Reduction in Cell Entry of Eimeria tenella (Coccidia) Sporozoites by Protease Inhibitors, and Partial Characterization of Proteolytic Activity Associated

with Intact Sporozoites and Merozoites Author(s): A. Lorraine Fuller and L. R. McDougald Source: The Journal of Parasitology, Vol. 76, No. 4 (Aug., 1990), pp. 464-467 Published by: The American Society of Parasitologists Stable URL: http://www.jstor.org/stable/3282822 Accessed: 23/09/2010 11:00
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J. Parasitol., 76(4), 1990, p. 464-467 ? American Society of Parasitologists 1990

TENELLA IN REDUCTION CELLENTRYOF EIMERIA (COCCIDIA) AND PARTIAL BY SPOROZOITES PROTEASEINHIBITORS, ACTIVITY OF CHARACTERIZATION PROTEOLYTIC AND MEROZOITES SPOROZOITES WITHINTACT ASSOCIATED
A. Lorraine Fuller and L. R. McDougald Department of PoultryScience, Universityof Georgia, Athens, Georgia 30602

The role of proteasesin the invasion of host cells by Eimeria tenella(Wisconsinstrain)was studied in vitro. Proteaseinhibitorswereused to treatsporozoitesbeforeinoculationor wereappliedto culturedchicken chlokidneycells beforeinfection.The inhibitorsantipain,leupeptin,aprotinin,L-l-tosylamide-2-phenyl-ethyl romethyl ketone (TPCK),or N-a-p-tosyl-L-lysinechloromethylketone (TLCK)reducedparasiteinvasion to of 16-66%of controlaftertreatmentof culturedcells or sporozoiteswith 5- or 50-,g/ml concentrations inhibitors fluoride(PMSF)reducedinvasion to 32-57.7% at concentrations in the culturemedium. Phenylmethylsulfonyl of 1-4 mM. The optimum pH for hydrolysisof azocaseinby intact sporozoitesor merozoiteswas determined over a rangeof pH 5.0 to pH 9.0. Sporozoiteswere highly active over a broad rangefrom pH 5.5 to pH 9.0, with an apparentoptimum at pH 8.0. Merozoiteshad a much lower specificactivitywith pH optima at 7.0 and 8.5. The proteaseactivity of sporozoitesor merozoitescould be inhibitedcompletelyby the addition of 50 ,ug/ ml of leupeptin,TPCK, or TLCK or of 4 mM PMSF. Antipain inhibited proteasesof sporozoitesbut not of merozoites. Pepstatin had little effect on either sporozoites or merozoites. The results suggest that parasite proteasesof Eimeria may be necessaryfor invasion of host cells.
ABSTRACT:

The apicomplexan sporozoa possess an intricate system at the anterior of the sporozoite or merozoite called the apical complex that is important to entry into the host cell. Associated with the apical complex is a system of rhoptries, saccules that empty their contents during cell entry and during the rupture of the schizont (Jensen and Hammond, 1975; Jensen and Edgar, 1976). There is good evidence that proteases are important in cell invasion and schizont rupture of Plasmodium knowlesi (Hadley et al., 1983). Recent work by Adams and Bushell (1988) demonstrated that penetration by Eimeria vermiformis sporozoites could be blocked or reduced by treatment of the cell monolayers and sporozoites with various inhibitors of proteolytic activity. In the present work we describe the effects of various protease inhibitors on cell invasion by Eimeria tenella sporozoites, protease activity of intact and lysed sporozoites and merozoites, and the effects of inhibitors on the protease activity of intact merozoites and sporozoites, and we have obtained an apparent pH optimum for proteolytic activity of sporozoites and merozoites.

rozoites were releasedby treatmentof the sporocysts for 60 min with 0.25%trypsinand 0.50%taurodeoxycholic acid in Hanks' balanced salt solution (HBSS). and Sporozoiteswerecleaned5 times by centrifugation resuspendedin HBSS. Furthercleaning was accomplished by vacuum filtrationthrough a Nalgene cellulose filter support pad (Nalgene, Rochester, New experiments, monolayers York).In the cell penetration were infected with 100,000 sporozoites/mlof culture media. Merozoites were obtained from 2-wk-old broiler E. chickensinoculatedwith 4.6 x 105 sporulated tenella oocysts. Ceca were removed on the seventh day, opened,and the contentsdiscarded.Cecaltissues were washedwith phosphate-buffered saline, pH 7.2 (PBS), mincedwith scissors,and incubatedat 40 C for 30 min in HBSS containing0.25% trypsin and 0.50% taurodeoxycholate.The digestatewas then filteredthrough cheese cloth to remove large debris and then passed, using vacuum, througha Nalgene cellulose filtersup(200 portpad. Penicillin(200 U/ml) and streptomycin were added to the cleaned merozoites, which ,Ag/ml) essentiallywere free of host cell and bacterialcontamination.
Cell cultures

Primary chick kidney cells were prepared from 7-day-oldchicksas previouslydescribed(McDougald, 1978). Cells were platedat a density of 200,000 cells/ ml in 6-well culture plates (Coming, Coming, New York) containing22-mm coverslips.Cells were incuAND MATERIALS METHODS bated in RPMI-1640, 5%fetal bovine serum, and 50 Parasites ,ug/mlgentamycin (GIBCO Laboratories,Grand Is85%conOocysts of the Wisconsin strain of E. tenella were land, New York)for 60 hr to approximately broken by shakingwith 1-mm glass beads, and spo- fluencybeforeuse in the penetrationassay.
Protease inhibitors

Received 11 August 1989; revised 29 March 1990; accepted5 April 1990.

464

Antipain, L-l-tosylamide-2-phenyl-ethyl chloromethyl ketone (TPCK), N-a-p-tosyl-L-lysinechloro-

AND AND FULLER McDOUGALD-PROTEASES PENETRATION TENELLA 465 INE.

methylketone(TLCK),pepstatin,leupeptin,and aprotinin (Sigma,St. Louis, Missouri)were preparedin 50 in and 5 ,ig/ml concentrations PBS. Phenylmethylsulfonyl fluoride(PMSF)(Sigma)was first solubilizedin dimethylformamide (DMF) and used in 4.0, 2.0, and 1.0 mM concentrationsin PBS. DMF was added to control culturein equivalentamounts. PBS at pH 6.0 was used to approximatethe pH of the ceca.
Testing the effect of protease inhibitors on penetration of cultured cells

points from pH 5.0 to 9.0. The effectsof various protease inhibitors on casein hydrolysiswere studied by incubating106sporozoitesand 107merozoiteswith 50 ,tg/ml of each inhibitor with azocasein in PBS at pH 7.0.
RESULTS

To test the effect of various proteaseinhibitors on the penetrationof cells, we incubatedsporozoitesfor 30 min with proteaseinhibitorsin PBS(pH 6.0), rinsed out any residualinhibitorby repeatedcentrifugation, resuspended sporozoitesin PBS,andinoculatedmonolayerswith 100,000 sporozoites/mlof culturemedium. To test the effects of proteaseinhibitors on monolayers,we incubatedmonolayerswith proteaseinhibitorsfrom 10 min priorto inoculationwith sporozoites to 2 hr postinoculation. were terminated2 hr postinoculation. Experiments Monolayerswerewashedwith HBSS,fixed for 10 min with ice-cold methanol, and then washed twice with HBSS.Coverslipswereincubatedat room temperature for 1 hr with mouse anti-sporozoite primaryantibody. After washing4 times with HBSS, coverslipswere incubatedfor 1 hr with secondaryantibodyconsistingof fluorescein goat isothiocyanate (FITC)-conjugated antimouse IgG. Coverslipswere washed with HBSS and mounted using a glycerol-PBS9:1 medium for fluorescence microscopy. Intracellularsporozoites were countedusinga LeitzOrtholuxmicroscopewith a 490nM dichroicfluorescencefilter.
Analysis

Incubation of tissue culture cells and sporozoites with the peptide protease inhibitors antipain, leupeptin, pepstatin, and aprotinin at 50 or 5 ,ug/ml resulted in invasion of 50-66% of controls (Table I). TPCK at 50 or 5 ,tg/ml inhibited invasion to 26.8 and 62.7% of control, respectively; whereas TLCK at 50 or 5 t,g/ml inhibited invasion to 16.1 or 28.8% of control. Incubation of sporozoites with inhibitors and washing before inoculation also reduced the numbers of intracellular sporozoites significantly (Table II). PMSF (when incubated with sporozoites) at 4, 2, or 1 mM inhibited invasion by 32.6, 40.6, or 50.8% of control, and incubation of tissue culture cells at the same concentrations inhibited invasion by 51.8, 37.5, or 22.1% of control (Table III). Intact sporozoites had 2.11 x 10-3 units of activity per 106 cells, whereas merozoites had 1.08 x 10-3 units/107 cells (Table IV). Antipain, leupeptin, TPCK, and TLCK at 50 jug/ml or PMSF at 4 mM reduced protease activity of sporozoites by 100%, whereas pepstatin reduced activity by about 39%. Neither antipain nor pep-

Twenty random fields each containing approximately 100 kidney cells were counted for each group, and a percentinvasion relative to control was calculated for each inhibitor.All estimates of cell penetration were based on 6 replicationsin 3 experiments. Data wereanalyzedby generallinearmodelsand Duncan's multiple rangetest (P < 0.05).
Protease activity associated sporozoites and merozoites with

statin appeared to inhibit merozoite protease activity, but leupeptin, PMSF, TPCK, and TLCK were 100% effective at the level tested. Proteolytic activity associated with sporozoites was prominent from pH 5.5 to pH 9.0 with a peak at about 8.0 (Table V). Activity associated with merozoites was much lower, with apparent peaks of activity at pH 7.0 and 8.5 (Table V).
DISCUSSION

Freshly preparedsporozoites and merozoites were washed extensively with HBSS with 0.025% sodium azide solution and diluted to either 106 or 107 cells, respectively,per milliliterof azocaseinsolution. Azocasein (Sigma E'1366= 36) was preparedin a concentrationof 10 mg/ml in eitheracetate-buffered saline or PBS depending upon the pH desired. Samples were incubatedat 41 C with constantagitationfor 180 min; then 1-ml samples were precipitatedon ice with 0.25 ml of 25%trichloroacetic acid (TCA) and centrifuged at 13,000 g for 4 min in a Beckmanmicrocentrifuge Palo Alto, California). The ab(BeckmanInstruments, sorbance of soluble azopeptides in the TCA-soluble supernatant portionwas recordedat 366 nm. The rate of casein hydrolysis was calculatedin units where 1 unit of activityis thatwhichreleased1 mg/hrof soluble azopeptides(Starkey,1977). The pH optimum for casein hydrolysiswas determinedafterincubationof 107 cells for both merozoites and sporozoites at pH half

In Apicomplexa, host cell invasion appears to occur in 2 steps; the apical end of the parasite attaches to the cell membrane, then the sporozoite quickly enters the cell (Bannister et al., 1975). In eimerians, rhoptry saccules associated with the apical complex appear to empty during invasion (Jensen and Edgar, 1976). In species of Plasmodium there is a specific attachment phase involving glycosylated proteins (Hadley et al., 1986; Perkins and Holt, 1988) and a concomitant invasion phase that requires a proteolytic activity (Hadley et al., 1983). From the present results and the study of the invasion

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VOL. THE OF JOURNAL PARASITOLOGY, 76, NO.4, AUGUST 1990

I. Effect of protease inhibitors on cultured TABLE III. Effect of phenylmethylsulfonyl fluoride (PMSF)on sporozoitepenetration(percent conof cells (percentof control + SE).
TABLE

trol + SE).
% Control* Treatmentt Control Antipain Pepstatin Leupeptin Aprotinin TPCK TLCK 50 tg/ml -t + + + + + + 5 #Ag/ml -t + + + + + + PMSF concentration Sporozoites incubated for 30 min 0 mM 1 mM 2 mM 4 mM Inhibitor added to culture medium 0 mM 1 mM 2 mM 4 mM % Control*

53.7 53.14 55.65 49.06 26.82 16.09

7.71? 5.22? 12.81? 8.61? 6.53? 3.62?

62.69 66.07 58.93 67.06 62.73 28.84

8.18? 3.39? 8.06? 7.03? 4.64? 8.34?

50.77 + 18.18 (3) 40.57 + 3.83 (3) 32.58 + 7.05 (2) -: 22.09 + 5.37 (6) 37.45 + 6.42 (6) 51.82 +?6.53 (5)

* Percent of control calculated using mean of 6 experiments. Twenty random fields of approximately 100 cells were counted and averaged. t TPCK, L- l-tosylamide-2-phenyl-ethyl chloromethyl ketone; TLCK, N-a-P-tosyl-L-lysine chloromethyl ketone. : Control mean = 5.73 sporozoites/100 cells. ? Significantly different from control at P < 0.05 (F = 9.08).

* Percentage of control calculated using mean of number of experiments in parentheses. Twenty random fields of approximately 100 cells were counted and averaged. t Control mean = 1.47 + 0.08. t Control mean = 5.73 + 0.36.

of E. vermiformis, there is strong evidence that a similar process is involved in the eimerian sporozoites, where possible proteolytic activity is essential to invasion. The lower protease activity seen in merozoites suggests either that the proteases are of less importance in invasion, that azocasein is not a suitable substrate for merozoite proteases, or that merozoites simply possess less proteolytic activity. Differences in azocasein hydrolysis between merozoites and sporozoites were evident in the effects of the inhibitor antipain and in the apparent pH optima of intact merozoites and sporozoites. Proteases are most commonly classified as to reactive groups within the specificity region of the protein. PMSF is an inhibitor of serine proteases and pepstatin inhibits carboxyl proteases. TPCK, TLCK, antipain, and leupeptin inhibit both serine and cysteine proteases (Barrett, 1977;

North, 1982). In the work presented here, all classes of inhibitors to these reactive groups reduced the invasion of host cells to some extent. TPCK, TLCK, and pepstatin appeared to be more effective when applied directly to host cells, whereas antipain was more effective when applied to sporozoites, although all inhibitors were effective with either application method. All inhibitors reduced hydrolysis of azocasein by 100%, except pepstatin, which inhibited hydrolysis by 39% in sporozoites and 25% in merozoites, and antipain, which inhibited hydrolysis by 14% in merozoites. From the information in these experiments, it would be difficult to classify the activity seen in sporozoites and merozoites to any 1 of these classes. The results of the present study with E. tenella agree generally with the

TABLE Effectof proteaseinhibitorson hydrolysis IV. TABLE Effect of protease inhibitors on sporo- of azocaseinby sporozoitesand merozoites.* II. zoites (percentof control + SE).
Units (x 10-3) of azocaseinolytic activity % Control* Treatmentt Control Antipain Pepstatin Leupeptin Aprotinin TPCK TLCK 50 ug/ml -30.05 57.7 72.6 55.18 64.4 41.24 5 jsg/ml -t + 11.76 + 31.93 + 6.89 + 7.05 + 7.36 + 0.24 Control Antipain Leupeptin Pepstatin PMSF TPCK TLCK 2.11 0 0 1.29 0 0 0 1.08 1.23 0 0.93 0 0 0 Inhibitort Sporozoites (106) Merozoites (107)

+ + + + + +

11.51 9.36 5.31 11.99 3.78 8.02

(3)? (3)? (3)? (2)? (3)? (2)?

40.6 73.67 64.01 32.58 40.8 29.13

(3)? (2)? (3)? (3)? (3)? (2)?

* Percent of control calculated using mean of number of experiments in parentheses. Twenty random fields of approximately 100 cells were counted and averaged. t TPCK, L-l-tosylamide-2-phenyl-ethyl chloromethyl ketone; TLCK, N-a-P-tosyl-L-lysine chloromethyl ketone. f Control mean = 1.47 + 0.08. ? Significantly different from controls at P < 0.05 (F = 3.27).

* Rate of casein hydrolysis was determined by incubation of intact sporozoites or merozoites with 10 mg/ml azocasein at 41 C, precipitation with TCA, and measurement of the absorbance of the acid-soluble supernatant at 366 nm. t Added to incubation mixture at 50 ug/ml, except for PMSF, which was used at 4 mM. PMSF, phenylmethylsulfonyl fluoride; TPCK, L-1tosylamide-2-phenyl-ethyl chloromethyl ketone; TLCK, N-a-P-tosylL-lysine chloromethyl ketone.

FULLER AND McDOUGALD-PROTEASESAND PENETRATION E. TENELLA IN

467

TABLE V.

pH optima for azocaseinolyticactivity of

Sporozoites (units x 10-3) 5.46 11.50 12.50 14.30 16.60 16.80 18.80 16.80 17.80 Merozoites (units x 10-3) 0.73 0.11 0.60 0.26 1.79 0.76 0.43 1.38 0.40

LITERATURE CITED
ADAMS,J. H., ANDG. R. BUSHELL.1988. The effect

sporozoites and merozoites.*

pH 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

of proteaseinhibitorson Eimeria vermiformis invasion of cultured cells. International Journal for Parasitology 18: 683-685. BANNISTER, L. H., G. A. BUTCHER, E. D. DENNIS, AND G. H. MITCHELL.1975. Structure and invasive

behavior of Plasmodiumknowlesimerozoites in vitro.Parasitology71: 483-491.

A. BARRETT, J. 1977. Introduction to the history and classification of tissue proteinases. In Proteinases in mammalian cells and tissues, A. J. Barrett (ed.). Elsevier/North Holland Publishing Company, Amsterdam, p. 1-55. ANDL. H. MILLER. 1983. HADLEY, T. J., M. AIKAWA,

* Casein hydrolysis was determined by incubation of sporozoites and merozoites at 41 C with 10 mg/ml azocasein in acetate of phosphatebuffered saline to obtain the desired pH, precipitation with trichloroacetic acid, and measurement of the absorbance of the acid-soluble supematant portion at 366 nm.

Plasmodiumknowlesi:Studieson invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors. Experimental Parasitology 55: 306-311. AND L. H. MILLER. 1986. In, F. W. KLOTZ, vasion of erythrocytes by malaria parasites: A cellular and molecular overview. Annual Review of Microbiology 40: 451-477. JENSEN, J. B., AND S. A. EDGAR. 1976. Possible se-

results of studies with E. vermiformis (Adams and Bushell, 1988) and P. knowlesi (Hadley et al., 1983). In E. vermiformis, the inhibitors TPCK, TLCK, and PMSF were the most effective with reductions of 39.8, 34.9, and approximately 75%, respectively, in intracellular sporozoites. In the present work, all of the inhibitors reduced penetration to 70% of control or greater at the highest levels tested and, with the exception of pepstatin, inhibited the hydrolysis of azocasein by 100%. The most potent inhibitor in both E. vermiformis and E. tenella was PMSF. The virulence of strains of Entamoeba histolytica proteases can be correlated with a protease activity that is inhibited by alpha-2-macroglobulin and alpha- 1-antiprotease (Lushbaugh et al., 1981). In Babesia bovis there is a similar relationship between parasitic protease content and the pathogenicity of the strain (Wright et al., 1981). Other proteases of protozoal parasites have been implicated in resistance to drugs (Mahoney and Eaton, 1981). Thus far we have not compared strains of Eimeria to correlate protease activity with resistance. The importance of protease activity for cell entry in the apicomplexans suggests a new target for drug development or use of these specific proteins for vaccine production. ACKNOWLEDGMENTS This work was supported by state and Hatch funds allocated to the Agricultural Experiment Stations of the University of Georgia. We are grateful to Xie Ming Quan, Tsuneo Fukata, and Jeff Gilbert for providing the merozoites used for this study.

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1981. Inhibition of Entamoeba histolyticacytotoxin by alpha- 1-antiprotease and alpha-2-macroglobulin. American Journal of Tropical Medicine and Hygiene 30: 575-585. MAHONEY, J. R., AND J. W. EATON. 1981. Chloroquine resistant malaria: Association with enhanced plasmodial protease activity. Biochemical and Biophysical Research Communications 100: 1266-1271. McDOUGALD,L. R. 1978. The growth of avian Ei-

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