Abstract
A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.
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Key words: Giardia lamblia, gap2 gene expression, encystation, luciferase, transfection
INTRODUCTION
Giardia lamblia, a protozoan pathogen which causes a diarrheal disease in man, has a simple life cycle that is composed of 2 forms, trophozoites and cysts. Upon ingestion of the cysts, its infection is initiated and this is followed by excystation to the trophozoite form usually in the proximal duodenum of the host. Trophozoites are able to multiply by binary fission and colonize the proximal small intestine using adhesive discs. Some trophozoites undergo encystation, i.e., differentiation of trophozoites to the cyst form, in the distal small intestine. The distinct structural differences between these 2 forms imply that a series of genes are differentially expressed during these differentiations.
Previous studies on the encystation of
G. lamblia identified several encystation-induced genes such as
cwp1,
cwp2 (
Mowatt et al., 1995),
bip/grp78 (
Lujan et al., 1996), and
enc1 and
enc6 (
Que et al., 1996). Knodler et al. (
1999) reported that the expression of
gln6pi-b for glucose-6-phosphate isomerase was also induced during encystation. In addition, the expression of the
myb2 gene encoding a putative transcriptional factor was found to be induced during encystation (
Sun et al., 2002;
Yang et al., 2003). Investigation of the 2
gap genes,
gap1 and
gap2 of
G. lamblia (
Rozario et al., 1996) indicated that Gap1 protein functions as a major glycolytic enzyme whereas
gap2 does not seem to encode a protein with GAPDH activity (
Yang et al., 2002). Despite the isolation of the
gap2 gene as an induced clone in a differential display of encysting cells of
G. lamblia, no detailed examination has been performed on its expression and function. In the present study, the expression of the
gap2 gene was monitored using transgenic
G. lamblia carrying a
gap2-luc reporter system.
MATERIALS AND METHODS
Organisms and culture conditions
Trophozoites of
G. lamblia WB strain (ATCC 30957, Washington DC., U.S.A.) were grown for 72 hr in normal TYI-S-33 medium (2% casein digest, 1% yeast extract, 1% glucose, 0.2% NaCl, 0.2% L-cysteine, 0.02% ascorbic acid, 0.2% K
2HPO
4, 0.06% KH
2PO
4 and 10% calf serum) (
Keister, 1983) containing 0.5 mg/ml bovine bile at pH 7.1, and then transferred into an encystation medium containing 10 mg/ml of bovine bile at pH 7.8 (
Kane et al., 1991). Portions of the encysting cells were sampled at various times by chilling on ice and centrifugation.
To obtain a reporter system to allow us quantitative measurement of activity of the
gap2 gene promoter in
G. lamblia, we made a new vector based on pGFP.pac (
Singer et al., 1998). A 1,682-bp DNA fragment containing the full ORF of the
luc gene was amplified from pGL2-Basic vector (Promega, Madison, U.S.A.). by PCR using 2 primers, LUC-F and LUC-R (
Table 1). An
NcoI and a
NotI site located at either ends of the resultant
luc DNA were used to clone this DNA into the corresponding sites of pGFP.pac, which resulted in plasmid pLUC.pac, in which the
gfp gene was replaced by the
luc gene (
Fig. 1).
Based on the sequence published by Rozario et al. (
1996) (GenBank database accession number, U31911), 3 different DNA regions of the
gap2 promoter were amplified and cloned into pLUC.pac (
Fig. 2A). First a 218-bp region of the DNA upstream of the
gap2 gene was amplified from the genomic DNA of
G. lamblia using 2 primers,
gap2P-R and
gap2P-F1 (
Table 1), and inserted into pLUC.pac pretreated with
HindIII and
NcoI, which resulted in a deletion of the
gdh promoter. The second promoter studied was a 112-bp DNA upstream region of the
gap2 gene, which was made from the genomic DNA of
G. lamblia with the primers,
gap2P-R and
gap2P-F2. The third
gap2-
luc reporter containing a 43-bp upstream region of ATG of the
gap2 ORF was constructed by cloning the annealed linker of 2 primers,
gap2P-F3 and
gap2P-R3, into the
HindIII and
NcoI site of pLUC.pac.
Trophozoites were grown for 72 hr in normal TYI-S-33 medium. Fifteen µg of a gap2-luc reporter plasmid
was transformed into 1 × 107 trophozoites by electroporation
under the following conditions; 350 volts, 1,000 µF, and 700 Ω (Biorad Genepulser II, Hercules, U.S.A.). Trophozoites harboring a gap2-luc plasmid were selected by adding puromycin to the TYI-S-33 medium to a final concentration of 100 µM.
Determination of luciferase activities
Luciferase activities of the harvested cells carrying one of the gap2-luc reporter plasmids were measured using a Luciferase Assay System (Promega). Briefly, collected cells were resuspended in a lysis buffer (25 mM Tris-HCl, pH 7.8, 2 mM EDTA, 2 mM DTT, 10% glycerol, and 1% Triton X-100), and frozen at -70℃ at least for 1 hr Twenty µl of cell extracts were reacted with 100 µl of luciferase substrate, and light emission was measured for 5 min in a luminometer (TD20/20 DLReady, Turner Designs, Sunnyvale, U.S.A.).
Southern blot analysis
Genomic DNAs were purified from untransfected trophozoites as well as from trophozoites transfected with pGAP2(218).pac. Ten µg of genomic DNA was digested with HindIII and loaded into a 0.8% agarose gel. Upon separation by electrophoresis, digested genomic DNA was transferred to a nytran filter (Millipore, Billerica, U.S.A.), and fixed to the filter by UV-crosslinking (Hoefer, San Francisco, U.S.A.). The filter was then hybridized using the 218-bp gap2 promoter region, which was labeled with 32P by using a Random labeling kit (Takara, Otsu, Japan).
RESULTS
Construction of 3 gap2-luc reporter systems
We used the luciferase reporter system to examine the expression of the
gap2 gene, which has been identified to be induced during encystation (
Yang et al., 2002). To define the essential
cis-acting elements required for the full expression of the
gap2 gene, 3 different
gap2-
luc fusions were constructed, which contained
gap2 gene promoter regions of different sizes, as described in
Fig. 2A.
All of the 3
gap2-
luc constructs were transfected into trophozoites as described above. For each construct, 2 independent transfectants were selected, and used for further studies. Maintenance of the
gap2-
luc reporter in puromycin-resistant
G. lamblia was verified by Southern blot analysis (
Fig. 2B). Genomic DNA of untransfected trophozoites displayed a band of ~1.7 kb in Southern blots with a
gap2 promoter region. In the case of genomic DNA of trophozoites transfected with pGAP2(218).pac, an additional band of 5.8 kb DNA was found in addition to the ~1.7 kb DNA band, which demonstrated that pGAP2(218).pac was maintained in puromycin-resistant
G. lamblia clones.
For each gap2-luc construct, 2 independent transfectants were selected and their luciferase activities were measured. After establishing them as stable clones, the transfectants were induced to encystation by cultivating them in TYI-S-33 medium with high bovine bile at alkaline pH. At 5 different time-points of postencystation, i.e, 12, 24, 36, 48, and 60 hr, portions of encysting cells were harvested.
In the trophozoite form, the 3
gap2-
luc fusions displayed differential luciferase activities (
Fig. 2C). Transfectants carrying pGAP2(43).pac showed lower luciferase activities than those of trophozoites carrying 1 of the other 2 constructs, pGAP2(112).pac and pGAP2(218).pac. Trophozoites of
G. lamblia carrying pGAP2(112).pac showed 40% more luciferase activity than those carrying pGAP2(43).pac. Trophozoites carrying pGAP2(218).pac showed the highest luciferase activity, namely, over 3 fold higher than that of
Giardia trophozoites containing pGAP2(43).pac.
Upon encystation, Giardia containing these 3 constructs showed significant increases in luciferase activity, even though the fold increases were different for each construct. Transfectants carrying pGAP2 (43).pac, also showed higher luciferase activities during encystation than that of trophozoites, i.e., gradual increase of up to 3-fold. Giardia containing pGAP2 (112).pac also showed a gradual increase in luciferase activity as cells proceeded to encystation (20-fold). G. lamblia carrying pGAP2(218).pac also displayed a dramatic increase in luciferase activity upon encystations; a 13-fold increase at 60 hr post-induction.
DISCUSSION
Despite the putative role of glyceraldehydes 3-phosphate dehydrogenase implied from the amino acid sequences of the
gap2 ORF, no enzymatic activity was demonstrated in the previous study (
Yang et al., 2002). To identify the role of this putative
gap gene, we confirmed encystation-induced expression of the
gap2 using a luciferase reporter system. There is no information on the
cis-acting elements required for transcription in
G. lamblia at the present level of knowledge. Therefore, we randomly constructed 3
luc fusions containing different sizes of the
gap2 promoter, i.e., 43, 112 and 218-bps.
When the constructed plasmids were transfected into
G. lamblia, we found that the efficiency of transfection was very low, i.e., below 10
-7. Therefore, an experiment to detect the presence of the
gap2-
luc reporter in tranfected
G. lamblia was performed to exclude a possibility that we could select puromycin-resistant clones of
G. lamblia during transfection (
Fig. 2B).
Luciferase activities of 3 different
gap2-
luc fusions clearly showed that 43-bp upstream region of the initiation codon of the
gap2 ORF did not contain all the essential elements required for expression of the
gap2 gene (
Fig. 2C). In addition, the 112-bp upstream region of the
gap2 gene seemed to have
cis-acting information for the full induction of this gene during encystation.
In this study, we constructed a reporter system in G. lamblia, which allows the quantitative measurement of the expression of a prescribed gene. This system was applied to monitor the expression of gap2, which was identified as one of the genes induced during encystation. Using reporter constructs containing different regions of the gap2 promoter, we defined the cis-acting elements required for full induction of the gap2 gene during encystation.
Notes
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This work was supported by the Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund) (R04-2003-000-10023-0).
ACKNOWLEDGMENTS
The authors are thankful to Dr. Singer (Department of Parasitology, Georgetown University, Washington DC, U.S.A.) for donating pGFP.pac and his tips concerning the transfection of G. lamblia.
References
Fig. 1Construction of pLUC.pac. The
gfp gene of a shuttle vector of
E. coli and
G. lamblia, pGFP.pac (
Singer et al., 1998), was replaced by a 1,682-bp
luc DNA fragment of pGL2-Basic.
Fig. 2Expression of the gap2 gene in G. lamblia. A: Construction of plasmids containing gap2-luc fusions. Promoter regions used for plasmid construction are indicated as lines. For each construct, the locations and names of primers, are also presented as arrowed lines, B: Southern blot analysis of G. lamblia DNA transfected with pGAP2(218).pac containing the 32P-labeled gap2 promoter region. Lane 1, DNA of untransfected Giardia; lane 2, DNA of Giardia containing pGAP2(218).pac. Genomic DNAs were digested with HindIII, and separated by 0.8% agarose gel electrophoresis, C: Determination of luciferase activities of G. lamblia using one of the gap2-luc fusions during encystation. Luciferase activities of G. lamblia carrying one of 3 plasmids, pGAP2(43).pac (open bars), pGAP2(112).pac (gray bars), or pGAP2(218).pac (closed bars) were monitored at various time-points after encystation induction (0, 12, 24, 36, 48, and 60 hr). Luciferase activities were expressed as percentages of the RLU (relative light unit) value of trophozoites containing pGAP2(43).pac.
Table 1.Oligonucleotides used in this study
Table 1.
|
Primer name and nucleotide sequences |
|
Replacement of the gfp gene by the luc gene |
|
LUC-F: 5´-GTAACCATGGCATTCCGGTACTGTTG-3´- NcoI site underlined |
|
LUC-R: 5´-GAATGCGGCCGCATTTTACAATTTGGACTTTCC-3´- NotI site underlined |
|
|
Construction of gap2-luc fusions |
|
gap2P-F1: 5´-CCCAAGCTTGCGTAGATCTCCTCCACGGA-3´- HindIII site underlined |
|
gap2P-F2: 5´-GCGCAAGCTTCGCTCCAGCGTTTCTCTTG-3´ - HindIII site underlined |
|
gap2P-R: 5´-GCGCCCATGGCTAATTAGAGTGTTTATTTC-3´ - NcoI site underlined |
|
gap2P-F3: 5´-AGCTTATTACACTAAAACAGGTTGGGGAAATAAACACTCTAATTAGC-3´ - NcoI site underlined and HindIII site double-underlined |
|
gap2P-R3: 5´-CATGGCTAATTAGAGTGTTTATTTCCCCAACCTGTTTTAGTGTAATA-3´ - NcoI site underlined and HindIII site double-underlined |
