is one of the few parasites that
is one of the few parasites that can be encysted and excysted and we tested the localization and role of HA-tagged gEno in both differentiations, using a recently developed integrated vector that introduces a C-terminal triple HA tag under the selection marker puromycin . The localization of HA-tagged gEno (D–F) was identical to that of native gEno with a specific antibody (A–C) . The introduction of the epitope tag did not affect the localization of enolase. Despite the absence of an N-terminal secretion signal, gEno localizes to the plasma membrane, as well as the cytoplasm, and caudal flagella of vegetative trophozoites (A, B, D and E). The presence of enolase on the plasma membrane has also been observed in other protozoan parasites, bacteria, helminths and fungi and is suggested to aid in completion of their life cycle and/or tissue invasion , , , , , , , , . Similar to , and , , , gEno associates with the cyst wall in mature water-resistant cysts (G and H). In addition, the HA-tagged gEno expression was analyzed by Western blotting using a specific gEno antibody . Two bands matching the predicted full-length native gEno (48kDa) and HA-tagged gEno (54kDa) show the preponderance of the wild-type protein (J). To identify a specific role of enolase, we used an integrated vector to obtain four independent constructs that each expressed either wild type or one of three mutated forms of gEno and assessed the capacity of the mutant to encyst and excyst. Untransfected (C6) and trophozoites expressing wild type HA-tagged gEno (called “gEno-pKS”) were used as controls. The mutant cell lines are: gEno-del-pKS, a deletion of G167-K169 ion channels involved in 2PGA conversion; gEno-mutI-pKS, in which the first Mg binding site D257 was mutated to K257; gEno-mutII-pKS, in which the active site residues H389 and R390 were mutated to Q389 and S390. Mutations of either H159 or H373 (corresponding to H170 and H389, respectively in the gEno sequence) resulted in significant decreases in yeast enolase activity , , . These gEno mutations and deletion did not affect the growth or localization of HA-tagged enolase in vegetative trophozoites (data not shown). Trophozoites were induced to encyst and ESV were counted at 20h and cysts at 48h after induction. The total number of ESV in gEno-del-pKS and gEno-mutII-pKS encysting trophozoites was only slightly, but significantly reduced (A). No significant differences were observed in the number of mature cysts (data not shown). This was surprising since enolase is important in encystation of and , . Although the transgene only replaces one of the four copies of enolase genes, allowing for expression of the wild-type enzyme, the gEno-del-pKS and gEno-mutII-pKS mutations significantly impaired the ability of parasites to excyst (43 and 66%, respectively) compared to the controls (B). The fact that two separate mutations, that likely inactivate enolase, cause the same phenotype leads us to conclude that this enzyme has an important role in excystation. In contrast, mutation of D257, one of the Mg (I) binding sites (gEno-mutI-pKS) does not have any apparent effect on excystation (B). Since only one of the three Mg (I) sites was mutated, it is possible that the overall conformation of the active site pocket and Mg binding are unaffected. Future studies will evaluate the enzymatic activities of these gEno mutants. Our finding that parasites expressing gEno mutants are impaired in excystation indicates that enolase might have crucial roles in cellular activation from dormancy. This opens the door for in depth investigations of the role of other metabolic enzymes in differentiation of and other protozoan parasites. Acknowledgements We thank Maya Millman-Gray and Cynthia Quindoza for technical support, and Zac Cande and Stephane Gourguechon for the giardial integration vector. This work was supported by NIH grants, AI42488 and UO1AI75527. Araceli Castillo-Romero was a scholarship recipient from UCMEXUS-CONACYT and we are grateful for their support.