1 Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA
2 Faculty of Genetics Program, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA
Correspondence
Guan Zhu
Gzhu{at}cvm.tamu.edu
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ABSTRACT |
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INTRODUCTION |
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Multiprotein bridging factor type 1 (MBF1) was first identified in Drosophila as a mediator of the transcriptional activator FTZ-F1 and found to be evolutionarily conserved among a wide range of eukaryotic organisms including humans, animals, plants, insects, fungi and protists (Kabe et al., 1999; Liu et al., 2003
; Takemaru et al., 1997
, 1998
; Zhu et al., 2000
). MBF1 acts as a transcriptional co-activator that recruits DNA-binding regulators and TATA-binding protein (TBP) for the conditional or differential activation of gene expression. Under the condition of histidine starvation in Saccharomyces cerevisiae, MBF1 is responsible for the GCN4-dependent activation of transcription for the de novo synthesis of this amino acid (Takemaru et al., 1998
). Although MBF1-deficient yeast cells can grow slowly without histidine, this growth relies solely upon the basal level expression of histidine synthetic genes and is sensitive to 3-amino-1,2,4-triazole (3-AT), an inhibitor of imidazoleglycerol-phosphate dehydratase encoded by the HIS3 gene. We have previously identified a C. parvum MBF1 gene (CpMBF1) and demonstrated that CpMBF1 could functionally restore the 3-AT-resistant phenotype in an MBF1-deficient S. cerevisiae strain when introduced and expressed in this yeast (Zhu et al., 2000
).
In this study, we expressed the CpMBF1 protein in Escherichia coli as a maltose-binding protein (MBP) fusion protein and demonstrated the interaction of CpMBF1 with a recombinant C. parvum TBP (CpTBP1) that was recently identified from the parasite's genome. Using semi-quantitative RT-PCR and Western blotting analysis, we found that the CpMBF1 gene was differentially expressed in association with the parasite cell cycle. We also characterized the DNA-binding property of the CpTBP1 protein and found that the CpTBP1 gene was also differentially expressed during the complex parasite life cycle, but in a different pattern in comparison to that of CpMBF1.
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METHODS |
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Semi-quantitative RT-PCR.
This was performed similar to a previously described protocol (Abrahamsen & Schroeder, 1999). However, the quantity of products was densitometrically (rather than radioactively) measured in agarose gels. Total RNA was isolated from C. parvum oocysts, free sporozoites and intracellular stages developed in HCT-8 cells in vitro for various times (372 h) using RNeasy kit (Qiagen). All RNA samples were subject to intensive RNase-free DNase digestion until no products could be amplified by PCR. Since RNA samples isolated from intracellular parasite were mixed with host and parasite RNA, all samples were first normalized using C. parvum 18S rRNA by a semi-quantitative RT-PCR with a pair of previously described primers (i.e. 995F and 1206R) (Abrahamsen & Schroeder, 1999
). Twenty thermal cycles were employed so that the densities of RT-PCR amplicons could be measured within linear ranges in agarose gels. The RNA concentrations from various samples were adjusted to produce comparable amounts of C. parvum 18S rRNA amplicons by RT-PCR. The adjusted amounts of total RNA were then used for RT-PCR amplification of CpMBF1 and CpTBP1 transcripts by 23 thermal cycles using appropriate primer sets, i.e. CpMBF1-004F (5'-AGT CAG GAT TGG AAT CAA GTG-3') and CpMBF1-380R (5'-CAT CTG GAC ATC TTT TGA ACC-3') for CpMBF1 and CpTBP1-F001 (5'-ATG AGT GAT GAC ATA CTA AGT TC-3') and CpTBP-750R (5'-TTA CTT TCT GTA TTG ACA AAG AA-3') for CpTBP1, respectively. The density of each amplicon was determined using GENETOOLS software v. 3.00.22 (Hitachi Software Engineering) and its relative level was normalized by comparison with that of 18S rRNA control. All experiments were repeated in triplicate.
Production of polyclonal antibody and immunolabelling of CpMBF1.
A short peptide (CpMBF1-1118, 11KKGGSRPKGISKEQDLNQ18) was synthesized by the peptide synthesis facility at the Department of Veterinary Pathobiology, Texas A&M University. The short peptide was freshly cross-linked to keyhole limpet haemocyanin (Sigma) prior to each immunization. Polyclonal antibodies to CpMBF1 were raised in a specific pathogen-free rabbit that was initially immunized with 200 µg of antigen. Booster immunizations (100 µg) were performed later at 30 and 60 days, respectively. Rabbit serum was collected prior to and after the immunization protocol.
C. parvum was grown in vitro as described previously (Hijjawi et al., 2001), except that, after inoculation into human HCT-8 cells (ATCC CCL-244), cultures were kept in a 5 % CO2 incubator and the maintenance medium contained 5 % fetal calf serum. HCT-8 cells were grown on poly-L-lysine-treated glass coverslips for 24 h in maintenance medium prior to infection with parasites. Freshly excysted sporozoites (at a ratio of one sporozoite per 10 cells) in medium were added to host cell monolayers and incubated at 37 °C for 4 h, at which time point the medium was replaced. Subsequent medium changes were carried out every 24 h. Infected and uninfected samples were fixed in a 10 % formalin solution balanced with PBS for 10 min at 2, 4, 8, 12, 16, 24, 48 and 72 h post-infection (p.i.). In addition to the parasites in cultured cells, intact oocysts and free sporozoites were prepared, fixed with formalin, applied onto the poly-L-lysine-treated coverslips and air-dried for immunofluorescence microscopy analysis. Immunolabelling of the CpMBF1 protein in C. parvum extra- and intra-cellular life cycle stages was performed as described previously, with slight modifications (Millership & Zhu, 2002
; Zhu & Keithly, 1997
). Fixed cells were extracted with 50 % methanol/50 % acetone (v/v) for 5 min at 20 °C. Samples were rehydrated with water and then PBS. Following blocking in 10 % fat-free dried milk in PBS for 1 h at room temperature, the samples were incubated with affinity-purified primary antibodies in PBS containing 3 % milk for 1 h at room temperature and treated with an FITC-conjugated monoclonal antibody against rabbit IgG (Sigma Chemical). Wash steps between stages were accomplished with PBS containing 0·05 % Tween 20. Slides were mounted using a SlowFade Light Antifade mounting medium (Molecular Probes) and examined using an Olympus BX51 Epi-Fluorescence microscope system equipped with FITC/TRITC filters.
Expression of recombinant CpMBF1 and CpTBP1 proteins.
The sequence of the CpMBF1 gene, which encodes 147 aa with an estimated molecular mass of 16·4 kDa, has been described previously (Zhu et al., 2000). The CpTBP1 gene was newly identified from the C. parvum genome project (University of Minnesota, MN, USA) using various eukaryotic TBPs as queries (Fig. 1
). The entire open reading frames (ORFs) of CpMBF1 and CpTBP1 were amplified from C. parvum gemonic DNA using a high-fidelity Pfu DNA polymerase (Stratagene) with primer sets CpMBF1-F01 (5'-tcg aat tcA TGA GTC AGG ATT GGA ATC AA-3') and CpMBF1-R01 (5'-tcc gaa ttc TTA ATC ATT ATT ATT ATT ATC AGG-3'), and CpTBP1-F01 (5'-gcg aat tcA TGA GTG ATG ACA TAC TAA GTT C-3') and CpTBP1-R01 (5'-gcg aat tcT TAC TTT CTG TAT TGA CAA AGA A-3'), respectively (lower-case letters represent artificially added EcoRI linkers). The PCR amplicons were cloned into a pMAL-c2x vector (New England Biolabs) and transformed into bacterial competent cells. After PCR-screening of bacterial transformants to confirm the presence of inserts and their orientation, plasmids were prepared from selected colonies and sequenced. The plasmids containing correctly oriented CpMBF1 or CpTBP1 sequences were transformed into E. coli Rosetta cells (Novagen). The IPTG-induced expression and amylose resin-based purification of MBP fusion proteins were performed according to the manufacturer's instructions (New England Biolabs).
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The interaction between CpMBF1 and CpTBP1 was assayed by incubating 100 ng MBPCpMBF1 or MBP tag with various concentrations of MBPCpTBP1 in 15 mM Tris/HCl buffer at 37 °C for 30 min. The reactions were separated on 6 % native polyacrylamide gels and transferred onto nitrocellulose membranes. Free CpMBF1 and CpTBP1-bound CpMBF1 proteins were labelled using rabbit antiserum against CpMBF1 as primary antibody and horseradish peroxidase-conjugated secondary antibody, followed by a colour development with 3,3'-diaminobenzidine.
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RESULTS |
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CpMBF1 and CpTBP1 are differentially expressed in different manners
Semi-quantitative RT-PCR analysis was performed to measure the relative levels of transcripts during C. parvum life cycle in vitro for both the CpMBF1 and the CpTBP1 gene. After the density of each cDNA amplicon was measured in agarose gels and normalized with respect to the 18S rRNA standard, apparent differential expression patterns were observed for both CpMBF1 and CpTBP1 transcripts. Although CpMBF1 mRNA transcripts were detectable in all parasite life cycle stages, relatively small amounts of transcripts were detected in free sporozoites, early (312 h p.i.) and late (4872 h p.i.) life cycle stages (Fig. 2). However, the transcription of CpMBF1 was much higher during the 24 and 36 h p.i. in comparison to other life cycle stages (Fig. 2
), which typically represented the life cycle stages between first and second generations of merogony. Such an observation is further supported by indirect immunofluorescence microscopy. Using rabbit anti-CpMBF1 antibody to probe various parasite intracellular life cycle stages, fluorescence was detectable only in well-developed meronts or gamonts (Fig. 3
). Little or no CpMBF1 protein was detectable among oocysts, sporozoites and merozoites during their early invasion stages (data not shown). Furthermore, Western blot analysis only detected CpMBF1 protein among intracellular parasite-infected host cells, but not in the protein extracts isolated from intact oocysts and excysted sporozoites (data not shown). The antibody, also, did not cross-react with the host-cell proteins in Western blot analysis (data not shown) or immunofluorescence microscopy (Fig. 3
).
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Interactions of CpTBP1 with CpMBF1 and DNA oligonucleotide
Both CpMBF1 and CpTBP1 were successfully cloned into pMAL-c2x vector and expressed as MBP fusion proteins. Using an amylose resin-based affinity chromatography, both recombinant proteins were purified to homogeneity (Fig. 4). Although full-length recombinant proteins were obtained at their expected sizes, some minor smaller protein fragments were observed among affinity-purified proteins, which might represent partial translation of foreign proteins in bacteria or limited degradation of these fusion proteins during the purification.
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DISCUSSION |
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We have also identified, cloned and expressed the CpTBP1 gene which is essential for all TATA-box-regulated gene transcriptions. This permits the study of the interaction between CpMBF1 and CpTBP1 proteins, as well as that between CpTBP1 and the TATA-box consensus sequence in vitro. Indeed, under appropriate conditions, we observed the formation of CpMBF1CpTBP1 and CpTBP1oligonucleotide complexes using MBP fusion proteins (Fig. 5a), which validated the functions of both proteins in C. parvum. The establishment of the CpTBP1DNA binding assay also has great potential for testing the inhibitory effects of other compounds in vitro. However, the TATA-box consensus sequence used in this study was adopted from other studies and may not represent the true consensus sequence in C. parvum. We are currently scanning the potential TATA-box candidate sequences within the C. parvum intergenic regions for investigating their TBP-binding properties.
CpTBP1 appears to be the only TBP-encoding gene in the parasite, since intensive BLAST searches using TBP homologues failed to identify any other homologues from the nearly complete C. parvum genome sequence. The binding of TBP to the TATA-box that is located upstream of the 5' untranslated region (5'-UTR) initiates the general downstream gene transcription machinery by recruiting a variety of transcription factors including TFIIA, B, E, F, H and J (Kraemer et al., 2001; Ouyang et al., 2000
; Pugh, 2000
; Vasanthi et al., 2003
; Wu & Chiang, 2001
). Most of these factors can be identified from the C. parvum genome (http://cryptodb.org/) (data not shown), suggesting that the general gene transcriptional machinery is conserved between this parasite and other eukaryotes. Although the CpTBP1 gene is differentially expressed during the C. parvum complex life cycle, its expression pattern differs significantly from that of CpMBF1, indicating that other transcriptional activators and/or co-activators in addition to CpMBF1 are present in regulating specific gene transcriptions in this parasite. Based on the transcription level of CpTBP1 (Fig. 2
), it appears that more genes are regulated by transcriptional factors in oocysts than other stages.
Cryptosporidium causes one of the opportunistic infections in AIDS patients for which no effective treatment is yet available (Okhuysen & Chappell, 2002; Tzipori & Widmer, 2000
). The pathogenesis of cryptosporidiosis is associated with the complex life cycle of this pathogen. However, little is known about the gene activation and DNA replication of this parasite. Analysis of TATA-box consensus sequences using model bioinformatics tools and the C. parvum genome sequence may be undertaken to identify potential genes whose activation may be regulated by transcriptional factors. The identification of C. parvum genes regulated by CpMBF1 and other transcriptional factors or co-activators will clearly provide a new direction to study the gene regulation associated with the complex parasite life cycle. If CpMBF1-dependent gene transcriptional activation is proved to be essential for the parasite's intracellular development, this pathway may be pursued as a novel molecular target for the development of drugs against C. parvum or other apicomplexan-based diseases.
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ACKNOWLEDGEMENTS |
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Received 6 November 2003;
revised 18 February 2004;
accepted 18 February 2004.