Glycolysis is important to maintain the vital functions of T

Glycolysis is important to maintain the vital functions of T. gondi. Extracellular T. gondii shift its iCRT 14 metabolism towards glycolysis, based on the observation that a relocation of glycolytic enzymes from the cytosol to the pellicle occurs at the time when tachyzoites egress from host cells. It has recently been suggested that tachyzoites are independent from external carbon sources within the first hour of their extracellular life, which is the most relevant time span for finding a new host cell, but rely on the glycolytic metabolisation of internal carbon sources for ATP maintenance, gliding motility and host cell invasion [34], [35]. Two glycolytic isoforms of enolase are encoded in the genome of T. gondii, the specific expression of enolase genes is linked to life-stage conversion. While enolase 1 is expressed exclusively in slow-growing bradyzoites; conversely, enolase 2 is expressed only in rapidly dividing tachyzoites [36], [37]. Both enolase 1 and enolase 2 play important nuclear functions in parasite stage development and appear to play a role in transcriptional regulation in the parasite [38]. In this study, we provide the data of metabolism, biological and immunity to provide insights into the multifunctional roles of enolases, with a special focus on the tachyzoite enolase 2. We used immunoproteomics to identify enolase 2 as an immunogenic ESA protein of T. gondii. The factors influencing the enzymatic activity of the purified recombinant T. gondii enolase 2, the characteristics of a monoclonal antibody against enolase 2, and the location of enolase 2 in both extracellular and intracellular tachyzoites were also investigated. The results of this study provide evidences that enolase 2 might participate in T. gondii survival and pathogenesis during infection and offer additional perspectives for both drug discovery and vaccine development.
Materials and methods
Results
Discussion Toxoplasma is an obligate intracellular parasite that must invade a vertebrate host cell for survival and replication. This process is rapid and dynamic and relies on the secretion of numerous secretory proteins, which have been documented as ESA [10], [11]. In this study, the ESA of T. gondii strain RH were profiled on 2-D gels and further analyzed by Western blotting using dog serum against T. gondii RH. The Western blot analysis results showed that serum from a dog with T. gondii acute infection reacted strongly with some ESA. Three specific immunogenic proteins were identified, namely, membrane attachment protein, protein disulfide isomerase and enolase 2. Enolase has been found to play important roles in parasite metabolism, and this protein is likely a parasitic virulence factor [17], [18]. However, the potential roles of enolase 2 in T. gondii infection are not well described to date. As a glycolytic enzyme, enolase is involved in a variety of normal cellular activities. Studies of the enzymatic and biological properties essential for parasite survival and the mechanisms by which T. gondii establishes an infection in an animal or a human host are important for the development of both drug targets and vaccines. In this study, after demonstrating the presence of enolase 2 in ESA from T. gondii strain RH, enolase 2 was cloned, sequenced and expressed. The deduced 444 amino acid sequence of T. gondii enolase 2 was compared with the enolases from other parasites. This comparison revealed a high degree of conservation (from 58.9% to 98.9% sequence identity), including full conservation of the metal- and substrate-binding motifs and the enolase signature (LLLKVNQIGSVTES). The results of our enzymatic assays indicated that the overexpressed Tg-enolase 2 successfully catalyzed the conversion of 2-PGE to PEP, suggesting that recombinant T. gondii enolase 2 retained its enzymatic activity. The maximal activity of Tg-enolase 2 was attained at 37 °C, with relatively broad thermostability at temperatures ranging from 25 °C to 45 °C, this property is beneficial for promoting infection-related processes at different temperatures. The optimum reactivity of Tg-enolase 2 was found at pH 7.5, though activity decreased below 50% when the pH was reduced below 5.0 or increased greater than 9.0. These results indicate that optimal T. gondii enolase 2 activity typically occurs under weakly alkaline conditions (pH = 7.5), which may be similar to the typical internal environments of host organisms. A previous report has indicated that the enolase of Lactobacillus crispatus is localized at the cell surface at pH 5 but that it is released into the medium at alkaline pH, indicating that lactobacilli rapidly modify their surface properties in response to changes in pH [41]. It is possible that the pH in the local environment has a similar effect on T. gondii enolase, and further investigation of the biological significance of this factor is warranted. Mg2+ is a natural auxiliary factor that is essential for the enzymatic reactions of all enolases [42]. In this study, common metal ions, such as Cu2+, Cr3+ and Ni2+, were also assessed for their influence on enzymatic activity and to ascertain whether they can act as cofactors for enolase 2. Our results showed that the activity of T. gondii enolase 2 was completely inhibited by the replacement of 10 mM Mg2+ with Na+, Cu2+ and Cr3+ in reaction buffer and that its activity was remarkably reduced when K+, Ni2+, or Al3+ was added. Other studies have reported that divalent metals, such as Co2+, Mn2+ and Cu2+, inhibit the enzymatic reactions of all tested enolases [16], [43]. Enolases are also generally inhibited by the monovalent ions Li+ and Na+; Trypanosoma brucei and yeast enolases are not activated by K+, whereas rabbit enolase is [16]. These results indicate that the enzymatic activity of T. gondii enolase 2 is ion dependent; thus, inhibiting its enzymatic activity by the addition of such divalent metals as Na+, Cu2+, or Cr3+ might represent a new strategy for the prevention of T. gondii infection in hosts.