Functional evaluation of the thrombospondin related anonymous protein 2 from Neospora caninum (NcMIC2-like1)

Author(s)

Luiz Miguel Pereira

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2013-08-30

Examining board members

Ana Patricia Yatsuda Natsui; Marcia Eliana da Silva Ferreira Balieiro; Munira Muhammad Abdel Baqui; Rosangela Zacarias Machado; Hilda Fátima de Jesus Pena

• Advisor: Ana Patricia Yatsuda Natsui

Abstract

The Apicomplexa protozoan Neospora caninum is an obligate and intracellular parasite that has the dog and other canides as definitive host and especially cattle as intermediate hosts. It causes respectively encephalopathy. abortions and loss of fertility causing deep economic impact at the world livestock. As Plasmodium and Toxoplasma gondii, N. caninum has a specific system of active secretion, which allows the invasion of the parasite into the host cell, the establishment of parasitophorous vacuole and replication. Due to the intracellular cycle of parasite, several strategies have been developed to characterize the invasion process of the phylum, such as deletion or control of expression of related genes. Among the genes with an important role in the invasive process, NcMic2-like1 stands out, once the serum against the recombinant form inhibited the in vitro invasion of N. caninum up to 69%. This microneme protein has adhesive domains (one integrin and six thrombospondins) acting between the host cell receptors and the actin/myosin motor of the parasite, allowing the active invasion. The main aim of this work was the development of genetic tools for deletion and overexpression of NcMic2-like1 and other proteins in N. caninum for a deep understanding of this mechanism, in addition to the characterization, localization and expression of NcMic2-like1. Therefore two models based on the drug resistance and integration into the genome of the parasite were constructed. One was based on the resistance against chloramphenicol (chloramphenicol acetyl transferase gene by - CAT) and the other against pyrimethamine (by mutation of dihydrofolate reductase-thymidylate synthase - DHFRM2M3). The sequences that confer these two drug resistance patterns have been ligated to the promoter and the 3\' UTR region of three genes from N. caninum, NcSAG1 (or SAG1-like); NcDHFR or NcHXGPRT, conferring resistance to the drugs after transfection. These vectors were ligated to two sequences that confer resistance controlled by tetracycline, the TetR / tetO system. The T. gondii system was adapted for N. caninum. TetR was expressed under the control of N. caninum tubulin promoter and tetO controlled the expression of Lac-Z (?-galactosidase), a reporter enzyme. TetR and tetO were stably inserted in N. caninum after transfection iv with the vectors based on CAT and DHFRM2M3.The system was responsive following tetracycline presence, in addition to a TetR independent mechanism in N. caninum . Moreover, it was possible to express Lac-Z in N. caninum enabling the development and standardization of invasion, growth and detection tachyzoite assays. It was also possible to detect through fluorescence microscopy the YFP (yellow fluorescent protein) in tachyzoites transfected with YFP fused to TetR.. The tools for gene insertion and deletion allowed the construction of vectors for hiperexpression or deletion of NcMic2-like1. For hiperexpression the vector was obtained by the replacement of the Lac-Z by the NcMic2-like1 gene. For the NcMic2-like1 deletion the vector was achieved after the ligation of its promoter and 3 \'UTR region to CAT or NcDHFRM2M3. This approach leads to the replacement of the target gene by the resistance gene through homologous recombination. Furthermore approaches for expression and purification of proteins in N. caninum were developed. Also, a soluble recombinant NcMIC2-like1 form was obtained with pET28 system added with a soluble tail amplified from a genome region of N. caninum. The development of genetic manipulation methods is an unprecedented event for N. caninum and will enable deeper molecular studies over mechanisms of invasion and replication, where NcMic2-like1 is the first candidate for these assays. (AU)

• FAPESP's process: 09/07713-7 - Functional evaluation of the thrombospondin related anonymous protein 2 from Neospora caninum (NcMIC2-like1)
• Grantee: Luiz Miguel Pereira
• Support Opportunities: Scholarships in Brazil - Doctorate