Complete Molecular and Immunoprotective Characterization of Babesia microti Enolase

Abstract

The apicomplexan Babesia microti is the primary causative agent of human babesiosis, one of the most broadly distributed tick-borne diseases worldwide. B. microti undergoes a complex lifecycle within both the mammalian host and the tick vector, and employs several different specific molecular mechanisms to enter host cells. Enolase, the key glycolytic enzyme in intracellular glucose metabolism, can also be expressed on the parasite’s outer surface, binds to human plasminogen, and coordinates apicomplexan parasite invasion of host cells, however, it lacks sorting sequences or lipoprotein anchor sites. In the present study, we isolated the coding gene of B. microti enolase (BmEno), expressed it within E. coli and purified the recombinant BmEno protein (rBmEno). Consequently, we confirmed cytoplasmic and surface localization of BmEno via immunofluorescence, and demonstrated that rBmEno catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. Moreover, our results showed that rBmEno binds to human plasminogen, and that the lysine analog ε-aminocaproic acid significantly inhibited this binding. Furthermore plasminogen bound to rBmEno converts to active plasmin. Additionally, actively immunizing mice with rBmEno could evoke a partial protective immunity against B. microti infection following challenge. In conclusion, B. microti enolase is a multifunctional cytoplasmic protein which is also expressed at the parasitic outer surface, facilitates binding to host plasminogen, and could partially protect hosts against parasite infection.

Expression, purification, and biological characterization of <i>Anaplasma phagocytophilum</i> enolase

“…Interestingly, rApEno demonstrated the highest enzymatic activity at 50°C and pH 8.0 (Figure 3). Moreover, rApEno presented high K m and V max values during 2-PGA to PEP conversion, when compared to other infectious intracellular pathogens (13,20). This suggests that ApEno is highly adaptable to a changing surrounding environment.…”

“…In order to evaluate the potential catalytic and plasminogen-binding motif, enolase amino acid sequences from different hosts were aligned using MUSCLE multiple sequence alignment tools. As shown in Figure 1, ApEno possesses both 2-PGA binding residues and catalytic loops when compared to Babesia microti enolase and Toxoplasma gondii enolase (13,16). Furthermore, ApEno also possesses a conserved internal plasminogen-binding motif "FYDGKIYKFSGSSMS" located between amino acids 254 and 268, when compared to Streptococcus pneumoniae surface enolase (17).…”

“…Many reports have demonstrated that enolase can also be expressed on the surface of organisms, and act as the plasminogen receptor (10,13,(21)(22)(23). In order to characterize rApEno plasminogen binding, ELISA experiments were performed.…”

“…ApEno was then cloned into the pGEX-4T-2 expression vector (TransGen Biotech, Beijing, China), and transformed into Escherichia coli BL21 Star (DE3) competent cells. Recombinant protein expression and purification were performed as previously described (13). In brief, E. coli cells were induced overnight with isopropyl-β-Dthiogalactopyranoside (IPTG), and then lysed with a sonicator.…”

“…Polyclonal antibodies against rApEno were generated as previously described (13). Briefly, 50 μg purified rApEno protein was mixed with MONTANIDE TM ISA 70 VG adjuvant (Seppic, Puteaux, France), and then subcutaneously injected into four-to six-week-old female Balb/c mice at two-weekly intervals.…”