Variation in primary sequence and tandem repeat copy number among i-antigens of Ichthyophthirius multifiliis

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Abstract

The immobilization antigens (i-antigens) of Ichthyophthirius multifiliis are potential vaccine candidates for the prevention of ‘white spot’ disease in freshwater fish. These antigens vary with respect to antigenicity and molecular mass, and at least five i-antigen serotypes have been identified among parasite isolates thus far. In previous studies, the gene and corresponding cDNA encoding a ∼48 kDa i-antigen from parasite isolate G1 (serotype A), had been cloned and sequenced. We now report on the isolation of two new genes, designated IAG52A[G5] and IAG52B[G5], encoding ∼52/55 kDa i-antigens from a parasite isolate representing a different serotype, namely, D. Based on their deduced sequences, the ∼52/55 kDa gene products have the same structural features as the 48 kDa protein including hydrophobic N- and C-termini, periodic cysteine residues with the potential for metal binding, and tandemly repetitive amino acid sequence domains that span their length. Nevertheless, the products of these genes vary in their tandem repeat copy number, and share only ∼50% homology overall. When expressed in heterologous systems, the products of the newly described genes react strongly with monospecific polyclonal antisera against the i-antigens of serotype D and are clearly i-antigens. It would nevertheless appear that mRNA transcripts from the two genes are present at widely different levels within parasites themselves. Analysis at the protein level using 2-D SDS-PAGE would further suggest that multiple i-antigens are expressed within the same serotype at any given time.

Introduction

As the etiologic agent of ‘white spot’ disease in freshwater fish, the parasitic ciliate, Ichthyophthirius multifiliis, is a major problem in commercial aquaculture worldwide [1], [2]. The parasite is highly virulent and readily kills fish. Nevertheless, animals that recover from infection develop acquired immunity, and recent work has identified a family of abundant GPI-anchored membrane proteins (referred to as immobilization antigens, or i-antigens) as important targets of the host immune response [3], [4], [5], [6]. Based on passive immunization experiments, monoclonal antibodies against these proteins confer strong protection against an otherwise lethal parasite challenge [4], [5]. Furthermore, the proteins themselves induce active immunity when administered directly to naive fish (Wang and Dickerson, unpublished).

While the i-antigens constitute important vaccine candidates, serotypic variation among these proteins exists within natural populations. Using antisera that immobilize Ichthyophthirius in a strain-specific manner, five i-antigen serotypes (referred to as A–E) have been now been identified [7], [8]. Antisera against a given strain cross-react on Western blots with i-antigens of all serotypes indicating that the proteins share linear epitopes in common [3], [7]. The determinants responsible for immobilization are nevertheless distinct for each serotype and are conformational in nature. Passive immunization studies have suggested that these epitopes are essential for the development of protective immunity [4], [5]. Along with differences in antigenicity, the proteins vary in size and number. Depending on serotype, i-antigens migrate on one-dimensional SDS-PAGE as either one or two polypeptides with apparent molecular mass in the range of ∼40–60 kDa [3].

Since differences among these proteins may be of critical importance in the development of effective vaccines, efforts to clone and characterize i-antigen genes of different parasite strains are currently underway. In this regard, the gene encoding a ∼48 kDa i-antigen of parasite isolate G1 (serotype A) has recently been isolated [9], [10]. Based on its deduced sequence, the product of this gene (IAG48[G1]) contains hydrophobic signal peptides at its N- and C-termini, as well as a series of tandem repeats of about 80 amino acids each that span its length [10]. The repeats themselves are characterized by periodic cysteine residues that fall into register when the homologous segments are aligned. The spacing of these cysteines, namely, C–X2,3–C, within the higher order framework, C–X2–C–X20–C–X3–C–X20–C–X2–C, has suggested that the i-antigens may be metal-binding proteins [10]. This type of structure is common to the i-antigens of free-living ciliates [11], [12], and is reminiscent of surface coat proteins of more distantly related parasitic protozoa (most notably the variant-specific proteins (VSPs) of Giardia lamblia [13], [14], [15]).

Although the gene for the ∼48 kDa i-antigen of serotype A offers useful information regarding the overall structure of these proteins, little is known about the features that distinguish i-antigens of different serotypes from one another. Furthermore, serotype A occurs relatively infrequently in nature, and the gene for the 48 kDa protein may be of limited use in vaccine development. We report here on the isolation of genes encoding two ∼52/55 kDa i-antigens from a member of what appears to be the most common serotype, namely D [8]. Isolation of these new genes provides a basis for the first comparison between i-antigens of different serotypes, and suggests that multiple i-antigens are coordinately expressed within the same serotype during at least one stage of the parasite life cycle.

Section snippets

Parasite strains

I. multifiliis strains G1 and G5 were maintained on juvenile channel catfish as previously described [10]. These strains are representative of i-antigen serotypes A and D, respectively [8]. Based on one-dimensional SDS-PAGE and Western blotting analysis, serotype A antigens migrate as two polypeptides of apparent Mr of 48- and 60-kDa, while serotype D proteins run as a single, relatively broad band estimated previously as ∼52/55 kDa [3], [4], [6].

Library construction and isolation of a partial IAG52A[G5] cDNA

A size-selected cDNA library in λZAPII

Isolation of a ∼52/55 kDa i-antigen gene from serotype D (IAG52A[G5])

To obtain i-antigen gene sequences from serotype D, a cDNA library from the G5 parasite strain was screened with a radiolabelled probe that hybridized with the gene encoding the 48 kDa i-antigen of parasite isolate G1 (serotype A). Of roughly 2×104 phage clones screened, only three scored positive, and one was subjected to further analysis. This clone was found to contain a 1.2 kb insert with an open-reading frame extending 1052 nt from its 5′ end. The coding region ended in consecutive TGA

Discussion

Initital attempts to isolate and characterize i-antigen genes from serotype D met with limited success. Although not described here, these efforts began with screens of G5 genomic DNA libraries using IAG48[G1] as a probe. A few weakly hybridizing clones were identified in such screens, but none showed homology to the IAG48[G1] gene itself. As indicated in this report, attempts to amplify i-antigen coding sequences from G5 genomic DNA using degenerate primers were also unsuccessful, giving rise

Acknowledgements

We thank Annemieke Jansens for her contributions during cDNA library construction. We also thank Georgina Cheng, Yelena Bisharyan and Jane Noe for their excellent technical assistance, along with Dr James Casey, Rebecca Murcek, and Carrie Markowski for their helpful suggestions in the use of TaqMan® Real Time PCR. This work was supported by USDA NRICGP grants #97-35204-4481 and #98-35204-6812.

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    Note: Nucleotide sequence data reported in this paper are available in the GenBank™ database under the accession numbers: AF324424; AF405431.

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