Análise estrutural e funcional de proteínas de grânulos densos de Toxoplasma gondii através da edição gênica por CRISPR/CAS9

Abstract

Apicomplexan pathogens are the greatest threats to human and animal health in the world. Toxoplasma gondii is one of the protozoa belonging to this phylum, capable of infecting any nucleated cell. As an obligate intracellular protozoan, entry into the host cell is a crucial event to its survival. The host cell invasion is an active process in which the parasite recruits part of the host cell membrane to form the parasitophorous vacuole (PV). Whereas the PV helps protect the parasite from host cell elimination, the PV membrane (PVM), forms a physical barrier which deprives parasites of an abundant source of nutrients from the host’s endocytic and exocytic system. To overcome this problem the parasite secretes proteins that associate with the PVM, modulating its permeability allowing bidirectional diffusion of small molecules, probably through pores in the PVM. On the other hand, the host cells are able to recognize parasitic molecules in contact with their membranes or that are released into their cytoplasm, inducing the activation of crucial mechanisms for their resistance to infection. However, so far, the molecular basis of this hypothetical pore has not been reported, as well as there is little knowledge about the parasite agonists of innate receptors, being these the central goals of investigation in this work. In a first approach, GRA17 was identified as a protein secreted to the PVM that mediates passive transport of small molecules across the PVM, and its deletion induces slow parasitic proliferation and abrupt decline in virulence. Besides strongly predicted α-helices in the GRA17 composition, these were not functionally characterized. In the first chapter, we predict eight α-helix domains in the secondary structure of GRA17, which were individually truncated using the system Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR/ CRISPR associated protein 9-CAS9 (CRISPR/CAS9), in order to functionally characterize them. Through this approach, we show that with the exception of α-8, all the others predicted α-helices are required for GRA17 normal function, localization and PV stability. The truncation of these 7 motifs results in GRA17 mislocalization on the PVM and an aberrant PV morphology. Furthermore, GRA17 truncated parasites proliferate slowly and shows a decrease viability, leading us to the conclusion that the 7 α-helix structures of GRA17 are crucial for protein function and consequent viability of the parasite.

Furthermore, we sought to understand the mechanisms of host resistance, arising from the antigens release during the replication process. We use Lewis rats that was identified to be completely resistant to Toxoplasma infection. Previous findings have established that this resistance is mediated by the activation of the cytosolic sensor NLRP1, which results in the rapid host cell death through piroptose, limiting parasitic replication. However, the targets involved in NLRP1 activation were not identified. To identify the parasite genes involved in the activation of this pathway, we used a chemical mutagenesis screen, which led us to identify three proteins secreted from dense granules, TGGT1_226380 (GRA35), TGGT1_237015 e TGGT1_236870, which are individually involved in NLRP1 inflammasome activation and pyroptosis induction. Deletion of these proteins (by CRISPR/CAS9) results in significantly less pyroptosis in Lewis macrophages. A more detailed search of the clones generated by the mutagenesis assay reveals that additional factors may be involved in the induction of pyroptosis. As GRA35 family is composed by three other genes (TGGT1_225160, TGGT1_213067, TGGT1_257970) we decided to test the involvement of these proteins in pyroptosis induction. Deletion of these genes in T. gondii does not alter the viability of Lewis BMDMs compared to WT strain, which shows that, unlike GRA35, these proteins are not involved in the activation of NLRP1 inflammasome and induction of pyroptoisis. In conclusion, we demonstrate that dense granule proteins are an essential part for the parasite survival within the parasitophorous vacuole, controlling the transport of molecules through the PV membrane, as well as for being recognized by the host innate sensors, initiating a response that control the protozoan, through inflammasome activation and pyroptoisis induction.