15 gene in genotype I and II isolates

Molecular & Biochemical Parasitology 119 (2002) 203– 215 www.parasitology-online.com

Expression of the highly polymorphic Cryptosporidium par6um Cpgp40 /15 gene in genotype I and II isolates Roberta M. O’Connor *, Cheleste M. Thorpe, Ana-Maria Cevallos 1, Honorine D. Ward Di6ision of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts Uni6ersity School of Medicine, Box 041, 750 Washington Street, Boston, MA 02111, USA Received 14 August 2001; accepted in revised form 15 October 2001

Abstract The enteric protozoan Cryptosporidium par6um infects intestinal epithelial cells in a wide range of hosts, causing severe gastrointestinal disease. The invasive sporozoite stage most likely attaches to and invades host cells through multiple host receptor/parasite ligand interactions. Preliminary evidence suggests that the glycoprotein products of the Cpgp40 /15 gene, gp40 and gp15, are involved in these interactions. In addition, the Cpgp40 /15 gene that encodes these glycopeptides is highly polymorphic in genotype I isolates, suggesting that the gene products may be subject to immune selection. In this study, we characterized the Cpgp40 /15 gene in a genotype I isolate and compared expression of the Cpgp40 /15 gene in isolates of both genotype. Cpgp40 /15 is a single copy gene in both TU502 (genotype I) and GCH1 (genotype II) isolates. However, Northern blot analysis revealed the presence of two transcripts, 2.3 and 1.5 kb in size, in mRNA from GCH1 as well as TU502-infected Caco-2A cells. Accumulation of the two Cpgp40 /15 mRNAs peaked 12 – 24 h post-infection. Using 3%RACE analysis, three polyadenylation sites were identified 371, 978 and 1002 bp downstream of the GCH1 Cpgp40 /15 stop codon. Two of these polyadenylation sites were also used in TU502. The sequences of the GCH1 Cpgp40 /15 3%untranslated regions (3%UTRs) were identical to genomic sequence and shared 96.7% homology with TU502 3%UTRs. Actinomycin D treatment of GCH1-infected Caco-2A cells followed by Northern blot analysis, revealed that the stability of the 1.5 kb message was considerably greater than that of the 2.3 kb transcript. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium; Cpgp40 /15 ; Polymorphisms; 3%UTRs; Alternate polyadenylation; mRNA stability

1. Introduction The enteric protozoan, Cryptosporidium par6um, first described by Tyzzer in 1912 [1], is now recognized as a Abbre6iations: gDNA, genomic DNA; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; GPI, glycophosphatidyl inositol; ORF, open reading frame; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction; SAP, single amino acid polymorphism; UTR, untranslated region.  Note: Nucleotide sequence data reported in this paper are available in the GenBank™ data base under the accession numbers AY048665, AY048666 and AY048667. * Corresponding author. Tel.: +1-617-636-8437; fax: + 1-617-6365292. E-mail address: [email protected] (R.M. O’Connor). 1 Present address: Departamento de Biologı´a Molecular y Biotecnologı´a, Instituto de Investigaciones Bome´dicas, UNAM, Ciudad Universitaria, Mexico 04510, Mexico.

pathogen of significant medical and economic importance. C. par6um causes gastrointestinal disease, particularly in humans and neonatal ruminants [2,3] and, in immunocompromised hosts, can establish a persistent, life-threatening infection [4–6]. It is a common contaminant of drinking water [7] and has been identified as the causative agent in several large outbreaks of waterborne diarrheal disease [8,9]. Despite the importance of this pathogen, effective therapies to prevent or treat the disease have not been developed [10]. The disease processes caused by C. par6um begin with excystation of sporozoites from the oocyst and subsequent invasion of epithelial cells lining the small intestine [11]. During the first half of the life cycle, the parasite replicates through two rounds of merogony, before undergoing sexual replication to produce oocysts that are either released to the environment or excyst

0166-6851/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 0 1 ) 0 0 4 1 6 - 9

204

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

within the same host to initiate the cycle again. The process of recognition, attachment to and invasion of epithelial cells is probably complex, most likely involving multiple molecular components. Merozoites and sporozoites share many of the same surface and apical complex molecules [12– 17] and thus, any therapies that disrupt the initial invasion, may also prevent merozoite reinvasion as well. Several sporozoite/merozoite antigens have been identified as putative adhesins, based on the ability of antibodies to disrupt infection and/or the ability of the native proteins to bind directly to epithelial cell lines [15–19]. Among these antigens are the glycoproteins gp40 and gp15 [15,20]. These glycoproteins are produced as a single precursor protein from the Cpgp40 /15 gene and subsequently processed into the two component polypeptides by an unknown protease [15,20]. The antigens are O-glycosylated with exposed Gal(b13)GalNAc (Thomsen-Freidenreich (T) antigen) and GalNAc a1-3 (Tn antigen; precursor to the T antigen) determinants [21,22]. Lectins that recognize these determinants block sporozoite attachment [21] and completely and irreversibly ablate sporozoite infectivity for Caco-2A cells [23]. The deduced amino acid sequence of Cpgp40 /15 contains an N-terminal signal sequence and a C-terminal glycophosphatidyl inositol (GPI) anchor site. A striking feature of the gene is the polyserine domain present in the N-terminal region of the mature gp40 polypeptide, which varies in length between geographic isolates. C. par6um isolates can be subdivided into two major genotypes: genotype II isolates, which infect animals and humans and genotype I isolates, which almost exclusively infect humans [24]. Most human infections are caused by genotype I isolates [24– 27]. One of the intriguing aspects of the Cpgp40 /15 gene is that genotype I isolates each carry a polymorphic variant of the Cpgp40 /15 gene found in genotype II isolates [20]. Analysis of Cpgp40 /15 polymorphisms divides genotype I isolates into five subcategories (Ref. [20] and Leav et al., submitted). This observation suggests that an understanding of the biological role of this gene and its products based solely on studies of genotype II organisms may not be relevant to the majority of human infections. However, since genotype I isolates do not readily infect animals that are routinely used for propagation, most of the studies of C. par6um biology have been performed on genotype II isolates. Recently, genotype I isolates have been successfully passaged in gnotobiotic piglets [28]. Subsequently, the genotype I isolate, TU502, was adapted for propagation in calves following passage in gnotobiotic piglets (S. Tzipori, personal communication), enabling more detailed studies of the biology of genotype I isolates. Because of the apparent importance of the Cpgp40 / 15 gene products in attachment and invasion, and the

highly polymorphic nature of the locus in genotype I isolates, the objectives of this study were to characterize the Cpgp40 /15 gene in the ‘model’ genotype I TU502 isolate, to investigate expression of the Cpgp40 /15 gene in GCH1, a genotype II isolate and, when possible, to compare it to expression of the gene in the TU502 isolate. 2. Methods and materials

2.1. Parasite isolates The GCH1 isolate is a genotype II isolate maintained by serial passage through calves [29]. TU502, a genotype I isolate, was initially obtained from a Ugandan patient, passaged in gnotobiotic pigs and then adapted for passage through calves (S. Tzipori, personal communication). TU502 oocysts from an early passage, as well as those obtained from a later (after 9 months) passage, were available for study. Both isolates were obtained from Dr Saul Tzipori, Tufts University School of Veterinary Medicine, Grafton, MA. Before excystation, oocysts were treated with 1.75% sodium hypochlorite for 10 min on ice, then washed with Dulbecco modified Eagle medium (DMEM, Life Technologies, Grand Island, NY) supplemented with 25 mM HEPES and 100 U of penicillin and 100 mg of streptomycin per milliliter. Oocysts were excysted for 2 h at 37 °C in the presence of 0.75% taurocholic acid.

2.2. DNA extraction and genotyping TU502 and GCH1 genomic DNA (gDNA) were isolated from excysted oocysts using the Bio 101 gDNA isolation kit (Quantum Biotechnologies, Carlsbad, CA). The genotypes of the two isolates were confirmed by PCR-RFLP of the COWP [30] and TRAP-C1 [31] loci, using previously described primers and conditions for PCR and restriction enzyme (Rsa-1) digestion.

2.3. Characterization of the TU502 Cpgp40 /15 gene For this experiment, gDNA isolated from the human isolate TU502 was the generous gift of Dr D. Akiyoshi and Dr S. Tzipori, Tufts University School of Veterinary Medicine, Grafton, MA. The complete coding sequence and some 5%UTR sequence was obtained by amplifying the TU502 gDNA with primer 7-F, specific for the sequences 5% of the GCH1 Cpgp40 /15 start codon and primer Stop-R, which matches the end of the GCH1 coding sequence (Table 1). This amplified a single 1144 bp fragment that was cloned into pCRIITOPO (Invitrogen, Carlsbad, CA) and sequenced by the dye-terminator method on a Perkin-Elmer ABI 377 sequencer at the Tufts University School of Medicine Core Facility.

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

2.4. Plasmids pAMC5.1 and pAMC10.1 were generated during cloning and characterization of the Cpgp40 /15 gene [15]. pAMC5.1 contains an 861 bp insert of Cpgp40 /15 sequence, beginning downstream of the polyserine domain cloned into pCR2.1-TOPO (Invitrogen). pAMC10.1 contains an 1177 bp insert which includes the full length Cpgp40 /15 gene and some 5% and 3% untranslated region cloned into pCRII-TOPO. To generate the plasmids pROC1.7 and pROC2.1, the coding sequences for the mature gp40 and gp15 polypeptides were amplified from GCH1 (pROC1.7) and TU502 (pROC2.1) gDNA and cloned into pCRII-TOPO.

2.5. Southern blotting TU502 and GCH1 gDNA were isolated from excysted oocysts using the Bio 101 gDNA isolation kit (Quantum Biotechnologies). The gDNA was digested with EcoRI, HindIII, PstI, SspI and EcoRI + HindIII and the fragments resolved on a 1% agarose gel. The gel was depurinated, denatured and neutralized and then blotted to a nylon membrane (Bright Star Plus, Ambion, Austin, TX) using a Posiblot 30-30 pressure blotter (Stratagene, La Jolla, CA). Because other C. par6um antigens [32] and ESTs [33] contain polyserine tracts, care was taken to design probes containing Cpgp40 /15 sequences without the polyserine domain, in order to obtain the greatest specificity. To probe the GCH1 Southern blots, the 861 bp insert from pAMC5.1 [15] was gel isolated and labeled with 32PdCTP by random priming. For probing blots of TU502 nucleic acids, the TU502 Cpgp40 /15 coding sequence was cut out of plasmid pROC2.1, gel isolated and subsequently digested with AluI. The 644 bp AluI fragment containing sequences downstream of the polyserine domain was gel isolated and labeled by random priming with 32P-dCTP. The blots were hybridized in 0.5 M Na2HPO4, pH 7.0, 10 mM

EDTA, 0.1% BSA and 7% SDS overnight at 60 °C and washed twice in 40 mM phosphate, 0.1% SDS and twice with 20 mM phosphate, 0.1% SDS at the same temperature. Reactive bands were detected by autoradiography.

2.6. mRNA isolation and Northern blotting Caco-2A cells, maintained as described previously [34], were seeded into T75 flasks and allowed to reach 80–90% confluence. Oocysts were bleached and washed as described above and incubated with the Caco-2A cells at a concentration of 1× 106 oocysts per milliliter at 37 °C. At various times post-infection, the cells were lysed in OL1 buffer (Qiagen, Valencia, CA) and mRNA extracted directly using an Oligotex Direct kit (Qiagen). In all experiments, mRNA isolated from uninfected Caco-2A cells was included as a control. mRNAs were resolved on 1% glyoxyl agarose gels and transferred to BrightStar Plus nylon membranes (Ambion) using the Northern-Gly blotting kit (Ambion). The Northern blots were probed as described for the Southern blots, except that hybridization and washing was carried out at 65 °C. The Cpgp40 /15 transcripts from TU502 were detected with a probe that contained the complete coding sequence of the TU502 Cpgp40 /15 gene. GCH1 gp40 and gp15 specific probes were generated by gel isolation of a 400 bp HincII fragment (gp40-specific) and a 200 bp HpaII fragment (gp15 specific) of the pAMC10.1 insert. To confirm that the observed transcripts were in the sense orientation, Northern blots were also probed with an anti-sense RNA probe. To make the RNA probe, pROC1.7 was linearized with XhoI and transcription primed with SP6 polymerase in the presence of 32P-UTP. Hybridization and washing conditions were the same as for the DNA probes, except that they were conducted at 72 °C. To assess loading, the blot was stripped by boiling in 0.1% SDS and re-probed for human b-actin. The actin probe was amplified by PCR from Caco-2A cDNA using primers

Table 1 List of primers used for 3%RACE and PCR procedures 7-F [15] Stop-R Oligo dT-adapter Adapter-R 3%RACE-F TU502/3%RACE-F 3%End-R 2Tran-F 2Tran-R PolyA1-R PolyA2-R PolyA3-R

205

5%-ATGCAAAAATACGTGGACTGGG-3% 5%-CAACACGAATAAGGCTGCAAAGATTGC-3% 5%-GACTCGAGTCGACATCGAT17-3% 5%GACTCGAGTCGACATCGA-3% 5%-AATGAGAACGGAGACTTGG-3% 5%GGCATACCAATGGTGTCT-3% 5%-CGCTCAAAATAATTAACCCGACAA-3% 5%-AGGTTTCATTTTTTTGTCGGGTTGTG-3% 5%-AAGTCCTTGCGATCGTACAAAGCAA-3% 5%-TTTTTTTTTTTTTTTCGTTA-3% 5%-TTTTTTTTTTTTTTTCCTGATA-3% 5%-TTTTTTTTTTTTTTTATCGAA-3%

Sequence 5% of the Cpgp40 /15 gene 3% end of the Cpgp40 /15 coding sequence Used to prime RT reaction for 3%RACE 3%RACE reverse primer GCH1 sequence, 108 bp upstream of the stop codon TU502 sequence, 170 bp upstream of the stop codon 135 bp downstream of the 3rd poly A site 17 bp downstream of 1st poly A site 27 bp upstream of the 2nd poly A site Specific for the 1st poly A site 373 bp from stop codon Specific for the 2nd poly A site 978 bp from stop codon Specific for the 3rd poly A site 1003 bp from stop codon

206

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

described elsewhere [35] and the amplicons labeled with 32 P-dCTP in a random primed reaction.

2.9. Re6erse transcriptase-polymerase chain reaction (RT-PCR)

2.7. Isolation of GCH1 Cpgp40 /15 clones from a gDNA library

Total GCH1 sporozoite RNA from excysted oocysts and total RNA from TU502 infected Caco-2A cells were isolated using the RNeasy kit (Qiagen), DNase I treated and reverse transcribed with oligo dT primers and Stratascript RT (Stratagene). PCR was performed with the forward primers 3%RACE-F or TU502/ 3%RACE-F and each of the primers specific for the three polyadenylation sites, PolyA1-R, PolyA2-R, PolyA3-R (Table 1). The annealing temperatures for these PCR reactions were 50 °C for PolyA1-R and 48 °C for PolyA2-R and PolyA3-R. Controls run in parallel were a reaction without RT and GCH1 gDNA.

The C. par6um GCH1 gDNA library was a generous gift from Dr Donna Akiyoshi, Tufts University School of Veterinary Medicine, Grafton, MA. This library contained GCH1 gDNA, partially digested with Sau3AI and cloned into the BamHI site in Lambda ZAP Express (Stratagene). For the initial screening, the library was plated on E. coli XL1-Blue MRF% at 50,000 plaques per 150 mm plate. The plaque lifts were screened by hybridization with a 782 bp HindIII-EcoRI fragment from pAMC10.1 labeled as described above. Positive plaques were purified by four rounds of plating and the phagemids excised as per the manufacturer’s directions (Stratagene). Because the clones contained overlapping sequence, the inserts were sequenced on one strand by primer walking.

2.8. Rapid amplification of cDNA ends (3 %RACE) Caco-2A cells were seeded into six well plates and infected with GCH1 oocysts, as described above. Postinfection (24 h), total RNA was isolated from the cells using the RNeasy kit (Qiagen). The RNA was treated with DNase-Free (Ambion) to remove contaminating gDNA, then reversed transcribed with an oligo dT primer that included an adapter sequence on the 5% end (Table 1, oligo dT adapter). A gene-specific primer, composed of sequences from the 3% end of the coding sequence of Cpgp40 /15, was used to prime the second strand synthesis (Table 1, 3%RACE-F). PCR was then performed with the 3%RACE-F and Adapter-R primers (Table 1). Controls run in parallel were Caco2A cells infected with heat-inactivated oocysts (shaminfected), as well as a reaction excluding reverse transcriptase (RT) and GCH1 gDNA. Bands containing Cpgp40 /15 sequences were identified by Southern blotting. The bands of interest were excised from the gel, cloned into pCRII-TOPO and sequenced. To obtain 3%UTR sequence from TU502, gDNA was amplified with the forward primer 3%RACE/ TU502-F (Table 1) and a reverse primer specific for sequence downstream of the third polyadenylation site in GCH1 (3%End-R, Table 1). The PCR products were sequenced directly. To confirm the identity of the polyadenylation sites identified with 3%RACE, PCR was used to amplify sequences 3% to the first polyadenylation site using primers 2Tran-F and 2TranR. The probe was labeled by priming with the specific primers in the presence of 32P-dCTP and hybridized to Northern blots of uninfected and GCH1-infected Caco2A cells.

2.10. Message stability assay Caco-2A cells grown in T75 flasks were infected with GCH1 oocysts, as described above. Post infection (18 h), 10 mg/ml of actinomycin D (Calbiochem-Novabiochem International, San Diego, CA) was added to the cultures. mRNA was extracted from cells collected 0, 0.25, 0.5, 0.75, 1, 2, 4 and 6 h after the addition of actinomycin D. The samples were subjected to Northern blot analysis using the 861 bp GCH1 Cpgp40 /15 probe, as described above. The blot was stripped and reprobed for human glyceraldehyde 3 phosphate dehydrogenase (GAPDH) to control for equal loading. The GAPDH probe was isolated from ATCC plasmid 57090 [36] and labeled by random priming.

2.11. Analysis of sequence data Sequences were analyzed with Vector NTI, Suite 6 (Informax, North Bethesda, MD). Putative N-glycosylation sites and predicted O-glycosylation sites were identified with the Motif [37] and NetOGlyc 2.0 [38] programs, respectively and promoter regions with PROSCAN 1.7 [39]. DNA sequences were compared to sequences in the database using the BLAST algorithm [40]. Sequences were also blasted against sequence data from the University of Minnesota C. par6um Genome (MCPG) sequencing project (http://www.cbc.umn.edu/ ResearchProjects/AGAC/Cp/).

3. Results

3.1. The TU502 isolate exhibits genotype I alleles at the COWP and TRAP-C1 loci To confirm the genotype of the TU502 isolate, PCRRFLP analysis of the COWP and TRAP-C1 loci was performed on gDNA isolated from different passages. Fig. 1(A) shows the Rsa-1 digestion of the COWP (Fig.

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

207

Fig. 1. The TU502 isolate exhibits genotype I alleles at the COWP and TRAP-C1 loci and the Cpgp40 /15 locus identifies TU502 as genotype Ia. (A) The RsaI fragments of the COWP (lanes 1 –3) and TRAP-C1 (lanes 4 – 6) PCR products were resolved on a 1% agarose gel and visualized with ethidium bromide. TU502 late passage, lanes 1 and 4; TU502 early passage, lanes 2 and 5; GCH1 lanes, 3 and 6. (B) Comparison of the GCH1 and TU502 Cpgp40 /15 deduced amino acid sequence. The sequences share 72% identity at the amino acid level and 80.5% identity at the nucleotide level (not shown). Italicized letters indicate the signal sequences and bold letters indicate predicted O-glycosylation sites. The putative N-glycosylation site is italicized and underlined. The underlined C-terminal amino acids indicate the putative glycophosphatidyl inositol anchor site. The bold, italicized letters show the cleavage site between gp40 and gp15. SAPs are indicated with asterisks. The TU502 Cpgp40 /15 sequence is most similar to genotype Ia Cpgp40 /15 sequences (not shown).

1A, lanes 1–3) and TRAP-C1 (Fig. 1A, lanes 4–6) PCR products amplified from gDNA of GCH-1 (Fig. 1A, lanes 1 and 4), TU502 from early passage (Fig. 1A, lanes 2 and 5) and TU502 from passage 9 months later (Fig. 1A, lanes 3 and 6). PCR-RFLP analysis of TU502 gDNA generated fragment sizes characteristic of genotype I isolates (285, 125 and 106 bp for COWP, and 341 and 114 for TRAP-C1) confirming that this isolate displays genotype I alleles at these loci. Further, there was no change in the genotype of the TU502 isolate after repeated passage through calves over a 9-month period. In contrast, the GCH1 isolate exhibited PCRRFLP fragments characteristic of genotype II isolates (410 and 106 for COWP, and 455 and 51 for TRAPC1).

quence and N-terminal sequences of the mature protein are conserved between the two isolates, but the sequences directly following the polyserine domains are highly divergent with four insertions and several single amino acid polymorphisms (SAPs). The single potential N-glycosylation site is conserved. However, most of the predicted O-glycosylation sites outside of those in the polyserine domain are divergent. The cleavage site between gp40 and gp15 is conserved, as has been observed for all Cpgp40 /15 sequences described [15,20,41]. The proposed attachment site for the GPI anchor in gp15 [42] is also conserved. Identical sequence was obtained from the Cpgp40 /15 PCR product amplified from TU502 gDNA obtained from later passage (after 9 months) oocysts (data not shown).

3.2. The genotype I TU502 Cpgp40 /15 gene is highly di6ergent from the genotype II Cpgp40 /15 sequence

3.3. Cpgp40 /15 is present in single copy in genotype I and II isolates

To begin the characterization of the TU502 Cpgp40 / 15 gene, gDNA from TU502 oocysts was amplified with all available primers specific for GCH1 Cpgp40 /15 sequences [15]. Primers 7-F and Stop-R amplified a single 1144 bp fragment that was subsequently cloned into pCRII-TOPO and sequenced. The sequence included the complete coding sequence of the TU502 Cpgp40 /15 gene and 112 bp of sequence 5% to the start codon (Accession No. AY048667). The TU502 sequence shared \ 95% homology with the Cpgp40 /15 Ia sequences, described by Strong et al. [20]. Fig. 1(B) compares the deduced amino acid sequences of TU502 and GCH1 Cpgp40 /15 gene products. The signal se-

Previous studies had shown that the GCH1 Cpgp40 / 15 gene was present in single copy in the C. par6um genome [15,20]. Since this observation had been made using genotype II isolates and the Cpgp40 /15 gene shows extensive polymorphism in genotype I isolates [20], we performed Southern blotting to determine copy number of the Cpgp40 /15 gene in the TU502 isolate. Genomic DNA was obtained from both isolates, digested with various restriction enzymes and analyzed by Southern blotting with isolate-specific Cpgp40 /15 probes (Fig. 2). Although the size of the restriction fragments containing Cpgp40 /15 sequence varied between the two isolates, the probes recognized a single

208

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

Fig. 2. Cpgp40 /15 is a single copy gene in both GCH1 and TU502 isolates. gDNA from GCH1 (panel A) and TU502 (panel B) oocysts was digested with EcoRI (lane 1), HindIII (lane 2), PstI (lane 3), SspI (lane 4) and EcoRI+ HindIII (lane 5), resolved on a 1% agarose gel, blotted and probed with isolate-specific Cpgp40 /15 sequences.

restriction fragment in each digest, suggesting that the gene is present as a single copy in both isolates.

3.4. Two transcripts are expressed from the Cpgp40 /15 gene in genotype I and II isolates To characterize expression of the Cpgp40 /15 gene, Northern blots were performed on mRNA from Caco2A cells that had been infected for 24 h with each C. par6um isolate. Surprisingly, these experiments demonstrated the presence of two transcripts of :1.5 and 2.3 kb in both GCH1 (Fig. 3A, lane 1) and TU502 (Fig. 3A, lane 2) infected Caco-2A cells.

Fig. 4. Expression of Cpgp40 /15 transcripts during intracellular development. mRNA from GCH1-infected Caco-2A cells was extracted 6, 12, 24 and 48 h after infection, subjected to Northern blotting and transcripts detected with a GCH1 Cpgp40 /15 probe (panel A). The same blot was stripped and probed for human b-actin to assess loading (panel B). mRNA from uninfected Caco-2A cells was included as a negative control (lane ‘Un’).

Further studies to investigate the significance of the two transcripts were performed on the GCH1 isolate, since large numbers of oocysts of this isolate (which are required for Northern blot analysis) are more readily available. Northern blots of mRNA from GCH1-infected Caco-2A cells were probed with gp40- and gp15specific sequences. Both transcripts contained gp40 (Fig. 3B, lane 1) as well as gp15 specific sequences (Fig. 3B, lane 2). Neither transcript was in the anti-sense orientation, as an anti-sense RNA probe identified the same transcripts as the DNA probes (not shown). None of the probes recognized any transcript in uninfected Caco-2A cells (Fig. 4, lane ‘Un’).

Fig. 3. C. par6um expresses two transcripts from the Cpgp40 /15 gene: mRNA from GCH1- (panel A, lane 1) and TU502- (panel A, lane 2) infected Caco-2A cells were subjected to Northern blotting and probed with isolate specific Cpgp40 /15 sequences. mRNA from GCH1-infected Caco-2A cells was probed with gp40 (panel B, lane 1) and gp15 (panel B, lane 2) specific sequences.

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

To determine if expression of the two transcripts differed over the course of in vitro infection, mRNA was isolated from Caco-2A cells and collected 6, 12, 24 and 48 h after infection with the GCH1 isolate (Fig. 4). The two transcripts were observed at 12 and 24 h and faintly at 48 h (Fig. 4A), which is similar to the results obtained by Strong et al. with RT-PCR analysis of time course infections [20]. The blot was probed with a human b-actin probe to assess loading of mRNA (Fig. 4B).

3.5. Characterization of the Cpgp40 /15 locus in a genotype II isolate To identify sequences surrounding the Cpgp40 /15 gene that could account for the generation of multiple transcripts, a GCH1 genomic library was screened by hybridization with Cpgp40 /15 coding sequences. Two clones, pBKCMV-11 and pBKCMV-17, contained sequences upstream of the Cpgp40 /15 gene (Fig. 5). Five other clones were analyzed by restriction digestion to choose one that would include 3% sequences. The phagemid pBKCM-10 was missing the 384 bp EcoRIPstI fragment upstream of the Cpgp40 /15 coding sequence (Fig. 5) and contained an insert large enough ( : 3500 bp) to include sequences 3% of the coding sequence. The portion of the pBKCMV-10 insert that was 3% of the Cpgp40 /15 coding sequence was sequenced through on both strands. In this way, we obtained : 5 kb of sequence surrounding the Cpgp40 / 15 coding sequence (Accession No. AY048665). Only one consensus polyadenylation signal sequence (ATTAAA) was observed 11 nucleotides downstream of the stop codon. Analysis of the sequence with PROSCAN [39] identified a putative promoter region on the reverse strand at nucleotides (nt) 2371-2621. A second open reading frame (ORF 2) was identified on the reverse strand (Fig. 5). Comparison of the Cpgp40 /15 locus to sequences in the database identified only previously

209

published sequences similar to Cpgp40 /15 [15,20,41] and an unpublished 3.1 kb sequence (Accession No. AF203016), which shared 99% identity with the GCH1 Cpgp40 /15 locus. Comparison of the 5 kb sequence containing the Cpgp40 /15 locus to sequences from the University of Minnesota C. par6um Genome (MCPG) project (http://www.cbc.umn.edu/ResearchProjects/ AGAC/Cp/) identified two contigs (C. par6um contigs 1389 and 1296) containing overlapping sequence. Assembly of all three sequences yielded 7.4 kb of sequence surrounding the Cpgp40 /15 locus (not shown). Only one copy of the Cpgp40 /15 gene was found within this sequence.

3.6. Multiple Cpgp40 /15 transcripts are generated by alternate polyadenylation in genotype I and II isolates A previous report had shown that C. par6um used alternate polyadenylation sites to generate multiple transcripts of the initiation translation factor eIF-4A [43]. To test the hypothesis that alternate polyadenylation might be the mechanism underlying the generation of the two Cpgp40 /15 transcripts, 3%RACE analysis was performed on RNA isolated from Caco-2A cells that had been infected for 24 h with the GCH1 isolate. cDNA synthesis was primed with oligo dT containing an adapter sequence on the 5%end (Table 1) and for second strand synthesis, a primer specific for sequences 126 bp upstream of the Cpgp40 /15 stop codon (3%RACE-F). The 3%UTRs were then amplified by PCR with the primers 3%RACE-F and Adapter-R (Table 1). Using this procedure, two bands, 1150 and 550 bp, were specifically amplified from cDNA obtained from GCH1-infected Caco-2A cells. There was no significant reaction with samples primed without RT or with cDNA generated from sham-infected Caco-2A cells. Southern blotting confirmed the presence of Cpgp40 /15 coding sequence in these bands (not shown). The bands were excised from the gel and cloned into pCRII-

Fig. 5. The Cpgp40 /15 locus. pBKCMV17, 11 and 10 were isolated from a GCH1 gDNA library and completely or partially sequenced to obtain sequences surrounding the Cpgp40 /15 gene.

210

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

Fig. 6. Comparison of the 3%UTR sequences of GCH1 and TU502 Cpgp40 /15 gene. The sequences share 96.7% identity. Single nucleotide polymorphisms and nucleotide insertions are indicated with asterisks. The polyadenylation sites are highlighted. The degenerate repeat, AC/UUAAUUUUUUA/UUU, is underlined.

TOPO. Since insert size differed slightly among the clones, two clones of each were sequenced on both strands. Both clones containing the 1150 bp insert had identical sequence except for insertion of poly A tracts 978 and 1002 bp downstream of the stop codon (Fig. 6). In both clones containing the 550 bp inserts, the poly A tracts were inserted 371 bp downstream of the Cpgp40 /15 stop codon. The GCH1 Cpgp40 /15 3%UTRs were collinear with respect to the genomic sequence. Examination of the sequences immediately upstream of the first and third polyadenylation sites identified the degenerate repeat AC/UUAAUUUUUUA/UUU (Fig. 6). The second polyadenylation site is immediately upstream of this sequence. The consensus polyadenylation signal sequence ATTAAA was not utilized for the generation of any of the Cpgp40 /15 transcripts. A probe specific for the 3% UTR of the larger transcript recognized only the 2.3 kb transcript on a Northern blot of GCH1-infected Caco-2A cells (data not shown), confirming the results of the 3%RACE analysis. Studies were then performed to determine whether both transcripts in the TU502 isolate were also generated

as a result of alternate polyadenylation. To obtain the sequence of the TU502 Cpgp40 /15 3%UTR, TU502 gDNA was amplified with primers 3’RACE/TU502-F and 3%END-R (Table 1) and the PCR product sequenced. In marked contrast to the coding sequences, the GCH1 and TU502 3%UTRs shared 96.7% identity (Fig. 6). To determine whether the same polyadenylation sites were used by the TU502 isolate, reverse primers specific for the three GCH1 polyadenylation sites were designed (Table 1) and used with TU502/3%RACE-F to test if all three transcripts were present in cDNA from TU502-infected Caco-2A cells. Transcripts of the appropriate size were obtained with the forward primer TU502/3%RACEF and primers PolyA1-R and PolyA3-R (Fig. 7A, lanes 1 and 2). There was no significant band of the appropriate size obtained with the reverse primer PolyA2-R (not shown), suggesting that either this site is not used by the TU502 isolate or the infection level was not great enough for detection of this transcript. The specificity of these reactions was confirmed by the lack of amplicons from the reactions in which RT was not added (Fig. 7A, lanes 3 and 4), and from TU502 gDNA (not shown).

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

211

3.7. All three transcripts are expressed in GCH1 sporozoites We then performed studies to determine if expression of the multiple transcripts was also present in sporozoites. Because sufficient sporozoite RNA for Northern blot analysis could not be obtained, reverse primers specific for the three polyadenylation sites (Table 1) were used with 3%RACE-F to test if all three transcripts were present in GCH1 sporozoite cDNA. Each of the primer combinations amplified a fragment of the appropriate size from sporozoite cDNA (Fig. 7B, lanes 1 – 3), but not from control reactions lacking RT (Fig. 7B, lanes 4– 6) or from GCH1 gDNA (not shown).

3.8. The two GCH1 Cpgp40 /15 transcripts ha6e different half-li6es Because 3%UTRs can contain sequences that determine the stability of a transcript [44– 46], we examined the relative stability of the Cpgp40 /15 transcripts. Caco-2A cells that had been infected for 18 h with the C. par6um GCH1 isolate were treated with actinomycin D to halt transcription. mRNA from cells that had been collected at various time points after the addition of actinomycin D was analyzed by Northern blotting (Fig. 8). The larger Cpgp40 /15 transcript exhibited a half-life of 30 min in marked contrast to the 2 h half-life of the smaller transcript (Fig. 8, panel A). The blot was re-probed for human GAPDH to assess loading of the samples (Fig. 8, panel B). The observed decrease in the size of the GAPDH mRNA (Fig. 8, panel B, 6 h) may be due to progressive deadenylation of the transcripts following transcription inhibition.

Fig. 7. The same Cpgp40 /15 polyadenylation sites are used in TU502 infected Caco-2A cells and in GCH1 sporozoite stages. (A) cDNA from TU502 infected Caco-2A cells (lanes 1 and 2) and control reactions lacking RT (lanes 3 and 4) were amplified with primers TU502/3%RACE-F and PolyA1-R (lanes 1 and 3) and TU502/ 3%RACE-F and PolyA3-R (lanes 2 and 4). (B) GCH1 sporozoite cDNA (lanes 1 – 3) and the control reaction minus RT (lanes 4 – 6) were amplified with primers 3%RACE-F and PolyA1-R (lanes 1 and 4), 3%RACE-F and PolyA2-R (lanes 2 and 5) and 3%RACE-F and PolyA3-R (lanes 3 and 6). The amplicons were resolved on a 1% agarose gel and the ethidium bromide stained bands visualized.

Fig. 8. Stability of the Cpgp40 /15 transcripts. Caco-2A cells that were infected for 18 h with the GCH1 isolate were treated with actinomycin D. mRNA was extracted from the cells at the indicated time points, subjected to Northern blotting and probed for Cpgp40 /15 (panel A) and GAPDH (panel B).

4. Discussion One of the most intriguing and little understood aspects of C. par6um biology is the existence of two major genotype populations with different transmission cycles, one zoonotic and the other anthroponotic [24]. A variety of factors could account for the differences in host specificity and identifying these factors may be essential for developing therapies effective against all human C. par6um infections. To date, the most striking molecular difference described between genotype I and II isolates is the contrast in Cpgp40 /15 gene characteristics [20]. The observation that the Cpgp40 /15 gene is highly polymorphic among genotype I isolates, but remains fairly invariant among genotype II isolates suggested the possibility that control of Cpgp40 /15 gene expression could also differ between the two isolates. These data also raised the possibility that C. par6um, similar to other apicomplexans [47,48], might be capable of antigenic variation. In this work, we took advantage of the availability of the TU502 isolate to explore these hypotheses. The gene encoding the TU502 Cpgp40 /15 is a member of the Ia group, as defined by Strong et al. [20] and is consistent in the characteristics described previously for genotype I Cpgp40 /15 sequences, with the greatest number of polymorphisms concentrated in a hypervariable region directly following the polyserine domain. The features that are conserved between GCH1 and TU502 Cpgp40 /15 sequences, the signal sequence, the polyserine domain, the proteolytic cleavage site between

212

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

gp40 and gp15 and the GPI anchor site, are conserved in all Cpgp40 /15 sequences described [15,20,41]. The predicted O-glycosylation sites within the polyserine domain were conserved, but within the hypervariable region following the polyserine domain, the O-glycosylation sites varied significantly between the two isolates. It is unclear if the observed polymorphisms translate into functional differences, such as recognition of different receptors or if the variation is a reflection of immune selection pressure, or both. Because many apicomplexan parasites generate polymorphic proteins from multiple copies of a gene [49– 53], we explored the possibility that Cpgp40 /15 gene variability also arose through a similar mechanism. Although Cpgp40 /15 had been previously described as a single copy gene in genotype II isolates, it was important to confirm this observation in polymorphic genotype I isolates. The Cpgp40 /15 gene appeared as a single band in all digests of TU502 and GCH1 gDNA suggesting that the gene is present as a single copy. It is possible that multiple tandem copies are present but not detected because of identical restriction sites surrounding each copy. This interpretation of the data seems unlikely for two reasons. First, in 7.4 kb of sequence surrounding the Cpgp40 /15 locus only one copy of the gene was observed, suggesting that the gene is not tandemly duplicated, at least in genotype II isolates. Second, most of the genotype I Cpgp40 /15 sequence data has been obtained from sequencing of PCR products (Ref. [20] and Leav et al., submitted) suggesting not only that Cpgp40 /15 is a single copy gene, but also that the parasites within an isolate are homogeneous at this locus. If there was a duplicate copy, or several copies, of the Cpgp40 /15 gene, there should be evidence of more than one sequence, especially in these nonclonal isolates. These data suggest that the polymorphisms observed at this locus are not generated through the use of multiple gene copies. These findings are not surprising in light of the biology of C. par6um infections. Some apicomplexans, such as Plasmodium falciparum and Babesia bo6is are capable of establishing persistent infections through rapid variation of surface antigens [47,48]. The genes encoding these antigens are present at a very high copy number [50,53], enabling expression of a diversity of molecules that are variant with regard to B-cell epitopes [47,54], as well as recognition of host receptors [55,56]. In contrast, Cryptosporidium elicits an immune response involving mucosal and serum antibodies, T-cells and IFN-g production that clears the infection within a short period [57]. Persistent infections are only observed in immunocompromised or immunosuppressed hosts [4– 6]. Human studies have shown that repeat infections can occur, but the number of oocysts required to initiate infection is greater and the disease is less severe [58,59]. It is possible that the Cpgp40 /15

variation arose to improve the parasites’ ability to reinvade an immune host, either through evasion of the existing immune response or through the utilization of alternate host receptors for invasion. Either way, defining the significance of variation in Cpgp40 /15 and possibly other antigens as well, may be essential for the development of effective strategies to prevent and treat infections in humans. In both genotype I and II isolates, multiple transcripts are generated from the Cpgp40 /15 gene through the use of alternate polyadenylation sites. This phenomenon has been previously observed for the C. par6um initiation translation factor eIF-4A [43] and may be a common occurrence during transcription of C. par6um genes. Northern blot analysis of the C. par6um ATP-binding cassette protein (CpABC) also revealed the presence of two transcripts, although the mechanism underlying the production of the two transcripts was not explored [60]. Plasmodium species also generate multiple transcripts of particular genes through both alternate polyadenylation [61,62] and transcription termination [61,63]. The end result of alternate polyadenylation is the generation of transcripts bearing different 3%UTRs that may be involved in the control of gene expression in a variety of ways. Many functions have been ascribed to 3%UTRs, such as regulation of stage-specific gene expression [45,64], stabilization and destabilization of message [44–46] and localization of mRNA to particular regions of the cell [65]. The functions of the 3%UTRs of apicomplexan genes are unknown but are likely to be similar. Sequences in the 3%UTR of the P. gallinaceum pgs28 gene were shown to affect translation efficiency of a reporter gene [62], demonstrating that 3%UTRs in apicomplexans can encode elements that affect gene expression. The function of the multiple Cpgp40 /15 transcripts remains unclear. None of the Cpgp40 /15 transcripts appeared to be stage specific. In intracellular stages (present at 6–48 h after infection) the transcripts appeared to follow the same temporal pattern of expression, although subtle differences in timing probably would not be detected due to the inability to synchronize the infection. All three transcripts were also detected in sporozoite cDNA, suggesting that transcription of the Cpgp40 /15 in this stage also involves the production of multiple transcripts. However, there was a striking difference in the stability of the messages. Although the global effect of actinomycin D on transcription makes determination of message halflife difficult, the half-lives of the transcripts with respect to each other should not have been affected. Because we only investigated message stability in GCH1 intracellular stages, the possibility remains that the relative stability of the two messages could vary between life cycle stages and could be quite different in the TU502 isolate. The 3%UTR sequences did not contain any of

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

the previously described signature sequences that are associated with message stability in other systems [44– 46]. As far as we can ascertain, this is the first investigation of message stability in an apicomplexan parasite. The Cpgp40 /15 3%UTRs may contain unique, possibly apicomplexan-specific elements affecting message stability that will need to be defined experimentally. There was no consistent sequence marking of the polyadenylation insertion sites, except for the degenerate repeat sequence AC/UUAAUUUUUUA/UUU, found immediately upstream of the first poly A site and between the second and third sites. Whether this motif has any functional significance remains to be determined. The 3%UTRs of the C. par6um eIF-4A initiation translation factor contain the eukaryotic consensus signal for polyadenylation, AATTAAA, just upstream of the polyadenylation sites [43]. This sequence occurred once in the Cpgp40 /15 3%UTRs directly following the stop codon and did not appear to function as a polyadenylation signal sequence for Cpgp40 /15 transcripts. The lack of a consensus sequence suggests that C. par6um can utilize a variety of sequences for polyadenylation. Alternately, these putative polyadenylation signal sequences could be serendipitous occurrences in the AT-rich genomes of apicomplexans. A considerable amount of research remains to be carried out in order to understand factors governing expression of the Cpgp40 /15 gene and the functional significance of Cpgp40 /15 gene variation. Because the glycoproteins products of the Cpgp40 /15 gene have been implicated in the attachment and invasion of sporozoites into epithelial cells and may thus be targets for preventative therapies, it is important to determine if Cpgp40 /15 polymorphisms translate into differences in host receptor specificity. Expression of different Cpgp40 /15 isotypes in eukaryotic expression systems could prove useful in determining the molecular specifics of gp40 and gp15 interactions with epithelial cells. Inclusion of the 3%UTR sequences in eukaryotic expression vectors has been shown to improve recovery of functional protein [66] and, in this case, may yield information concerning the function of the Cpgp40 /15 3%UTRs. Information from such studies would improve our understanding of C. par6um biology, possibly identifying novel means for control of this protozoan parasite.

Acknowledgements We wish to thank Dr Saul Tzipori and Dr Donna Akiyoshi for the GCH1 and TU502 isolates, the TU502 gDNA and the GCH1 gDNA library, Smitha Jaison for excellent technical assistance and Anne Kane for helpful comments and careful review of the manuscript. This work was supported by Grant AI46299 from the

213

NIAID, NIH; Grant 2000-02247 from the USDA; and Grant P30 DK34928 from the NIDDK, NIH to the GRASP Digestive Diseases Center at New England Medical Center. R.M. O’Connor was supported by training Grant AI07389 from the NIAID, NIH.

References [1] Tyzzer EE. Cryptosporidium par6um (sp. nov) a coccidium found in the small intestine of the common mouse. Arch Protistenk 1912;26:394 – 412. [2] O’Donoghue PJ. Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol 1995;25:139 – 95. [3] Griffiths JK. Human cryptosporidiosis: epidemiology, transmission, clinical disease treatment, and diagnosis. Adv Parasitol 1998;40:37 – 85. [4] Current WL, Reese NC, Ernst JV, Bailey WS, Heyman MB, Weinstein WM. Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies of an outbreak and experimental transmission. New Engl J Med 1983;308:1252 –7. [5] Colford JM Jr, Tager IB, Hirozawa AM, Lemp GF, Aragon T, Petersen C. Cryptosporidiosis among patients infected with human immunodeficiency virus. Factors related to symptomatic infection and survival. Am J Epidemiol 1996;144:807 – 16. [6] Petersen C. Cryptosporidiosis in patients infected with the human immunodeficiency virus. Clin Infect Dis 1992;15:903 –9. [7] Guerrant RL. Cryptosporidiosis: an emerging, highly infectious threat. Emerg Infect Dis 1997;3:51 – 7. [8] Brasseur P. Waterborne cryptosporidiosis: a major environmental risk. J Eukaryot Microbiol 1997;44:67S – 8S. [9] Fricker CR, Crabb JH. Water-borne cryptosporidiosis: detection methods and treatment options. Adv Parasitol 1998;40:241 –78. [10] Blagburn BL, Soave R. Prophylaxis and chemotherapy. In: Fayer R, editor. Cryptosporidium and Cryptosporidiosis. Boca Raton, FL: CRC Press, 1997:111 – 23. [11] Fayer R, Speer CA, Dubey JP. The general biology of Cryptosporidium. In: Fayer R, editor. Cryptosporidium and Cryptosporidiosis. Boca Raton, FL: CRC Press, 1997:1 – 41. [12] Bjorneby JM, Riggs MW, Perryman LE. Cryptosporidium par6um merozoites share neutralization-sensitive epitopes with sporozoites. J Immunol 1990;145:298 – 304. [13] Tilley M, Upton SJ. Sporozoites and merozoites of Cryptosporidium par6um share a common epitope recognized by a monoclonal antibody and two-dimensional electrophoresis. J Protozool 1991;38:48S – 9S. [14] Riggs MW, McNeil MR, Perryman LE, Stone AL, Scherman MS, O’Connor RM. Cryptosporidium par6um sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a beta-mannosylated glycolipid. Infect Immun 1999;67:1317 –22. [15] Cevallos AM, Zhang X, Waldor MK, et al. Molecular cloning and expression of a gene encoding Cryptosporidium par6um glycoproteins gp40 and gp15. Infect Immun 2000;68:4108 –16. [16] Barnes DA, Bonnin A, Huang JX, et al. A novel multi-domain mucin-like glycoprotein of Cryptosporidium par6um mediates invasion. Mol Biochem Parasitol 1998;96:93 – 110. [17] Riggs MW, Stone AL, Yount PA, Langer RC, Arrowood MJ, Bentley DL. Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium par6um sporozoites and merozoites. J Immunol 1997;158:1787 – 95. [18] Langer RC, Riggs MW. Cryptosporidium par6um apical complex glycoprotein CSL contains a sporozoite ligand for intestinal epithelial cells. Infect Immun 1999;67:5282 – 91.

214

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

[19] Perryman LE, Kapil SJ, Jones ML, Hunt EL. Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium par6um recombinant protein. Vaccine 1999;17:2142 –9. [20] Strong WB, Gut J, Nelson RG. Cloning and sequence analysis of a highly polymorphic Cryptosporidium par6um gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products. Infect Immun 2000;68:4117 – 34. [21] Cevallos AM, Bhat N, Verdon R, et al. Mediation of Cryptosporidium par6um infection in vitro by mucin-like glycoproteins defined by a neutralizing monoclonal antibody. Infect Immun 2000;68:5167 – 75. [22] Gut J, Nelson RG. Cryptosporidium par6um sporozoites deposit trails of 11A5 antigen during gliding locomotion and shed 11A5 antigen during invasion of MDCK cells in vitro. J Eukaryot Microbiol 1994;41:42S –3S. [23] Gut J, Nelson RG. Cryptosporidium par6um: lectins mediate irreversible inhibition of sporozoite infectivity in vitro. J Eukaryot Microbiol 1999;46:48S –9S. [24] Peng MM, Xiao L, Freeman AR, et al. Genetic polymorphism among Cryptosporidium par6um isolates: evidence of two distinct human transmission cycles. Emerg Infect Dis 1997;3:567 – 73. [25] Patel S, Pedraza-Diaz S, McLauchlin J, Casemore DP. Molecular characterization of Cryptosporidium par6um from two large suspected waterborne outbreaks. Outbreak Control Team South and West Devon 1995, Incident Management Team and Further Epidemiological and Microbiological Studies Subgroup North Thames 1997. Commun Dis Publ Health 1998;1:231 – 3. [26] Widmer G, Tzipori S, Fichtenbaum CJ, Griffiths JK. Genotypic and phenotypic characterization of Cryptosporidium par6um isolates from people with AIDS. J Infect Dis 1998;178:834 – 40. [27] McLauchlin J, Amar C, Pedraza-Diaz S, Nichols GL. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. J Clin Microbiol 2000;38:3984 –90. [28] Widmer G, Akiyoshi D, Buckholt MA, et al. Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium par6um. Mol Biochem Parasitol 2000;108:187 –97. [29] Tzipori S, Rand W, Griffiths J, Widmer G, Crabb J. Evaluation of an animal model system for cryptosporidiosis: therapeutic efficacy of paromomycin and hyperimmune bovine colostrumimmunoglobulin. Clin Diagn Lab Immunol 1994;1:450 –63. [30] Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A. PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. par6um, and between C. par6um isolates of human and animal origin. FEMS Microbiol Lett 1997;150:209 – 17. [31] Spano F, Putignani L, Guida S, Crisanti A. Cryptosporidium par6um: PCR-RFLP analysis of the TRAP-C1 (thrombospondin-related adhesive protein of Cryptosporidium-1) gene discriminates between two alleles differentially associated with parasite isolates of animal and human origin. Exp Parasitol 1998;90:195 – 8. [32] Spano F, Putignani L, Naitza S, Puri C, Wright S, Crisanti A. Molecular cloning and expression analysis of a Cryptosporidium par6um gene encoding a new member of the thrombospondin family. Mol Biochem Parasitol 1998;92:147 – 62. [33] Strong WB, Nelson RG. Preliminary profile of the Cryptosporidium par6um genome: an expressed sequence tag and genome survey sequence analysis. Mol Biochem Parasitol 2000;107:1 – 32. [34] Joe A, Verdon R, Tzipori S, Keusch GT, Ward HD. Attachment of Cryptosporidium par6um sporozoites to human intestinal epithelial cells. Infect Immun 1998;66:3429 –32. [35] Jung HC, Eckmann L, Yang SK, et al. A distinct array of proinflammatory cytokines is expressed in human colon epithe-

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50]

[51]

[52]

[53]

[54]

lial cells in response to bacterial invasion. J Clin Invest 1995;95:55 – 65. Tso JY, Sun XH, Kao TH, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 1985;13:2485 – 502. Yada T, Totoki Y, Ishikawa M, Asai K, Nakai K. Automatic extraction of motifs represented in the hidden Markov model from a number of DNA sequences. Bioinformatics 1998;14:317 – 25. Hansen JE, Lund O, Tolstrup N, Gooley AA, Williams KL, Brunak S. NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconj J 1998;15:115 – 30. Prestridge DS. Predicting Pol II promoter sequences using transcription factor binding sites. J Mol Biol 1995;249:923 –32. Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389 – 402. Priest JW, Kwon JP, Arrowood MJ, Lammie PJ. Cloning of the immunodominant 17-kDa antigen from Cryptosporidium par6um. Mol Biochem Parasitol 2000;106:261 – 71. Priest JW, Xie LT, Arrowood MJ, Lammie PJ. The immunodominant 17-kDa antigen from Cryptosporidium par6um is glycosylphosphatidylinositol-anchored. Mol Biochem Parasitol 2001;113:117 – 26. Spano F, Crisanti A. The initiation translation factor eIF-4A of Cryptosporidium par6um is encoded by two distinct mRNA forms and shows DNA sequence polymorphism distinguishing genotype 1 and 2 isolates. J Parasitol 2000;86:777 – 82. Chen CY, Shyu AB. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci 1995;20:465 – 70. Di Noia JM, D’Orso I, Sanchez DO, Frasch AC. AU-rich elements in the 3%-untranslated region of a new mucin-type gene family of Trypanosoma cruzi confers mRNA instability and modulates translation efficiency. J Biol Chem 2000;275:10218 – 27. Peng SS, Chen CY, Shyu AB. Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements. Mol Cell Biol 1996;16:1490 – 9. Allred DR, Cinque RM, Lane TJ, Ahrens KP. Antigenic variation of parasite-derived antigens on the surface of Babesia bo6isinfected erythrocytes. Infect Immun 1994;62:91 – 8. Saul A. The role of variant surface antigens on malaria-infected red blood cells. Parasitol Today 1999;15:455 – 7. al-Khedery B, Barnwell JW, Galinski MR. Antigenic variation in malaria: a 3% genomic alteration associated with the expression of a P. knowlesi variant antigen. Mol Cell 1999;3:131 – 41. Allred DR, Carlton JM, Satcher RL, et al. The 6es multigene family of B. bo6is encodes components of rapid antigenic variation at the infected erythrocyte surface. Mol Cell 2000;5:153 –62. Baruch DI, Pasloske BL, Singh HB, et al. Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 1995;82:77 – 87. Smith JD, Chitnis CE, Craig AG, et al. Switches in expression of Plasmodium falciparum 6ar genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 1995;82:101 – 10. Su XZ, Heatwole VM, Wertheimer SP, et al. The large diverse gene family 6ar encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 1995;82:89 – 100. Newbold CI, Pinches R, Roberts DJ, Marsh K. Plasmodium falciparum: the human agglutinating antibody response to the

R.M. O’Connor et al. / Molecular & Biochemical Parasitology 119 (2002) 203–215

[55]

[56]

[57] [58]

[59]

[60]

infected red cell surface is predominantly variant specific. Exp Parasitol 1992;75:281 –92. Gardner JP, Pinches RA, Roberts DJ, Newbold CI. Variant antigens and endothelial receptor adhesion in Plasmodium falciparum. Proc Natl Acad Sci USA 1996;93:3503 –8. Baruch DI, Gormely JA, Ma C, Howard RJ, Pasloske BL. Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1. Proc Natl Acad Sci USA 1996;93:3497 –502. McDonald V. Host cell-mediated responses to infection with cryptosporidium. Parasite Immunol 2000;22:597 –604. Okhuysen PC, Chappell CL, Sterling CR, Jakubowski W, DuPont HL. Susceptibility and serologic response of healthy adults to reinfection with Cryptosporidium par6um. Infect Immun 1998;66:441– 3. Chappell CL, Okhuysen PC, Sterling CR, Wang C, Jakubowski W, Dupont HL. Infectivity of Cryptosporidium par6um in healthy adults with pre-existing anti-C. par6um serum immunoglobulin G. Am J Trop Med Hyg 1999;60:157 –64. Perkins ME, Riojas YA, Wu TW, Le Blancq SM. CpABC, a Cryptosporidium par6um ATP-binding cassette protein at the host– parasite boundary in intracellular stages. Proc Natl Acad Sci USA 1999;96:5734 –9.

215

[61] Ruvolo V, Altszuler R, Levitt A. The transcript encoding the circumsporozoite antigen of Plasmodium berghei utilizes heterogeneous polyadenylation sites. Mol Biochem Parasitol 1993;57:137 – 50. [62] Golightly LM, Mbacham W, Daily J, Wirth DF. 3%UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum. Mol Biochem Parasitol 2000;105:61 – 70. [63] Lanzer M, de Bruin D, Ravetch JV. Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates. EMBO J 1992;11:1949 – 55. [64] Weston D, La Flamme AC, Van Voorhis WC. Expression of Trypanosoma cruzi surface antigen FL-160 is controlled by elements in the 3% untranslated, the 3% intergenic, and the coding regions. Mol Biochem Parasitol 1999;102:53 – 66. [65] Decker CJ, Parker R. Diversity of cytoplasmic functions for the 3% untranslated region of eukaryotic transcripts. Curr Opin Cell Biol 1995;7:386 – 92. [66] van de Wiel DF, van Rijn PA, Meloen RH, Moormann RJ. High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system. J Mol Endocrinol 1998;20:83 – 98.