J. Parasitol., 94(6), 2008, pp. 1269–1274 ᭧ American Society of Parasitologists 2008 IDENTIFICATION AND CHARACTERIZATION OF A SERPIN FROM EIMERIA ACERVULINA R. H. Fetterer, K. B. Miska, M. C. Jenkins, R. C. Barfield, and H. Lillehoj Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, U.S. Department of Agriculture, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, Maryland 20705. e-mail: [email protected] ABSTRACT: Serpins are serine protease inhibitors that are widely distributed in metazoans, but they have not been characterized previously in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed, and characterized. Random screening of an E. acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame consisting of 1,245 nucleotides that encode a peptide of 413 amino acids, with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55-kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 and 48 kDa in sporozoites (SZ) and merozoites (MZ), respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa that may correspond to secreted serpin. By immunostaining, the serpin was located in granules distributed throughout both the SZ and MZ, but granules seemed to be concentrated in the parasites’ anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis, the endogenous serpin in MZ extracts did not form the expected high-molecular-weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown, the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes. Although both coccidostats and live vaccines are widely used processes (Huber and Carrell, 1989; Irving et al., 2000; Roberts as control measures, coccidosis caused by several species of et al., 2004). However, there are members of the serpin family, Eimeria remains an economically important disease of poultry. such as chicken ovalbumin, thyroxin-binding globulin, and cor- Reduced weight gain, and decreased efficiency of feed conver- tisol-binding globulin, that do not inhibit proteases (Silverman sion associated with the disease, results in significant economic et al., 2001). Maspin, which blocks the motility of tumor cells, loss to the poultry industry (Williams, 1998; Allen and Fetterer, is included in this group of serpins (Sheng et al., 1996). 2002). The development of drug resistance that has been re- A gene encoding a 43-kDa serpin protein has been reported ported for many years continues to reduce the efficacy of com- from the parasitic protozoan Entamoeba histolytica. The recom- mon anticoccidials (Chapman, 1993; Stephen et al., 1997; Wil- binant serpin was not able to inhibit a panel of serine proteases, liams, 2006). Available vaccines, although effective, are costly, but the native 48-kDa protein secreted by the parasite interacted and they require the use of live parasites; due to antigenic strain with cathepsin G produced by lymphocytes in vitro (Riahi et variation, vaccine efficacy can differ geographically (McDonald al., 2004). et al., 1988; Fitz-Coy, 1992; Innes and Vermeulen, 2006; Morris Inhibitors of the serpin family have not been characterized et al., 2007). Although several recombinant vaccine candidate in Apicomplexa, although protease inhibitors have been report- proteins have been proposed, effective recombinant subunit ed. Multidomain Kazal type serine protease inhibitors (TgPI-1 vaccines have not yet been developed (Jenkins, 1998; Vermeu- and TgPI-2) were identified in Toxoplasma gondii (Morris et len et al., 2001; Innes and Vermeulen, 2006). al., 2002; Morris and Caruthers, 2003). TgPI-1, which inhibits The search for targets for development of new controls re- many serine proteases, is secreted from dense granules into the quires a detailed understanding of genes and proteins and their parasitophorous vacuole upon invasion of the host cell, and it expression during development of Eimeria species. The quest may play a role in regulation of invasion events. A 79-amino to characterize genes has been greatly enhanced by the produc- acid inhibitor with selectivity for inhibition of subtilisin, which tion of an 8.4-fold shotgun sequence of the approximately 55 may correspond to one domain of a Kazal type inhibitor, was megabase pairs of E. tenella genome (Shirley et al., 2004; identified in Neospora caninum (Bruno et al., 2004). http://www.Sanger.ac.uk/Projects/E࿞tenella). In addition, efforts Serpins have not been characterized previously from Eimeria to characterize expressed sequence tags (ESTs) from various species. In the present study, we present the cloning, sequenc- stages of E. tenella (Wan et al., 1999; Ng et al., 2002, Miska ing, and partial characterization of a serpin from E. acervulina. et al., 2004; Klotz et al., 2005) and more recently E. acervulina (Miska et al., 2008) have identified genes that may be important MATERIALS AND METHODS in understanding development of Eimeria spp. Host and parasites Examinations of ESTs from E. tenella (Ng et al., 2002; Miska Chickens (80–100 sex-sals, Moyers Hatcheries Inc., Quakertown, et al., 2004) have identified genes with strong homology to Pennsylvania), 4–5 wk of age, were infected with 1.0 ϫ 105 E. acer- serpin, although serpin was less than 2% of the total ESTs ex- vulina (strain 12) oocysts per bird, placed in feed. On days 6–7 post- amined (Ng et al., 2002). Serpins are part of a large superfamily immunization (PI), unsporulated oocysts were collected from feces of of proteins, usually consisting of 350–400 amino acids, which E. acervulina-infected birds. For studies of sporulation time course, un- sporulated oocysts were suspended in phosphate-buffered saline (PBS) often function as inhibitors of serine proteases and, by regulat- containing an antibiotic/antimycotic mixture (Invitrogen, Carlsbad, Cal- ing proteolytic cascades, are essential to many physiological ifornia) and incubated under aeration at 29 C. At the desired time in- terval (ranging from 0 to 24 hr), an aliquot containing about 1 ϫ 108 oocysts was removed from the incubation flask, centrifuged, and the Received 26 December 2007; revised 4 April 2008; accepted 4 April pellet containing oocysts was resuspended in 1.0 ml of 40 mM Tris and 2008. stored at Ϫ70 C. 1269 1270 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 6, DECEMBER 2008 Sporozoites (SZ) were prepared from fully sporulated oocysts as de- nitrilotriacetic acid column. The column was washed 5 times with native scribed previously (Fetterer and Barfield, 2003), and merozoites (MZ) binding buffer, followed by 5 washes with native wash buffer (12.3 mM were collected at 89 hr PI from the duodenum from birds inoculated NaH2PO4, 7.8 mM Na2H2PO4, 0.5 M NaCl, pH 6.3, and 8 M urea). with 1 ϫ 105 sporulated oocysts per bird and isolated as described Recombinant protein was eluted 5 times with 1 ml of elution buffer (20 previously (Fetterer et al., 2007). Isolated SZ and MZ were resuspended mM NaH2PO4, 0.5 M NaCl, pH 4.0, and 8 M urea). Recombinant pro- in 40 mM Tris and frozen at Ϫ70 C. tein was electrophoresed on 12.5% sodium dodecyl sulfate-polyacryl- amide gel electrophoresis gel, and visualized by staining with Coo- Cloning and expression of serpin gene massie Blue (Sigma). Escherichia coli Rosetta cells harboring nonre- cDNA library construction: Total RNA was isolated from frozen E. combinant pET28(a) or pQE30 vectors were used as negative controls. acervulina sporozoites using TRIzol (Invitrogen) by following manu- For use in protease inhibition assays, recombinant serpin (expressed in facturer’s recommended protocol. Total RNA was resuspended in 100 pQE30 vector) in elution buffer (3 ml) was made 5 mM with dithio- ␮l of diethyl pyrocarbonate-treated water. The integrity of the RNA was threitol (DDT) and dialyzed versus 20 mM Na2PO4 and5mMDTT, determined by electrophoresing 1.3 ␮g of denatured total RNA on 1% pH 7.5, for 3 hr at room temperature and then overnight at 4 C. The agarose gel. Approximately 1.3 ␮g of total RNA was used as template protein was then dialyzed overnight versus 20 mM Na2HPO4. Samples for cDNA synthesis. Double stranded cDNA was generated using the were concentrated to about 0.5 ml and stored at 4 C before use in assays. SuperScript Choice System for cDNA synthesis (Invitrogen) and oli- Protein concentration was estimated from predicted extinction coeffi- go(dT) primers. The ends of the double stranded cDNA were blunted cient. by incubating for 45 min at 37 C with 20 U of DNA Ligase and 8 U of T4 DNA polymerase (Hoffman-La Roche, Nutley, New Jersey). The Antibody production cDNA was ligated for 1 hr at room temperature into pBluescript II KSϩ Female New Zealand White rabbits, 9–11 kg and specific pathogen- vector (Stratagene, La Jolla, California) using T4 DNA ligase (Hoff- free (Charles River Laboratories, Inc., Wilmington, Massachusetts), man-La Roche). Vector DNA was digested with EcoRV (New England were bled via the central auricular artery, before immunization to obtain Biolabs, Ipswich, Massachusetts) and dephosphorylated with alkaline baseline control sera. Rabbits were immunized using the ImmuMax SR phosphatase (New England Biolabs) before ligation. Ligated cDNA was adjuvant system (Repros Therapeutics, Inc., The Woodlands, Texas; Fet- transformed into TOP10 Escherichia coli (Invitrogen).