INFECTION AND IMMUNITY, OCt. 1994, p. 4667-4670 Vol. 62, No. 10 0019-9567/94/$04.00+0 Copyright X) 1994, American Society for Microbiology

Arginine Aminopeptidase, an Integral Membrane Protein of the Cryptosporidium parvum Sporozoite PABLO C. OKHUYSEN,1 HERBERT L. DuPONT,1 CHARLES R. STERLING,2 AND CYNTHIA L. CHAPPELL'* Center for Infectious Diseases, The University of Texas Health Science Center-Houston Medical School and School ofPublic Health, Houston, Texas,1 and Department of Veterinary Science, University ofArizona, Tucson, Arizona2 Received 20 April 1994/Returned for modification 22 June 1994/Accepted 1 August 1994

Cryptosporidium parvum oocysts were studied for the expression of aminopeptidase by using amino acids bound to the synthetic fluorescent substrate 7-amino-4-trifluoromethyl coumarin. After 1 h of incubation, intact oocysts showed no activity; however, homogenization and solubilization with Triton X-114 followed by phase separation yielded a 22-fold increase in aminopeptidase activity in the detergent fraction. With arginyl-6-amino-2-styrylquinoline as a substrate, aminopeptidase activity was observed in permeabilized oocysts and freshly excysted sporozoites but not on intact oocysts or empty oocyst membranes after excystation. These results suggest that C. parvum expresses an arginine aminopeptidase that is an integral protein of the sporozoite membrane.

Cryptosporidium parvum, a small coccidian parasite, is a by using detergent-compatible reagents (Bio-Rad, Hercules, frequent cause of chronic diarrhea in individuals with ad- Calif.) (7). vanced human immunodeficiency virus infection (16). Immu- AP activity was evaluated with amino acids (Arg, Phe, Ala, nocompetent hosts experience self-limited diarrhea when they Leu, Gly) bound to the fluorescent substrate 7-amino-4- become infected through contact with animals (12), during trifluoromethyl coumarin (AFC; Enzyme Systems Products, foreign travel (22, 29), or in large-scale outbreaks in the Dublin, Calif.). Fluorometer tubes contained a total volume of community (13, 15, 19). 500 ,ul consisting of 480 ,ul of 0.15 M PBS (pH 7.2), 10 ,ul of C. Parasite proteases are important in degradation of nutrient parvum extract, and 10 RI of substrate (final concentration of proteins (8), adhesion and invasion of host tissues (4, 18), 20 mM). Tubes containing buffer solution, extracts, and the evasion of host immunity (18, 24), and resistance to antimicro- NH-blocked substrate z-Arg-AFC were used as negative con- bial agents (23). Aminopeptidases (APs) are important in the trols. Tubes were incubated at 37°C for 1 and 5 h, and activity life cycles of Schistosoma mansoni (31) and Brugia pahangi (20) was measured with a fluorometer (McPhearson, Acton, Mass.). and have been described for Babesia, Eimeria, and Plasmodium Fluorescent units were compared with a standard curve cre- spp. (2, 9, 17, 26, 28, 30). Differences in alanine AP expression ated with known amounts of AFC, and the AP activity was differentiate pathogenic from nonpathogenic trichomonads converted to nanomolar AFC per hour per milliliter per (21) and may be a virulence marker. The purpose of this study microgram released. was to test for AP activity in a C. parvum strain known to be The topographic localization of AP was investigated with an pathogenic to calves and humans (14). insoluble fluorescent substrate that precipitates at the site of C. parvum oocysts (Iowa strain) from infected newborn calves were purified as previously described (3) and kept in 2% potassium dichromate. Oocysts were washed in sterile phos- phate-buffered saline (PBS), and the concentration was esti- 4- mated by a hemacytometer. Sporozoites were isolated as INTACTOOCYSTS described previously (27). Bacterial contamination was ex- cluded by incubating aliquots (25 ,ul) of the extracts into FROZENWHAWED OOCYSTS blood-agar plates for 48 h at 37°C. 3- HOMOGEN0 OOCYSTS Proteins were extracted by freeze-thawing and homogeniza- tion in a tissue grinder on ice in 10 mM PBS with or without detergents. For phase partition studies, preparations were E 0 2- centrifuged for 20 min at 10,000 x g at 5°C. The superna- U- tant was removed, and the-pellet was resuspended in PBS with c) 1% Triton X-114 and then centrifuged at 100,000 x g for 1 h m at 5°C to remove particulate material. The supernatant was 1 placed over a 6% sucrose cushion and subjected to phase partitioning (6). Protein extraction for each step was assessed

0 rI 0 1 5 * Corresponding author. Mailing address: The University of Texas Health Science Center-Houston, School of Public Health, W 736, Hours 1200 Hermann Pressler Dr., Houston, TX 77030. Phone: (713) 792- FIG. 1. Arginine AP activity of C. parvum oocysts. Experiments 4451. Fax: (713) 792-4403. were performed in triplicate.

4667 4668 NOTES INFECT. IMMUN.

TABLE 1. Substrate specificity of C. parvum AP: results of duplicate experiments in triplicate Substrate AP activity (mean ± SD) Relative % (AFC) (nmol of AFC h- ml-l) activity Arginine 23.64 ± 1.88 100 Alanine 19.63 ± 1.88 83 Phenylalanine 12.73 ± 3.22 54 Glycine 11.72 ± 1.67 49 Leucine 7.095 ± 2.63 30

cleavage (arginyl-6-amino-2-styrylquinolone [Arg-6-SAQ]; En- zyme Systems Products). C. parvum preparations were incu- bated with 20 mM Arg-SAQ substrate for 30 to 45 min at room temperature. After incubation, the solution was transferred to a slide and counterstained with 10% Evans blue prior to right and fluorescent microscopy. Preparations incubated with 10% commercial bleach or without the substrate were used as negative controls. After oocyst purification, the visually estimated bacterial contamination of oocyst preparations was less than 0.1% and was nonviable after culture. Optimal protein yield was ob- tained by homogenizing oocysts previously permeabilized by a FIG. 2. Localization of arginine AP in C. parvum oocysts. Oocysts freeze-thaw cycle. Homogenization in the presence of Triton were incubated with the insoluble substrate arginine 6-amino-2- X-100 and Nonidet P-40 was not inhibitory; however, these styrylquinolone and visualized with fluorescent microscopy as de- detergents did not solubilize the AP. Solubilization in the scribed in the text. (A and B) Intact oocysts under light (A) and presence of sodium dodecyl sulfate was inhibitory to the fluorescent microscopy (B). (C and D) Oocyst permeabilized by freeze-thawing under light (C) and fluorescent microscopy (D). Bar, enzyme. 5 ,Lm. After 5 h of incubation, oocyst homogenates yielded the most activity, while the solutions containing intact oocysts and permeabilized oocysts demonstrated 32 and 60% of this activ- ity, respectively (Fig. 1). No activity was seen for N-blocked empty oocyst walls demonstrated minimal residual fluores- z-Arg-AFC substrate, confirming the lack of endopeptidase cence (Fig. 3C). activity. C. parvum AP preferentially cleaved Arg-AFC (Table Members of the Apicomplexa produce proteases that are 1). vital to parasite survival and host cell invasion (1, 17). How- No proteolytic activity was found when detergent-free su- ever, no proteases have previously been described for C. pernatants of C. parvum homogenates were assayed for up to parvum. 5 h. Partial solubilization of the enzyme was achieved by We have demonstrated the presence of a C. parvum-specific homogenization of oocysts in Triton X-114. After phase sepa- AP that preferentially cleaves N-terminal arginine from a ration (Table 2), the detergent-rich fraction yielded 19% of the synthetic substrate. The activity is associated with sporozoites total activity, representing a 22-fold increase in specific argi- during and after excystation but is not found associated with nine AP activity. Thirty-seven percent of the total activity was the outer wall of intact oocysts or the inner structures of empty solubilized by this method, and the remainder was accounted oocyst walls. It is unlikely that bovine host proteases were for in the detergent-insoluble pellet. acquired by the oocysts during their passage in the hosts' Intact oocysts did not demonstrate SAQ activity (Fig. 2A gastrointestinal tracts since intact oocysts failed to demon- and B); however, time-dependent SAQ deposition was ob- strate external proteolytic activity. The low-level proteolytic served for freeze-thaw permeabilized oocysts (Fig. 2C and D). activity seen in the preparations containing intact oocysts after During early excystation, structures in the apical complex lengthy incubation at 37°C likely reflects partial excystation, a could be discerned. As sporozoites exited the oocyst wall, AP phenomenon known to occur at 37°C in the absence of further activity was localized to the sporozoite (Fig. 3A). Sporozoite treatment (10, 11). These results strongly suggest that the AP rounding was seen after 45 min of incubation (Fig. 3B). The activity is expressed by sporozoites and not on the external

TABLE 2. Triton X-114 phase separation of C. parvum membrane proteinsa

Preparation Total AP activity Protein content % Protein Specific AP activity Fold increase in (nM AFC h-1) (jg) recovery (nM AFC h- jLg-') AP activity Oocysts homogenized in Triton X-114 420,559 1,894 100 222 1 Aqueous phase 0 181 9.5 0 0 Sucrose interface 69,600 31 1.63 2,245 10.1 Detergent phase 78,639 16 0.84 4,914 22.1 Resuspended pellet 279,999 871 46 321 1.4 a Approximately 108 oocysts were used. VOL. 62, 1994 NOTES 4669

proteinase activity is necessary for parasite adherence to epithelial cells. Infect. Immun. 57:2991-2997. 5. Bouvier, J., C. Bordier, H. Vogel,R Reicheelt, and R. Etges. 1989. Characterization of the promastigote surface protease of Leish- mania as a membrane-bound zinc endopeptidase. Mol. Biochem. Parasitol. 37:235-245. 6. Bovier, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256:1604-1607. 7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 8. Chappell, C., and M. Dresden. 1986. Schistosoma mansonii: pro- teinase activity of "Hemoglobinase" from the digestive tract of adult worms. Exp. Parasitol. 61:160-167. 9. Charet, P., E. Assisi, P. Maurois, S. Bouquelet, and J. Biguet. 1980. Aminopeptidase in rodent Falciparum. Comp. Biochem. Physiol. 65B:519-524. 10. Current, W. 1986. Cryptosporidium: its biology and potential for environmental transmission. Crit. Rev. Environ. Control 17:21- 51. 11. Current, W. L 1990. Techniques in laboratory maintenance of Cryptosporidium, p. 31-49. In J. P. Dubey, C. A. Speer, and R. Fayer (ed.), Cryptosporidiosis of man and animals. CRC Press, Inc., Boca Raton, Fla. 12. Current, W. L, N. C. Reese, J. V. Ernst, W. S. Bailey, M. B. Heyman, and M. D. Weinstein. 1983. Human cryptosporidiosis in immunocompetent and immunodeficient persons: studies of an FIG. 3. Localization of arginine AP during C parvum excystation. outbreak and experimental transmission. N. Engl. J. Med. 308: (A) AP activity as sporozoites exit the oocyst. (B) Increased fluores- 1252-1257. cence and swelling of a free sporozoite. (C) Free sporozoites after 13. D'Antonio, R G., R E. Win, J. P. Taylor, T. L Gustafson, W. L excystation with minimal residual fluorescence of the empty oocyst Current, and M. M. Rhodes. 1986. A waterborne outbreak of wall. Bar, 5,um. cryptosporidiosis in normal hosts. Ann. Intern. Med. 103:886- 888. 14. DuPont, H., C. Chappell, C. Sterling, P. Okhuysen, J. Rose, and of the oocyst. It should be noted, however, that internal W. Jakubowski. 1994. Infection of Cryptosporidium parvum for surface adult humans. Clin. Res. 42:285. of the sporozoite may also contribute to membrane structures 15. Edwards, D. 1993. Troubled waters in Milwaukee. ASM News 59: the AP activity seen in parasite extracts. 342-345. The preferential Triton X-114 detergent phase partitioning 16. Fayer, R, C. A. Speer, and J. P. Dubey. 1990. General biology of suggests that C. parvum arginine AP is an integral membrane Cryptosporidium, p. 1-29. In J. P. Dubey, C. A. Speer, and R. Fayer protein. Similar hydrophobic separations have been described (ed.), Cryptosporidiosis of man and animals. CRC Press, Inc., for surface proteins important in the parasite-host interaction, Boca Raton, Fla. such as the major surface metalloprotease of Leishmania spp. 17. Fuller, A., and L. McDougald. 1990. Reduction in cell entry of (5) and the variant surface glycoprotein surface proteins of Eimeria tenella (coccidia) sporozoites by protease inhibitors, and Trypanosoma spp. (25). partial characterization of proteolytic activity associated with The role of AP activity in C. parvum is as yet unclear. intact sporozoites and merozoites. J. Parasitol. 76:464-467. 18. Hadley, T., M. Airkawa, and L Miller. 1983. Plasmodium knowlesi: excystment are consis- Temperature and pH requirements for studies on invasion of rhesus erythrocytes by merozoites in the tent with the involvement of proteases located within the presence of protease inhibitors. Exp. Parasitol. 5:306-311. oocyst (16). It is possible that the sporozoite AP may partici- 19. Hayes, E. B., T. D. Matte, T. R O'Brien et al. 1989. Large pate in hatching, i.e., excystation. At present, there is no community outbreak of cryptosporidiosis due to contamination effective therapy for human immunodeficiency virus-related of a filtered public water supply. N. Engl. J. Med. 320:1372- cryptosporidiosis. The identification of enzymes that mediate 1376. potential virulence traits or essential metabolic pathways 20. Hong, X., J. Bouvier, M. Wong, G. Yamagata, and J. McKerrow. may provide new targets for the treatment of cryptospori- 1993. Brugia pahangi: identification and characterization of an diosis. Further studies of this unique C. parvum AP are aminopeptidase associated with larval molting. Exp. Parasitol. warranted. 76:127-133. 21. Lockwood, B., M. North, K. Scott, A. Bremmer, and G. Coombs. 1987. The use of a highly sensitive electrophoretic method to This study was sponsored in part by a grant from the Environmental compare the proteinases of trichomonads. Mol. Biochem. Parasi- Protection Agency. tol. 24:89-95. 22. Ma, P., D. L Kaufman, C. G. Helmick, A. J. D'Souza, and T. R Navin. 1985. Cryptosporidiosis in tourists returning from the REFERENCES Caribbean. N. Engl. J. Med. 312:647. 1. Adams, J., and G. Bushell. 1988. The effect of protease inhibitors 23. Mahoney, J., and J. Eaton. 1981. Chloroquine resistant malaria: on Eimeria vermiformis invasion of cultured cells. Int. J. Parasitol. association with enhanced plasmodial protease activity. Biochem. 18:683-685. Biophys. Res. Commun. 100:1266-1271. 2. Aissi, E., and P. Charet. 1981. Proteolytic systems in Babesia 24. Marikovski, M., R Arnon, and Z. Fishelson. 1988. Proteases hylomysci. Comp. Biochem. Physiol. 15:491-495. secreted by transforming schistosomula mansoni promote resis- 3. Arrowood, M., and C. Sterling. 1987. Isolation of Cryptosporidium tance to killing by complement. J. Immunol. 141:273-278. oocysts and sporozoites using discontinuous sucrose and isopycnic 25. McConville, M. 1991. Glycosylated phosphatidylinositols of percoll gradients. J. Parasitol. 73:314-319. trypanosomatidae, p. 286-304. In G. Coombs and M. North (ed.), 4. Arroyo, R, and J. Aldrete. 1989. Trichomonas vaginalis surface Biochemical parasitology. Taylor & Francis, London. 4670 NOTES INFEcr. IMMUN.

26. Riddles, P., and R. Blakely. 1991. Proteinases of Babesia, p. dium chabaudi and Babesia hylomusci. J. Protozool. 30:376-382. 281-285. In G. Coombs and M. North (ed.), Biochemical parasi- 29. Soave, R., and P. Ma. 1985. Cryptosporidiosis: traveler's diarrhea tology. Taylor & Francis, London. in two families. Arch. Intern. Med. 145:70-72. 27. Riggs, M., and L. Perryman. 1987. Infectivity and neutralization 30. Vander Jagt, D., B. Baack, and L. Hunsaker. 1984. Purification of Cryptosporidium parvum sporozoites. Infect. Immun. 55:2081- and characterization of an aminopeptidase from Plasmodium 2087. falcipanum. Parasitology 10:45-54. 28. Slomianny, C., P. Charet, and G. Prensier. 1983. Ultrastructural 31. Xu, Y., and M. Dresden. 1986. Leucine aminopeptidase and localization of enzymes involved in the feeding process in Plasmo- hatching of Schistosoma mansoni eggs. J. Parasitol. 72:507-511.