Cryptosporidium parvum: Functional Complementation of a Parasite Transcriptional Coactivator CpMBF1 in Yeast

Experimental Parasitology 96, 195–201 (2000) doi:10.1006/expr.2000.4574, available online at http://www.idealibrary.com on

Cryptosporidium parvum: Functional Complementation of a Parasite Transcriptional Coactivator CpMBF1 in Yeast1

Guan Zhu,*,†,2 Michael J. LaGier,* Susumu Hirose,‡ and Janet S. Keithly* *Wadsworth Center, New York State Department of Health, P.O. Box 22002, Albany, New York 12201-2002 U.S.A.; †Department of Veterinary Pathobiology, Texas Veterinary Medical Center, Texas A&M University, College Station, Texas 77843-4467 U.S.A.; and ‡Department of Developmental Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan

Zhu, G., LaGier, M. J., Hirose, S., and Keithy, J. S. 2000. Cryptosporidium parvum: Functional complementation of a parasite transcriptional coactivator CpMBF1 in yeast. Experimental Parasitology 96, 195–201. We report here the identification of a novel multiprotein bridging factor type 1 from the apicomplexan Cryptosporidium parvum (CpMBF1), one of the opportunistic pathogens in AIDS patients. In slime molds, insects, and humans, MBF1-regulated systems have been associated with cell differentiation, which indicates that CpMBF1 could be responsible for the activation of similar systems in C. parvum during its complex life cycle. Because of the difficulties and high cost in obtaining sufficient and purified C. parvum material for molecular and biochemical analyses, well-characterized yeast genetic systems may be useful for investigating the functions of C. parvum genes. In this study, the function of CpMBF1 as an interconnecting element between a DNAbinding regulator and TATA-box-binding protein (TBP) was confirmed using a yeast complementation assay. Under conditions of histidine starvation, an MBF1-deficient strain of Saccharomyces cerevisiae was unable to activate the HIS3 gene, which encodes imidazoleglycerolphosphate dehydratase (IGPDH), and thus became sensitive to 3-amino triazole, an inhibitor of this enzyme. Upon introduction of parasite CpMBF1 into S. cerevisiae, 3-amino triazole resistance of the MBF1deficient strain was restored to wild-type levels, and Northern blot analysis revealed that CpMBF1 was able to activate HIS3 transcription in response to histidine starvation. 䉷 2000 Academic Press Index Descriptors and Abbreviations: Cryptosporidium parvum; multiprotein bridging factor 1 (MBF1); transcriptional coactivator; yeast; complementation; 3-amino triazole (3-AT); expressed sequence tag (EST); genomic sequence survey (GSS); open reading frame (ORF);

polymerase chain reaction (PCR); reverse transcription-polymerase chain reaction (RT-PCR).

INTRODUCTION

The protist Cryptosporidium parvum (Apicomplexa) is a parasite infecting both humans and animals. Its pathogenesis starts with the invasion of sporozoites into intestinal epithelial cells following ingestion of oocysts by a host. Within host cells and just underneath epithelial cell membranes, this parasite begins a complex life cycle, which typically includes two asexual stages of multiplication and one sexual stage of gametogony to form a new generation of oocysts (Fayer et al. 1997). The oocysts rapidly initiate sporulation and a percentage of them undergo excystation, releasing infectious sporozoites within the intestinal lumen of the same host. This autoinfection is a contributing factor to prolonged disease in AIDS patients, which can be life-threatening since there is no effective treatment for cryptosporidiosis (Tzipori 1998). The importance of C. parvum has recently been recognized by both government agencies and the global scientific community, resulting in a significant increase in studies on the biology of this parasite. With the rapid advancement in the fields of molecular biology and biochemistry, the search

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The sequence data reported herein have been submitted to GenBank and assigned the Accession No. AF247975. 2 To whom correspondence should be addressed. Fax: (979) 8459972. E-mail: [email protected].

0014-4894/00 $35.00 Copyright 䉷 2000 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Alignment of MBF1 proteins from C. parvum and other eukaryotes. Identical amino acids between C. parvum and other species are highlighted in black, while other conserved residues (identical among six or more species) are in gray. Dots represent gaps introduced to achieve optimal alignments. Above the alignment, triangles indicate aa conserved among all species, and the open arrow at Asp125 indicates the TBP-binding site in yeast. Nucleotide sequences encoding these proteins may be obtained from GenBank using accession Nos. AF247975 (C. parvum), AI759422 (Eimeria tenella), AW053826 [Mesembryanthemum crystallinum (ice plant)]; Z49698 (Ricinus communis); AI856508 (Glycine max); AW057095 (Zea mays); AW010338 (Pinus taeda); AU029264 (Oryza sativa); AL035536 (Schizosaccharomyces pombe); AB017593 (Saccharomyces cerevisiae); AL114245 (Botryotinia fuckeliana); AF132156 (Drosophila melanogaster); AB001078 (Bombyx mori); and AB002282 (Homo sapiens).

for drug targets has led to the identification and characterization of enzymes or metabolic pathways unique to C. parvum (Barnes et al. 1998; Doyle et al. 1998; Entrala and Mascara 1997; Keithly et al. 1997; Spano et al. 1998; Vasquez et al. 1996; Zhu et al. 1999, 2000). Recent expressed sequence tag (EST) and random genomic sequence survey (GSS) projects have resulted in the discovery of numerous new genes in this parasite (Liu et al. 1999; Strong and Nelson 2000). However, when compared with other parasitic protists, including the apicomplexans Toxoplasma gondii and Plasmodium falciparum, both the limited supply of purified parasite material and the lack of a transfection system restrict sophisticated functional analyses of genes and proteins in C. parvum (Coombs 1999). Multiprotein bridging factor type 1 (MBF1) is a transcriptional coactivator that connects DNA-binding regulators and the TATA-box-binding protein (TBP), the function of which is to activate amino acid synthetic pathways in eukaryotic cells (Takemaru et al. 1997, 1998). Under histidine starvation, the yeast Saccharomyces cerevisiae undergoes a GCN4/

MBF1-dependent activation of transcription both for de novo synthesis of this amino acid and for the HIS3 gene encoding imidazole glycerol-phosphate dehydratase (IGPDH). Although MBF1-deficient yeast cells can grow slowly without histidine, this growth relies solely upon the basal level expression of HIS3 genes and is sensitive to inhibition by amino-triazole (3-AT), an IGPDH-specific inhibitor (Hope and Struhl 1986; Takemaru et al. 1997, 1998). Therefore, any deficiency in yeast cells of HIS3 gene function, or its activation, can be detected by cultivation in 3-AT histidinedropout medium. Here we describe a novel gene encoding MBF1 in C. parvum (CpMBF1), the function of which as a transcriptional coactivator has been confirmed by complementation of an MBF1-disruptant S. cerevisiae strain (⌬mbf1) using both a 3-AT sensitivity assay and Northern blot analysis. This study also demonstrates that a C. parvum protein can effectively restore this cellular function in yeast, thus suggesting that S. cerevisiae genetic systems may be useful for elucidating C. parvum gene functions.

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Cryptosporidium parvum TRANSCRIPTIONAL COACTIVATOR CpMBF1

FIG. 1.—Continued

MATERIALS AND METHODS Nucleic acid methods. An MBF1 homologue was identified in the genome of C. parvum as a part of our search for novel drug targets. The CpMBF1 gene is located a short distance downstream of a P-type ATPase gene (CpATPase2, GenBank Accession No. AF134591). A 444-bp complete open reading frame (ORF) of CpMBF1 was obtained within a DNA fragment derived from a C. parvum (KSU-1 strain) pBluescript SK(⫹) genomic DNA library. Both strands of the fragment were sequenced to confirm identity. The transcription of CpMBF1 in C. parvum sporozoites (Iowa strain) and intracellular stages (KSU-1 strain) was analyzed by a 33-cycle reverse transcription-polymerase chain reaction (RT-PCR). In addition to standard RT-PCR reagents specified by the manufacturer for the Access RT-PCR kit (Promega Co., Madison, WI), each reaction also included 10 ng total RNA isolated from parasites or HCT-8 cells using the RNeasy Mini kit (Qiagen Inc., Valencia, CA) as previously described (Zhu and Keithly 1997) and a pair of CpMBF1-specific primers (5⬘ AGT-CAG-GAT-TGG-ATT-CAA-GTG 3⬘ and 5⬘ CATCTG-GAC-ATC-TTT-TGA-ACC 3⬘). Automated sequencing and oligonucleotide synthesis were conducted by the staff of Molecular Genetics Core Facility at the Wadsworth Center. Sequence analyses were performed using the Unix version of GCG Wisconsin package v10 (Genetic Computing Group, Madison, WI). Yeast strains and plasmids. An mbf1-deficient yeast mutant (⌬mbf1) has been generated from wild-type (WT) KT130 strain (trp1⌬1 ura3-52 leu2-P1) by gene replacement of yeast MBF1 with a LEU2 selectable marker (Takemaru et al. 1998). To express CpMBF1 in the ⌬mbf1 strain, the complete CpMBF1 ORF was PCR-amplified from

C. parvum DNA using Pfu DNA polymerase and a pair of primers (5⬘ ccg-aat-tcc-ATG-AGT-CAG-GAT-TGG-ATT-CAA-G 3⬘ and 5⬘ ccgaat-tcT-GTT-AAT-CAT-TAT-TAT-TAT-TAT-C 3⬘; letters in lower case indicate artificially added EcoRI linkers). Amplicons were digested with EcoRI to release cohesive ends and were then ligated into an EcoRI-linearized pYES2 vector containing a URA3 selectable marker (Invitrogen Co., Carlsbad, CA). After transformation into Escherichia coli XL10-Gold ultracompetent cells (Stratagene Inc., La Jolla, CA), plasmids were isolated from several transformants and were sequenced to confirm the orientation and identity of the CpMBF1 insert within pYES2. Plasmids containing correctly oriented CpMBF1 were transformed into yeast ⌬mbf1 competent cells using a frozen-EZ Transformation II kit (Zymo Research, Orange, CA). The subsequent strain ⌬mbf1/CpMBF1 was obtained by selecting yeast transformants on agar plates containing a uracil-free synthetic medium (Sigma Chemical Co., St. Louis, MO). For the purpose of quality control, the genotype of yeast MBF1 (ScMBF1) was reconfirmed by PCR in WT, ⌬mbf1, and ⌬mbf1/CpMBF1 using two pairs of primers specific for ScMBF1: 5⬘ AGC-ATC-AAC-GAC-AAC-AAC-GAG 3⬘ and 5⬘ CGA-TCCATC-TAC-CAC-CAG-AAC 3⬘, as well as 5⬘ TCT-CAA-GGC-CAAATT-AAT-GCT-G 3⬘ and 5⬘ AGG-CGA-ACC-GAT-GTT-GTT-AC 3⬘. Complementation assays. Since MBF1 mediates GCN4-dependent transcriptional activation in yeast, the ⌬mbf1 strain under conditions of histidine starvation is sensitive to 3-AT, which inhibits IGPDH encoded by the HIS3 gene (Hope and Struhl 1986; Takemaru et al. 1997, 1998). This 3-AT sensitivity in ⌬mbf1 can be overcome if the yeast (or silkworm) MBF1 gene is reintroduced (Takemaru et al. 1997). In this experiment, the functional complementation of CpMBF1 in yeast ⌬mbf1/CpMBF1 strain was evaluated for its sensitivity to 20 mM 3-AT and compared to both ⌬mbf1 and WT KT130 (mbf1*) strains in a histidine-free synthetic medium. Low-level expression of

198 CpMBF1 from the pYES2 vector was maintained using 20% raffinose as a carbon source, which does not repress or induce transcription from the GAL1 promoter in pYES2. All three strains were cultured in histidine-free medium for ⬎5 passages to deplete histidine before testing for 3-AT sensitivity. Yeast grown on agar plates was incubated with 20 mM 3-AT at 30⬚C for 7 days or for 3 days without 3-AT. Northern blot analysis. To directly test whether CpMBF1 could activate transcription of the yeast HIS3 gene in the presence of 3-AT, the level of transcription of both the HIS3 and the CpMBF1 genes was assessed by Northern blot analysis. Briefly, total RNA was isolated from yeast treated with 20 mM 3-AT for 6 h at 30⬚C using the RNeasy isolation kit (Qiagen, Santa Clarita, CA), fractionated in a 1.2% agarose/ formalin gel (5 ␮g RNA per lane), and blotted onto a Zeta-Probe nylon membrane using an alkaline-transfer protocol as suggested by the manufacturer (Bio-Rad, Hercules, CA). The yeast DED1 gene, which is not dependent upon regulation by GCN4/MBF1, was used as a control. The relative amount of transcription of the HIS3 gene in all three strains was determined by densitometric measurements of the Northern blots normalized using DED1, which was performed on a Macintosh Power PC using the public domain NIH Image v1.62 program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). All three probes were amplified by PCR from yeast or C. parvum DNA using the following pairs of oligonucleotides: HIS3, 5⬘ CAT-AGA-CGA-CCATCA-CAC-CAC 3⬘ and 5⬘ CTT-GCC-TCG-CAG-ACA-ATC-AAC 3⬘; CpMBF1, 5⬘ AGT-CAG-GAT-TGG-AAT-CAA-GTG 3⬘ and 5⬘ CATCTG-GAC-ATC-TTT-TGA-ACC 3⬘; and DED1, 5⬘ AGC-ATC-AACGAC-AAC-AAC-GAG 3⬘ and 5⬘ CGA-TCC-ATC-TAC-CAC-CAGAAC 3⬘. All probes were labeled by a 5-cycle PCR method in a standard reaction solution containing 20 ␮Ci of [␣-32P] dATP. Blots were hybridized overnight at 42⬚C in 7% SDS/0.5 M NaH2PO4/1 mM EDTA (pH 7.2), washed twice at 55⬚C for 30 min with 5% SDS/40 mM NaH2PO4/1 mM EDTA (pH 7.2), and exposed to a BioMax film (Kodak, Rochester, NY) at ⫺80⬚C for 7 days (DED1) or 21 days (HIS3 and CpMBF1). Film images were digitized using an AlphaImager (Alpha Innotech Co., San Leandro, CA) and documented using Photoshop 5.5 and Illustrator 8.0 software packages (Adobe Systems, Inc., San Jose, CA).

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residue (superscript indicates residual position within the alignment in Fig. 1), which is known to be critical for the function of TBP binding during yeast transcriptional activation (Takemaru et al. 1998; Ozaki et al. 1999). As expected, 377-bp products were observed by RT-PCR only in reactions containing parasite RNA (Fig. 2, lanes 5 and 7), thus confirming parasite-specific expression of CpMBF1 in both sporozoites and intracellular stages. As mentioned previously, MBF1 mediates the GCN4dependent activation of yeast histidine-biosynthetic genes including HIS3, which encodes enzyme IGPDH. Mutants lacking either MBF1 or GCN4 are sensitive to 3-AT, an inhibitor of IGPDH (Hope and Struhl 1986; Takemaru et al. 1997, 1998). In this study, the CpMBF1 gene was successfully cloned into the yeast expression vector pYES2 and then was introduced into the yeast ⌬mbf1 strain. The 3-AT sensitivity of transformed ⌬mbf1/CpMBF1 was assayed and compared with that of the parenal ⌬mbf1 and the WT (KT130) strains in a histidine-dropout medium with raffinose as a carbon source. By relying upon a basal level of expression of histidine biosynthetic genes, all three yeast strains were able to grow on plates containing the synthetic medium without histidine (Fig. 3a). The WT strain was resistant to inhibition by 20 mM 3-AT due to its capacity to overexpress the HIS3 gene, whereas the ⌬mbf1 strain was highly sensitive to 3AT due to a deficiency of MBF1 (Fig. 5b). However, by acquiring CpMBF1, the ⌬mbf1/CpMBF1 strain was able to

RESULTS AND DISCUSSION

MBF1 mediates transcriptional activation of various genes in eukaryotes by interconnecting a TATA-binding protein with sequence-specific regulators. This evolutionarily conserved protein has been previously found in yeast, insects, humans, and plants (Takemaru et al. 1997) and is known to differentially activate or down-regulate gene transcription during development. Here we describe an MBF1 gene from the parasitic protist C. parvum (CpMBF1) containing a 444bp ORF of 147 amino acids that shares the greatest degree of homology with other eukaryotic MBF1 proteins (Fig. 1). Like these proteins, CpMBF1 is a highly charged protein with a predicted pI of 10.7 that also contains an Asp125

FIG. 2. RT-PCR showing CpMBF1 transcription in uninfected HCT-8 cells (lanes 2 and 3), C. parvum-infected cells 48 h postinfection (lanes 4 and 5), and excysted sporozoites (lanes 6 and 7). Reactions without RNA (lane 1) or reverse transcriptase (lanes 2, 4, and 6) were included as additional negative controls. CpMBF1 cDNA was amplified from both samples containing C. parvum RNA with an expected size at 377 bp (lanes 5 and 7), but not from negative controls. Sizes of DNA ladders (L) are indicated on the right.

Cryptosporidium parvum TRANSCRIPTIONAL COACTIVATOR CpMBF1

FIG. 3. Growth of yeast strains in a synthetic medium without histidine in the absence (a) or in the presence (b) of the inhibitor 20 mM 3-AT. Cells of WT, ⌬mbf1, and the transformed ⌬mbf1/CpMBF1 were grown at 30⬚C for 3 days (a) or 7 days (b). Functional complementation of yeast by CpMBF1 is measured by the lack of 3-AT sensitivity in ⌬mbf1/CpMBF1.

grow at a rate similar to that of WT even in the presence of 3-AT (Fig. 3b). This growth rate in the presence of drug is similar to that noted for the ⌬mbf1 yeast transformed with WT ScMBF1 (Takemaru et al. 1998), suggesting that HIS3 has been activated in response to histidine starvation. Since the expression of cloned genes in vector pYES2 can be either induced by galactose or suppressed by glucose, the phenotypes of the three yeast strains (WT, ⌬mbf1, ⌬mbf1/ CpMBF1) were also determined using these two sugars as a carbon source. As expected, all three yeast strains cultivated in galactose showed similar rates of growth and sensitivity (or resistance) to 3-AT as did those cultivated in raffinose. Unexpectedly, however, yeast MBF1 strains became much less sensitive to the inhibition of 3-AT when grown in glucose (data not shown). It is as yet unclear whether this “glucose effect” is due to an unknown metabolic mechanism in the yeast or to universal histidine contamination of glucose, since this sugar, purchased from three manufacturers, yielded identical results. Unlike WT yeast, PCR genotyping reconfirmed that the

FIG. 4. Genotypes of the three yeast strains. The presence of ScMBF1 in WT, as well as the absence of ScMBF1 in both ⌬mbf1 and ⌬mbf1/CpMBF1 strains, was confirmed by PCR using two pairs of ScMBF1-specific primers (lanes 2 and 3). A negative control without DNA (lane 1) and a positive control using DED1 primers (lane 4) were included in all experimental groups.

199 yeast ScMBF1 was not present in ⌬mbf1/CpMBF1 (Fig. 4). Therefore, the activation of the HIS3 gene in ⌬mbf1/ CpMBF1 is most probably due to the newly acquired CpMBF1 gene. To test this hypothesis, the expression levels of both HIS3 and CpMBF1 genes in the presence of 3-AT were evaluated by Northern blot analysis. The ⌬mbf1 3-ATsensitive strain expressed a low (basal) level of the HIS3 gene (relative amount ⫽ 59), whereas both the 3-AT-resistant WT and ⌬mbf1/CpMBF1 strains expressed HIS3 at levels significantly higher than that of the ⌬mbf1 strain (relative amounts ⫽ 100 and 82, respectively) (Fig. 5). These data are consistent with the drug-screening data. In controls, all three yeast strains expressed similar levels of the DED1 gene, which is GCN4- and MBF1-independent (Fig. 5a). Therefore, the C. parvum factor CpMBF1 expressed in transformed ⌬mbf1/CpMBF1 yeast was able to activate HIS3 gene transcription and reverse 3-AT sensitivity. This confirms its function as a transcriptional cofactor. It is not yet

FIG. 5. Northern blot analysis revealed the correlation between the expression level of HIS3 and 3-AT sensitivity among the three yeast strains. The transcription of the yeast DED1 gene, which is not dependent upon regulation by GCN4/MBF1, was used as a control (a). Under conditions of histidine starvation, both WT and ⌬mbf1/CpMBF1 expressed significantly higher levels of HIS3 than ⌬mbf1 (b). The activation of HIS3 in ⌬mbf1/CpMBF1 is correlated with the expression of CpMBF1 (c). The relative levels of HIS3 transcription were determined by densitometric measurements of blots normalized using DED1 (d).

200 known, however, from the Northern blot analysis presented here, whether the initiation of transcription for CpMBF1 is at position ⫹13, which is typical for most GCN4/MBF1dependent HIS3 genes or at an atypical site since yeast may also transcribe HIS3 from positions ⫹1 or ⫹22 (Takemaru et al. 1998). In addition to the GCN4-dependent activation of HIS3 gene in WT yeast, MBF1 homologues are also known to mediate the following processes: (1) developmentally regulated activation of the division protein FTZ-F1 in Drosophila melanogaster and Bombyx mori (Takemaru et al. 1997); (2) down-regulation of H7 in Dictyostelium discoideum, during stalk formation (Singleton et al. 1991); (3) down-regulation of EDF-1 in humans during endothelial cell differentiation (Dragoni et al. 1998), and (4) activation of Ad4BP/SF-1 (a mammalian counterpart of FTZ-F1) and ATF1 (Kabe et al. 1999). All of these observations suggest that CpMBF1 might play a role in transcriptional activation for the differential development of meronts, merozoites, and/ or gametes during the life cycle of C. parvum. Additional experiments would be necessary to test this hypothesis. C. parvum is a parasitic protist causing one of the opportunistic infections in AIDS patients. Since there is no effective treatment against cryptosporidiosis, this disease can be lifethreatening in immunosuppressed persons. To date, little is known about the regulation and mechanism of gene transcription in this parasite. If CpMBF1-mediated systems are essential for the survival and/or development of C. parvum, the yeast complementation system described here could be used for high-throughput screening of potential CpMBF1specific inhibitors. Furthermore, CpMBF1-associated proteins and pathways may represent a new group of molecular targets for drug-development against this parasite.

ACKNOWLEDGMENTS

We thank Ms. Mary J. Marchewka at the Wadsworth Center, NYS DOH, for her technical assistance, and the staff of the Molecular Genetic Core Facility for the synthesis of oligonucleotides and automatic sequencing.

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