Importance of enolase in Giardia lamblia differentiation - PubMed (original) (raw)

Importance of enolase in Giardia lamblia differentiation

Araceli Castillo-Romero et al. Mol Biochem Parasitol. 2012 Aug.

Abstract

The ability of Giardia to differentiate into cysts which survive in the environment and release the virulent trophozoites after ingestion in the small intestine is essential for transmission and disease. We examined the role of enolase, a glycolytic enzyme, in Giardia differentiation. The sequence of Giardia lamblia enolase (gEno) is most similar to enolases in Homo sapiens and Leishmania mexicana, and shows the conserved catalytic and metal-binding residues. We used an integration vector to stably express wild type and mutant gEno. In trophozoites, wild type gEno localized to the cell membrane, caudal flagella and cytosol. gEno is present on the wall of mature cysts, but not in encystation secretory vesicles (ESV). The expression of gEno with a deletion of residues G167-K169, or mutations H389Q/R390S significantly inhibited excystation while mutation of residue D257K had no effect. These results suggest a role for enolase in regulation of Giardia excystation.

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Figures

Fig. 1. Sequence analyses of Giardia lamblia enolase (gEno)

Giardia lamblia enolase (gEno, GL50803_11118; GenBank Accession No. XP_001709336.1) was aligned with the enolase genes of Entamoeba histolytica (Ehist) (GenBank Accession No. AAA80166.1), Leishmania mexicana (Lmex) (GenBank Accession No. DQ221745), Trypanosoma brucei (Tbru) (GenBank Accession No. AAF73201) and α and γ enolase of Homo sapiens (AHomo and GHomo) (GenBank Accession No. NP_001419.1 and GenBank Accession No. NP_001966.1). Active site residues are marked with a star and metal-binding residues are labeled with a black circle. The alignment was made using Vector NTI Advance software, version 9 (Life Technologies). Black arrows indicate the residues that were deleted or mutated in this study. The underline identifies the highly conserved first loop involved in the protonation of 2PGA by H159 [4].

Fig. 2. Immunolocalization of enolase in Giardia lamblia trophozoites and cysts

Using genomic Giardia DNA and the primer pairs Eno-pKS-F and Eno-pKS-R (supplementary Table 1), a 925 bp coding region of gEno was amplified by PCR and cloned into an integration vector (called “gEno-pKS”) that introduces a C-terminal triple HA tag under puromycin selection [24]. Plasmid gEno-pKS was linearized using ApaI (Promega), and introduced into Giardia lamblia WB (clone C6, ATCC 50803). Transfected cells were cultured [25] and encysted as described [26]. gEno localization was analyzed using a fluorescence microscope (Nikon Eclipse E800). Vegetative trophozoites (A–F) and mature water-treated cysts (G–I) were stained with a specific gEno antibody (A, B) [15] and anti-HA-FITC antibody (green; Roche) (D and E, G and H). Mature cysts were also stained with an anti-CWP1-TRITC antibody (red; A300cy3R-1X, Waterborne) [27] (H). DIC images are shown on the right. Nucleic acid staining is shown in blue (DAPI). Arrows indicate trophozoite or cyst surface, and arrowheads show caudal flagella. Transfected cells were harvested and prepared for Western blotting using a specific gEno antibody (J) [15]. Black arrows indicate native and HA-tagged gEno. Bar = 10μm.

Fig. 3. Enolase participates in Giardia lamblia excystation

The overlapping-extension PCR was used to generate three mutant forms of gEno. We designed two flanking primers: Eno-pKS-F and Eno-pKS-R and three pairs of internal primers to generate the desired alterations: Eno-Del-F, Eno-Del-R, Eno-Mutl-F, Eno-MutI-R, Eno-MutII-F and Eno-MutII-R (Supplementary Table I). The first pair of internal primers was designed to produce a deletion of G167-K169, the second pair produced a single mutation D257K and the third pair produced a double mutation H389Q/R390S. Fragments were sequenced (EtonBio) using primers pKS-F and pKS-R, and cloned into the integration vector that introduces a C-terminal triple HA tag under puromycin selection [24]. The resulting plasmids, gEno-del-pKS, gEno-mutI-pKS or gEno-mutII-pKS, were linearized by ApaI digestion and introduced into Giardia trophozoites. Puromycin selected parasites were induced to encyst [26] and excyst [28]. Bars represent the average numbers of ESV/50 cells (A) and the % of inhibition of excyzoites (B). Data were statistically analyzed by ANOVA (StatPlus ® 2009) and P values of ≤ 0.05 were considered significantly different. Error bars represent the standard deviation (n=3; *p-value=0.02, **p-value=0.017, ***p-value=0.00011).

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Importance of enolase in Giardia lamblia differentiation - PubMed (original) (raw)