Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

Abstract

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite's molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite's surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein's bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4 c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4 c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

Cloning and expression of Neospora caninum dense-granule 7 in E. coli

“…So far, cloning and expression of NcGRA7 has performed in different expression systems such as pCMViluc1 eukaryotic expression vector (Liddell et al 2003) and in bacterial expression vectors, pGEX-4T-3 (Hara et al 2006) and pRSET-C (Aguado- Martínez et al 2010). In this study we cloned and expressed NcGRA7 for the first time in Iran with new and different primers which have sites for EcoR1 restriction enzymes.…”

“…So far, three important N. caninum recombinant antigens have been determinated for diagnostic purposes (Chahan et al 2003) namely, NcSAG1, NcSRS2 (Howe and Sibley 1999) and the dense granule protein NcGRA7 (AlvarezGarcia et al 2007;Aguado-Martínez et al 2010). The aim of this study was to produce recombinant NcGRA7 protein in an efficient prokaryotic expression system, in order to standardize an ELISA based serodiagnostic procedure in Iran in near future.…”

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress

“…Polyclonal antibodies (PAbs) against rNcNTPase were raised in two female New Zealand White rabbits as previously described [ 32 ]. Polyclonal and monoclonal antibodies (MAbs) against rNcGRA7 were obtained as previously described [ 10 , 19 ]. Affinity purified antibodies were prepared following standard procedures [ 19 ].…”

“…The PV acts as a metabolically active compartment designed to favour parasite replication [ 7 , 8 ]. More than 20 GRA proteins have been reported for T. gondii [ 9 ], and 15 have been identified in N. caninum at protein and transcriptional levels [ 10 – 12 ]. Nevertheless, only a limited number of GRA proteins have been studied in N. caninum , despite the fact that some GRA proteins such as TgGRA7, TgGRA15, TgGRA16, TgGRA24, TgGRA25 and TgNTPase I, among others, contribute to virulence in T. gondii [ 13 – 17 ].…”

“…The N. caninum GRA7 protein was extensively characterized during the last few years [ 10 , 18 , 19 ], whereas little information is available for the NcNTPase [ 20 ]. This protein appears to be more abundant in virulent isolates, suggesting that its function could be related with parasite virulence [ 21 ].…”