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  • dimethyl Host cell invasion by tachyzoites relies on the


    Host cell invasion by tachyzoites relies on the secretion of numerous secretory proteins and plays a crucial role in maintaining infection [6]. ESA released by tachyzoites are highly immunogenic and induce either antibody-dependent or cell-mediated protective immunity [11], [44], [45]. To investigate the immunological characteristics of T. gondii enolase 2, an immunization was performed, and one hybridoma cell line with a relatively high-affinity mAb (1D6) against enolase 2 was isolated. This mAb was characterized, and our immunological data revealed that T. gondii enolase 2 is a potent antigen that stimulates a robust immune response. The recombinant enolase 2 protein reacted strongly with serum from a dog infected with T. gondii, showing that the protein can trigger an antibody response over the course of infection. Regarding the vaccination trial, the recombinant enolase 2 was seen to be immunogenic and induced high titres of specific antibodies, and an ELISA titer of 1: 64000 was achieved. Western blot analysis further revealed that the acquired 1D6 mAb recognized both the recombinant antigen and the native enolase in ESA, thus confirming the secretion of this protein to an extracellular fluid. Our results also suggest that enolase 2 may be an infection-associated antigen and that the 1D6 mAb, with its high specificity and sensitivity, could be used to detect T. gondii acute infection and to potentially inhibit infection by this parasite. Enolase is one of the highly conserved “moonlighting” proteins, which perform multiple functions that differ from their “classical” well-known activities. These “moonlighting” functions are often revealed when these proteins are found at cellular locations that differ from those where the primary functions are exerted [16], [46]. Using the 1D6 mAb in the indirect immunofluorescence assays, our results demonstrated that enolase 2 could be specifically detected on T. gondii infecting dimethyl and T. gondii collected from the abdominal cavities of infected mice. Immunogold staining of T. gondii revealed the subcellular localization of this protein; more importantly, surface-associated localization of enolase 2 was observed in T. gondii tachyzoites. Although enolases have been shown to be secreted and translocated to the surface in many prokaryotic and eukaryotic pathogenic organisms, a detectable secretion signal or membrane anchor region that would explain its membrane localization is lacking. At this time, it remains unclear how the T. gondii enolase 2 is released into the ESA. However, there are several possible explanations, as enolase has been found on cell surfaces and has been identified as an excretory/secretory product of many parasites [23], [24], [25]. It is known that the majority of ESA are released by the apical portion of tachyzoites as micronemes and rhoptry secretions but that dense granules are released by the lateral portion [10], [11], [47]. As we found that enolase 2 is an ESA protein, we suspect that it may be released by one of these organelles. Similar results have been obtained for Leishmania mexicana, for which enolase was found to be present in the microsomal fraction that predominantly contains the plasma membrane [48]. In addition, enolase 2 may be present if some cytosolic enolase 2 is released into the ESA fraction as a result of inadvertent parasite lysis. Additionally, enolase expression in Entamoeba invadens is induced by environmental factors, in association with cytoplasmic vesicle-like structures that transport the protein to the cell wall [49]. Nevertheless, the exact mechanism by which enolase 2 is translocated to the parasite surface and secreted into the ESA fraction of T. gondii remains unknown. Our future study will focus on these issues. Enolases from several pathogens have been identified as important immunogenic proteins and protective antigens [50], [51], [52], [53]. For instance, our previous study demonstrated that an enolase of V. parahaemolyticus can be used as an immunogen to protect mice from infection [12]. Moreover, mice immunized with a recombinant Plasmodium falciparum enolase (r-Pfen) protein showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues [54]. In addition, BLAST analysis revealed high similarity between the enolase 2 protein of T. gondii and those of other parasites. It is likely that inhibitors developed in this manner against this T. gondii enzyme may also affect the activity of the enzymes of H. hammondi, N. caninum, B. darlingi, and M. balamuthi, among others. It is also likely that vaccines developed in this manner can confer cross-protection for the control of these parasites, which share many common features with T. gondii, including N. caninum. Our immunological data have revealed strong antigenicity and immunogenicity of T. gondii enolase 2, although whether this protein could effectively serve as a vaccine candidate will be confirmed using a challenge study in our future work.