ANTISERUM AGAINST RECOMBINANT NCTRAP-2 DECREASES THE IN-VITRO INVASION PROCESS OF NEOSPORA CANINUM

BM03

Pereira, LM (1); de Vries, E (2); Yatsuda Natsui, AP (1)

(1)Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,  Universidade de São Paulo, Ribeirão Preto, Brazil; (2)Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.

The protozoan Neospora caninum is responsible for infecting a wide range of animals and in bovines it can induce abortions, which represents huge economical losses. N. caninum is an Apicomplexan parasite, phylum of intracellular parasites where the invasion step is crucial for survival. One important group of proteins secreted prior to invasion is the TRAP (Thrombospondin Related Anonymous Protein) family. Upon contact with host cells the TRAPs undergo exocytosis onto the apical surface of the parasite where they initiate tight binding. The adhesive complexes are translocated towards the posterior pole of the parasite via actin–myosin-based motility machinery, resulting in invasion of the host cell. In N. caninum one member, NcTRAP1 was previously described. A second possible member called NcTRAP2 was detected by non-annotated EST clustering. The aim of the present work was the cloning of the NcTRAP-2 full-length sequence, production of recombinant antisera, localization of native form in 2D western blot and in vitro invasion inhibition assay. The full-length gene was obtained with a predicted protein sequence with 39% of identity and 53% of similarity with its homologues of Toxoplasma gondi (TgMIC-2); 39% and 53% with Neospora caninum (NcTRAP-1). The TRAP homologues have a signal peptide, two adhesive domains (an integrin-like domain and one or more thrombospondin type I repeats) and a transmembrane domain. Two recombinant fragments (fragments 1 and 2, both without signal peptide and transmembrane region) of NcTRAP-2 were generated (pET28 vector), MW of 50 and 78 kDa (fragment 2 is 163 aa longer towards the C-terminal end). Antisera against the recombinant forms were obtained and monitored by ELISA. The antiserum against recombinant fragment 1 recognized the native NcTRAP-2 (80 KDa, acidic PI) and putatively two isoforms (50 KDa, neutral PI) in 2D western blot.  In vitro assays were performed to test inhibition of the invasion with the sera against recombinants 1 and 2 which were estimated both by: microscopic counting (10 fields/well in Giemsa stain) on 8 well plates and Real Time PCR on 24 well plates. The sera against recombinants forms of NcTRAP-2 (fragments 1 and 2) decreased the in-vitro invasion process in 46% and 27% respectively.