Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6363-6367, July 1992 Developmental Biology

Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multiftlis THEODORE G. CLARK*, ROYAL A. MCGRAWt, AND HARRY W. DICKERSON* *Department of Medical Microbiology, and tDepartment of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

Communicated by John R. Preer, Jr., April 6, 1992

role in the evasion of host immunity (6, 7). Parasite surface antigens and the i-antigens of ciliates share an interesting structural feature-namely, the presence of tandemly repetitive amino acid sequence domains (10-14). Nevertheless, they are clearly the products of different genes, and the molecular mechanisms responsible for their expression appear to be different. Whereas in the free-living ciliates surface antigen expression is readily reversible and controlled by changes in environmental conditions, in the parasites it occurs during the normal life cycle (either in a stage-specific manner or sequentially during chronic infection) and is developmentally regulated. In this regard, the common fish parasite Ichthyophthirius multiflujis (Ich) represents a bridge between the free-living ciliates and parasitic protozoa. As a holotrich ciliate, it is taxonomically related to both Paramecium and Tetrahymena (order Hymenostomatida) and, in addition to its obvious phenotypic similarity, has i-antigens directly analogous to those found on the free-living ciliates (15, 16). At the same time, Ich is an obligate parasite of fish and has a life cycle reminiscent of the protozoan parasites cited above. It was therefore of interest to examine the nature of the expression of i-antigen genes in this system. The work presented here shows that RNA transcripts encoding a predominant i-antigen of Icht undergo a dramatic change in abundance during the parasite life cycle, indicating that, in addition to control through environmental stimuli (as in Paramecium and Tetrahymena), i-antigen genes can be developmentally regulated. The fact that transcript levels increase in parallel with the infectivity of the organism bears on the functional role of surface antigens in this system and is consistent with previous observations suggesting that the i-antigens of Ich are involved in the development of protective immunity in fish (15, 17).

ABSTRACT A 1.2-kilobase (kb) cDNA encoding a major surface antigen of the holotrich ciliate Ichthyophthirius multiflUis (an obligate parasite of fish) has been isolated and used as a probe to examine the expression of immobilization antigen (i-antigen) genes in this system. The cDNA encodes a predicted protein of 394 amino acids with a tandemiy repeated structure characteristic of the i-antigens of the related free-living ciliates Paramecium and Tetahymena. As shown by Northern hybridization analysis with both total and poly(A)+ RNAs, the 1.2-kb cDNA recognizes distinct transcripts of 1.6 and 1.9 kb which are differentially expressed through the parasite life cycle. During the transition from the host-associated trophozoite stage to the infective tomite stage, steady-state levels of the 1.9-kb RNA undergo a marked increase of 250-fold, while the 1.6-kb transcript increases only slightly. The absolute amounts of RNA encoding the i-antigen have been quantitated and were found to reach extremely high levels equivalent to -6% of the poly(A)+ RNA of 1. mudifihiis tomites. Southern hybridization analysis with I. mulkifhlis genomic DNA suggests that at least two genes encode the i-antigen transcripts. In experiments to examine the effects of temperature on the expression of I. mulifiWis i-antigen genes, levels of the 1.6- and 1.9-kb transcripts were found to remain relatively constant in cells maintained at different temperature extremes. These studies indicate that genes encoding i-antigens of 1. multfilfis are developmentally regulated, and they suggest the existence of alternative mechanisms for the control of surface antigen expression in ciliates.

Variation in surface antigen expression in the free-living ciliates Paramecium and Tetrahymena is a well-studied phenomenon (for review, see refs. 1-3). The antigens themselves, known as immobilization antigens (or i-antigens), are encoded by a repertoire of genes that are expressed in a mutually exclusive fashion; only one type of antigen is made at any given time. While the expression of a particular antigen type is relatively stable under a given set of conditions, antigen switching can occur in response to environmental stimuli, most notably shifts in temperature. For example, Tetrahymena thermophila can express any of seven different antigen types under selected culture conditions, three being expressed at different temperatures (1). Although the mechanisms which control these variations are unknown, i-antigen synthesis appears to be regulated at the level of RNA transcription and/or processing (4, 5), and experimental alteration in i-antigen expression provides an extremely useful approach to the analysis of gene regulation in lower eukaryotes. The phenomenon takes on further significance in light of the ability of parasitic protozoa (including the trypanosomes, malarial plasmodia, etc.) to alter their surface antigens as well (6-9). In these systems, antigen diversity plays an important

MATERIALS AND METHODS Parasite Culture. Ich was maintained on channel catfish, Ictalurus punctatus, as previously described (15). For RNA isolations, trophozoites were obtained by gently scraping the surface of infected fish and collecting the freed parasites individually with a glass pipette. Tomites were obtained by allowing mature trophozoites to come off fish and go through normal rounds of cell division to form infective cells (see Fig. 2) (15). Isolation and Characterization of i-antigen cDNA. A custom-made cDNA library was prepared from trophozoite poly(A)+ RNA in A ZAP II phage by Stratagene and screened with an oligonucleotide probe derived from the N-terminal amino acid sequence of a predominant 48-kDa i-antigen from Ich. The antigen itself was isolated from Ich tomites by Abbreviations: i-antigen(s), immobilization antigen(s); Ich, Ich-

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thyophthirius multififfis.

*The sequence reported

in this paper has been deposited in the GenBank data base (accession no. M92907).

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affinity chromatography with a mouse monoclonal antibody (designated 1OH3) that strongly immobilized the Ich isolate used in these studies (18). After purification by SDS/PAGE, the N-terminal amino acid sequence of the 48-kDa antigen was determined by Edman degradation using Applied Biosystems 470A/477A automated protein sequencers (see Fig. 1). An "antisense" 24-mer oligonucleotide probe (5'AGCAGCACCAACATCAGTCAAACC-3') corresponding to eight amino acids (Gly-Leu-Thr-Asp-Val-Gly-Ala-Ala) near the N terminus of the protein was synthesized, endlabeled with 32p, and used to screen the cDNA library (19). Among a number of positive recombinants isolated, one (designated clone 2-3) contained a 1.2-kilobase (kb) EcoRP insert and was further analyzed. The full-length EcoRI insert and individual Pst I restriction fragments were subcloned in M13 phage, and the 1.2-kb cDNA was sequenced in its entirety off both strands by the dideoxy method of Sanger et al. (20). Northern Hybridization Analysis. Parasites were concentrated by centrifugation for 2 min at 1000 x g and total RNA was isolated from cell pellets after lysis in guanidine thiocyanate (21). Poly(A)+ RNA was purified by two rounds of chromatography on oligo(dT)-cellulose. Final concentrations of total and poly(A)+ RNA were determined by absorbance at 260 nm. RNA was fractionated by electrophoresis on 1.2% agarose gels containing 2.2 M formaldehyde and transferred to Biotrace nylon filters (Gelman) in 20x SSC (3 M NaCl/0.3 M sodium citrate, pH 7.0). Filters were hybridized with alkali-denatured probe (.106 cpm/ml), then washed at 65°C as described by Mahmoudi and Lin (22). Final wash buffer was 40 mM Na2HPO4/1% SDS/1 mM EDTA, pH 7.2. For

preparation of the probe, the 1.2-kb cDNA insert was purified by agarose gel electrophoresis and labeled to >109 cpm/,ug with [a-32P]dATP by using random oligonucleotide priming

(23).

QuaDitation of RNA Transcripts. In all cases, RNA levels were measured under hybridization conditions of probe excess. Changes in transcript prevalence during development were determined from densitometry scans of autoradiographic exposures of Northern blots in which equal amounts of RNA from different stages of the parasite were loaded. X-ray film strips with exposures in the linear range of response of the film were scanned spectrophotometrically at 470 nm and individual peak areas were measured and compared. Results were normalized to ethidium fluorescence in ribosomal RNA bands or to signals generated by hybridization of the same filters with a heterologous actin cDNA under conditions of reduced stringency (24). Absolute levels of RNA were determined by using a single-stranded RNA probe as described in Fig. 3. For probe synthesis, the 1.2-kb cDNA was subcloned in pBluescript by in vivo excision from A ZAP II DNA according to the supplier (Stratagene), and the orientation of the cDNA insert was determined by restriction endonuclease analysis. Antisense RNA was then synthesized under standard conditions from the T7 promoter flanking the insert (25) and the template was removed by digestion with RQ1 DNase (Promega). Southern Hybridization Analysis. Total genomic DNA was isolated from Ich tomites by methods previously described for Tetrahymena (26). This DNA was considered to be predominantly macronuclear in origin by analogy with other holotrich ciliates (27). DNA was digested to completion and

- P - P D O S Q T Q A O L S DV O A a D L O V P C P D G T Q T Q A G L T D V G A A D L G T C V N C R P N F Y Y N G G AA Q G I GAGCTGTTICCATGTCCTGATGGTACMTAGACTCAAGCTGGATCTGATGTAGGTGCTGCTGATICTGGTACTTGTGTTAATTGCAGACCTAAIT1TACTATAATGGTGGTGCTGCTrAAGGA I +XCV I V P C QI N R V G S V T N A G D L A T L A T E A N G N Q P F A A N N A A R AAAT TCVTCKPTTACTGACTTTAGCA GDCTAGVT VCTC CGCA T 126 GMGCTM1TGGTACTTT

GL IC

246

O C 8 T Q C P T G T A L D D G V T D V F D R 8 A A Q C V X C K P N F Y Y N aG TAGCCGCATAA70 TMATGCAGTACITTMTGTCCTACTGGCACCACTTACTOATGAA7GNACKAG

366

CCTTMGGTGAAGCCTGGCTTTAC I

486

GVAATLITATLCVKCR GCAGATCAGAANLGTACCAMTATnATQCSNQCPTGACTAVL

606 726 846

S

120

A G V A A V T S Q C V P C Q L N I N D 8 P A T Al ATGCAGGIECOGTACTAOZ&Glizh"TCATAQTA&qaAACgTIGCKAT

160

A G A Q A N L A T O C S. N Q C P T G S V L D D G V T L V F N T 8 A T L C V R C R

200

P N F Y Y N G G S P Q G E A P G V Q V F AA GA A A A G V A A V T S Q C V P C Q VS CMGTGTAIQDG CCTAAACTKANTATMTGGAACACCTTS

240

P Q G E A P G V Q V F A A G A

29M OIMG

L N K N D S P A T A G A Q A N L A T Q C 8 T Q C P T G T A I Q D G V T L V F S N 280 IXTGGCCOATCAAGCGGACACTTGvlTTlTAGTAAT CTMAACAAAAACGATTCTCCTGCCACTGCAGGTGCC TAGCTAATTAGCCACATAATGCAGTAiCTTAATGTCCAAC S S T Q C S Q C I A N Y F F N G N L E A G K S Q C L K C P V S R T T P A H A P G 320 C TACINTTGCATTIC NTAAAGKS CLKTTTAAGCCGTASTTAC ACPCAGGT TCATC

NTACTGCTQCATQCITTCCACAAGTACVDTAGAT ATCSCT

F

1086

80

T

N T A T Q A T Q C I T T C P A G T V L D D G T S T N F V A S A T E C T I C S A G 966

40

VTAGC

ATC

360

SACTG

394 F A S K T T G F T A G T D T C T E C T KX L T S G A T A V N I H Q CTTCTGT CCACAGCTAAACCTATGAAOTTCGAAAACAT mlGATCAAACACTGGTTACAMvCAGTACTGATACATG TACTGAMTTACTAAAATTA

1206

Jr rr%^T7mrT.A6,XZJ: ZuuuOzMV' unwv AI UV 1V ?gMACE§AUATQCSTQCPTGTALDVVD ICVPCGBIIM A1JCVK NFYYNGGSPQGEAPGVQVFAAGAA AAGVAAVTSQCVPCQLNKNDSPATAGAQANLATQCS00CPTG DDGV lV

AAGVAAVTSQCVPCQLNKNDSPATAGAQANLATQCSTQCPTGT

IDGVTLV!1SI

A

I

III Ill

FIG. 1. Nucleotide and deduced amino acid sequence of the Ich i-antigen cDNA. The complete nucleotide and deduced amino acid sequence ofthe 1.2-kb i-antigen cDNA is shown at the top. The N-terminal amino acid sequence of the 48-kDa antigen is on the first line; hyphens represent amino acids that could not be identified with certainty. The areas in the sequence that are boxed and designated by Roman numerals show the regions of extended homology within the deduced protein structure, and they are aligned relative to one another at the bottom part of the figure. Domains I and II, and II and III share 82% and 83% identity at the amino acid sequence level, respectively.

Developmental Biology: Clark et al. fractionated by electrophoresis in 0.7% agarose gels. Gels were stained in ethidium bromide, and the DNA was transferred to Zeta-Probe nylon membranes (Bio-Rad) as described by Rigaud et al. (28). Filters were prehybridized 5 hr at 650C in 6x SSC/10x Denhardt's solution (0.2% bovine serum albumin/0.2% Ficoll/0.2% polyvinylpyrrolidone)/ 0.1% SDS containing 10 ;kg of denatured herring sperm DNA per ml, then hybridized with alkali-denatured probe (.106 cpm/ml) for 24 hr under the same conditions. Filters were washed in two changes of 2x SSC/0.1% SDS at room temperature, followed by two changes of 0.2x SSC/0.1% SDS at 650C (15 min each).

RESULTS Isolation of i-antigen cDNA. i-antigens have recently been isolated from Ich tomites and identified as a pair of integral membrane proteins on the surface of the parasite. On affinity chromatography with immobilizing mouse monoclonal antibodies as the ligand, the two antigens copurify and have approximate molecular masses of 48 and 60 kDa on SDS/ PAGE (18). Oligonucleotide probes based on the N-terminal amino acid sequence of the predominant 48-kDa protein were used to screen an Ich cDNA library in A ZAP II, and a 1.2-kb cDNA encoding this antigen was isolated. The nucleotide sequence and corresponding amino acid sequence of the 1.2-kb cDNA are shown in Fig. 1. Beginning with a proline (one residue from the N terminus of the protein) there is exact correspondence between the amino acid sequence predicted from the cDNA and the actual amino acid sequence determined chemically from the purified i-antigen. The cDNA itself contains 1249 nucleotides and includes a long open reading frame (nucleotides 3-1187) encoding a deduced protein of 394 amino acids (estimated mass = 39,429 Da). There are 23 UAA triplets and 1 UAG triplet interspersed throughout the correct reading frame. The presence of a glutamine (residue 8) at a position which corresponds to a UAG triplet in the cDNA indicates that Ich uses a nonstandard genetic code shared by a number of other ciliates, in which UAA and UAG specify glutamine rather than acting as stop codons (for review, see ref. 29). The nucleotide sequence of the 1.2-kb cDNA contains 23 repeats of 10 nucleotides or more. Not surprisingly, this translates into a protein with a repetitive structure that includes three regions of extended homology (=80 amino acids in each) arranged in tandem within the central part of the deduced protein (Fig. 1). This type of repeat structure is characteristic of the G and the SerH3 surface antigens of Paramecium tetraurelia and Tetrahymena thermophila, respectively (10, 11). Differential Expression of i-antigen Genes. To examine the expression of i-antigen genes in this system, RNA from Ich was subjected to Northern hybridization analysis using the 1.2-kb cDNA as a probe. The results of these studies are shown in Fig. 2. Under conditions of high stringency (