2 by RNA interference significantly decreased infection. Moreover, the participation of host cell actin and intracellular calcium in TLR2-mediated invasion was assessed. Taken together, these results suggest that activation of host cell TLR2 by T. cruzi molecules may play a dual, paradoxical role during the infection by stimulating microbicidal responses and increasing the parasite infectivity. It also begins to reveal previously unrecognized mechanisms of host cell signaling subversion that may be exploited by other intracellular pathogens.
In the second part of this research, a novel and improved approach to study in vitro models of host cell-parasite interaction and more specifically, T. cruzi infection, was developed. Conventional manual counting of parasite infection rate is extremely time-consuming and subjective. Hence, an approach based on high-content imaging and automated analysis in a multiwell plate format was developed to generate multiparametric data on a cell-by-cell basis which was further explored to precisely and quickly determine several parameters associated to in vitro infection of host cells. Statistical analysis confirmed that there was substantial agreement between the data acquired manually and by applying the automated analysis. Moreover, to further assess the applicability of this novel methodology, the effects of specific compounds on T. cruzi intracellular proliferation were evaluated. The results not only demonstrated the potential of the tested compounds as anti-T. cruzi agents, but also confirmed the effectiveness and uniqueness of high-content imaging in the determination of cytotoxic effects of the tested compounds as well as changes in T. cruzi infection rates and intracellular proliferation in a single experiment. Notably, this novel automated method may contribute to accelerate the discovery of potential drugs as well as the elucidation of molecular events related to the interaction between host cell and human intracellular pathogens.
Received from ProQuest
Lilian Lie Nohara
Nohara, Lilian Lie, "The Interplay Between Host Cells And The Human Pathogen Trypanosoma Cruzi: Role Of Toll-Like Receptors" (2010). Open Access Theses & Dissertations. 2743.