Serine Protease Activity in Developmental Stages of Eimeria Tenella

J. Parasitol., 93(2), 2007, pp. 333–340 ᭧ American Society of Parasitologists 2007 SERINE PROTEASE ACTIVITY IN DEVELOPMENTAL STAGES OF EIMERIA TENELLA R. H. Fetterer, K. B. Miska, H. Lillehoj, and R. C. Barfield Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, United States Department of Agriculture, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, Maryland 20705. e-mail: [email protected] ABSTRACT: A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor- sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)–sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined. Infection of chickens by several species of Eimeria causes rozoites and involved in cellular invasion. Results of a recent malabsorption, reduced weight gain, and decreased efficiency characterization of genes differentially expressed during spor- of feed conversion, resulting in significant economic losses to ulation of E. tenella indicated transcripts of several proteases the poultry industry, which are estimated to be over 800 million present in E. tenella (Miska et al., 2004). These include the dollars annually (Williams, 1998; Allen and Fetterer, 2002). Be- serine proteases subtilisin, rhomboid protease, prolyl endopep- cause they are intracellular parasites, poultry coccidians, like tidase and a metallo-protease, aminopeptidase N. Recently, a other apicomplexans, must have highly developed mechanisms rhomboid protease from E. tenella was cloned, expressed, and to invade and survive in host cells (Kim, 2004; Caruthers and localized to sporozoites (Li et al., 2006). In addition, an ami- Blackman, 2005). Along with intracellular invasion, other com- nopeptidase related to aminopeptidase N was purified and char- plex mechanisms are involved in Eimeria spp. survival, includ- acterized during sporulation of E. tenella, confirming a role for ing control of sporulation in the environment, evasion of host this protease in parasite development (Fetterer and Barfield, immune responses, successful reproduction, and nutrition (Al- 2003). Proteases are, therefore, potentially critical to many fac- len and Fetterer, 2002; Klemba and Goldberg, 2002). ets of the parasite’s survival including excystation, differentia- Recent research on the role of serine proteases in apicom- tion, immune invasion, and nutrition (Klemba and Goldberg, plexans has centered primarily on protein processing and other 2002). However, occurrence and function of proteases during functions related to intracellular survival. These have been rel- sporulation and in the sporozoite and merozoite stages are atively well investigated in Toxoplasma gondii and Plasmodium largely unknown. The current study presents an initial charac- species (Shaw et al., 2002; Kim, 2004; Withers-Martinez et al., terization of serine protease activity in E. tenella developmental 2004; Dowse and Soldati, 2005; O’Donnell and Blackman, stages. 2005). Recent studies have shown that serine protease inhibitors can prevent invasion of T. gondii tachyzoites (TZ) into host MATERIALS AND METHODS cells both in vitro and in vivo and that serine proteases related Bioinformatic analysis to rhomboid proteases or subtilisins are involved in protein processing of micronemes and other proteins that are essential for Sequence homologies to known serine proteases in the E. tenella invasion (Miller et al., 2001, 2003; Kim, 2004; Dowse and Sol- genome were investigated by performing a BLAST (Altschul et al., 1997) search using protein sequences representative of the serine dati, 2005). protease families from the MEROPS database (Rawlings et al., 2004) Presumably the invasion events in Eimeria spp. intracellular as a query against the sequence of the partial E. tenella genome stages are similar to those observed in other apicomplexans, located at the SANGER Institute (http://www.sanger.ac.uk/cgi-bin/ but the details of protease involvement in molecular events con- blast/submitblast/e-tenella/omni). trolling sporulation and other developmental processes are lack- ing. A study by Fuller and McDougald (1990) showed that ser- Host and parasites ine proteases related to trypsin may play a role in sporozoite Chickens (80–100 sex-sals, Moyers Hatcheries, Quakertown, Penn- invasion of host cells since classical serine protease inhibitors sylvania), 4–5 wk of age, were infected with 1.0–1.25 ϫ 105 E. tenella reduced invasion of E. tenella sporozoites into cells in vitro. In (Wampler strain) oocysts per bird, mixed in the feed. On day 7 post- inoculation (PI), birds were killed by cervical dislocation, and the cecae addition, a 20 kDa trypsin-like protease was partially purified were removed. Oocysts were recovered from infected cecae and spor- from E. tenella sporulated oocysts (Michalski et al., 1994), fur- ulated as previously described (Fetterer and Barfield, 2003). ther suggesting that a serine protease may be present in spo- To obtain oocysts in various stages of sporulation, unsporulated oocysts were suspended in PBS containing an antibiotic/antimycotic mix- ture (GIBCO, Gaithersburg, Maryland) and incubated under aeration at Received 28 December 2005; revised 3 August 2006; revised 21 Sep- 41 C. At the desired time interval (ranging from 0 to 72 hr), an aliquot tember 2006; accepted 21 September 2006. containing about 1 ϫ 108 oocysts was removed from the incubation 333 334 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 2, APRIL 2007 flask and centrifuged. The oocyst pellet was resuspended in 1.0 ml 40 of the protease inhibitor 4-(2-Aminoethyl) benzenesulphonyl fluoride mM Tris, pH 8.0, and stored at Ϫ70 C. (AEBSF, Sigma) dissolved in AB. The assay was incubated from 2 to Sporozoites (SZ) were prepared from fully sporulated oocysts (less 14 hr at 37 C. After incubation, 100 ␮l of cold 4.5% trichloroacetic than 30 days postharvest) as previously described (Fetterer et al., 2004). acid with 0.5 M NaCl was added to each reaction and the sample kept Merozoites (MZ) were collected from cecae at 110 hr PI from birds at 4 C for 30 min. The sample was centrifuged at 10,000 g for 5 min inoculated with 3 ϫ 105 sporulated oocysts per bird. MZ were isolated at 4 C. The supernatant was diluted with 180 ␮l of 0.5 M Tris, pH 8.0. and purified as described for SZ. Isolated SZ and MZ were resuspended The fluorescence was read in a microtiter plate fluorometer at an exci- in 40 mM Tris and frozen at Ϫ70 C. tation wavelength of 485 nm and emission wavelength of 538 nm. The amount of fluorescein released was estimated from a standard curve, Reverse transcription polymerase chain reaction (RT-PCR) and results expressed as nmol released per mg protein. All parasite material used in RNA isolation was snap frozen follow- Electrophoresis ing purification and stored at Ϫ70 C until use. Total RNA was isolated from MZ, SZ, unsporulated oocysts (0 hr), and sporulating oocysts (12– Protein samples were analyzed by polyacrylamide gel electrophoresis 48 hr), as well as fully sporulated oocysts (72 hr) using TRIzol (Invi- using 1-mm-thick gradient gels (8 ϫ 9 cm, 4–12% Bis-Tris, Invitrogen) trogen, Carlsbad, California). Each sample of oocysts or SZ was com- as described (Fetterer and Barfield, 2003). Western blot analysis was bined with approximately 3 g of diethylpyrocarbonate (DEPC) treated performed using the basic method previously described (Fetterer and Pyrex beads (3 mm diameter) (Corning, New York, New York) and 10 Barfield, 2003), except that the primary antibody consisted of a 1:500 ml of TRIzol. The samples were vortexed for 1 min, then incubated on dilution of mouse monoclonal antibody against a recombinant E. tenella ice for 1 min (4ϫ). MZ samples were treated directly with TRIzol microneme protein, MIC2 (Tomley et al., 1996; Lillehoj et al., 2005). without employing beads. The remainder of the total RNA isolation Substrate gels consisted of casein imbedded 12% Tris-glycine gels protocol was carried out using the

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