Cultured Host Cells* E

189

ENHANCEMENT BY LYSOZYME AND HYALURONIDASE OF THE PENETRATION BY TOXOPLASMA GONDII INTO CULTURED HOST CELLS* E. LYCKE, EBBA LUND AND 0. STRANNEGARD From the Municipal Virological Laboratory, Gothenburg, Sweden

Received for publication August 17, 1964 LYSOZYME, a basic polypeptide, is lytic for certain strains of bacteria and is antivirally active. It occurs in tissues and body fluids, e.g. in serum, and is supposed to be of importance in the mechanism of native immunity. In connection with a study on the influence of different serum factors on the infectivity of toxoplasma parasites for cultured cells (Lycke, Lund, Strannegard and Falsen, 1964) the effect of crystallized egg-white lysozyme on the parasites was examined. No antiparasitic activity of lysozyme could be demonstrated but in the presence of lysozyme the number of parasites which penetrated the host cells was increased. A similar enhancement of the penetration of parasites was demonstrable with another mucolytic enzyme tested, hyaluronidase. The present report describes and discusses the penetration-promoting effects exerted by lysozyme and hyaluronidase.

MATERIAL AND METHOD The parasite suspensions. The RH strain of Toxoplasma gondii was used. The preparation of suspensions of toxoplasma parasites from mouse peritoneal exudate has been described previously (Lycke and Lund, 1964a). The suspensions employed contained 4-8 x 106 to 1-44 x 107 parasites per ml. If not otherwise stated, the suspending medium was the same as the cell culture medium described below. The HeLa cell cultures.-HeLa cells were cultured as monolayer cultures in Gey chambers. The medium employed consisted of 0 5 per cent lactalbumin hydrolysate in Hanks' salt solution, to which 20 per cent dye test negative human serum, 100 i.u. penicillin and 100 pg. streptomycin per ml. were added. The culture technique and the standardization of the cultures have been reported (Lycke and Lund, 1964a, b). The cell culture test.-The parasite suspension was mixed with an equally large volume of a solution in Hanks' buffer of the compound to be tested. A set of three cultures was inoculated with the mixture, each culture receiving 0 4 ml. as replacement for the culture medium. The cultures were incubated at 370 for 19 hr. unless otherwise stated. At the readings, the relation between the number of parasites in the inoculum having penetrated the host cells and the number of host cells exposed was determined. The figure expressing this relation is referred to as the relative number of infective units (RNIU). The accuracy of the RNIU determinations, the relation of the RNIU to the concentration of infective parasites and the use of the cell culture method for determination of the growth rate of parasites have been described previously (Lycke and Lund, 1964b). The compounds tested.-The lysozyme used was an egg-white preparation. (Muramidase twice crystallized, Worthington Biochem. Corp. Freehold, New Jersey, U.S.A.). The other compounds tested were obtained from the following sources: Hyaluronidase: Nutritional Biochem. Corp. Cleveland, Ohio, U.S.A.; Hyaluronic acid: A.B. Leo, Halsingborg, Sweden; Polymyxin: Novo Industri A/S, Copenhagen, Denmark; Protamine sulphate: L. Light * This study was supported by a PHS research grant, AI 05074-02, from the National Institute of Allergy and Infectious Diseases, Public Health Service, U.S.A. 190 E. LYCKE, EBBA LUND AND 0. STRANNEGARD

and Co. Ltd., Colnbrook, England; Sodium ethylenediamine tetra-acetate (EDTA), Titriplex: E. Merck AG, Darmstadt, Germany; RDE (Receptor destroying enzyme) Vibrio cholera filtrate: N. V. Philips-Duphar, Amsterdam, Holland; fl-amylase: L. Light and Co. Ltd., Colnbrook, England; Phospholipase: Venom (Crotateus Adamanteus), Sigma Chem. Co., St. Louis, U.S.A. The absorption with bentonite.-Bentonite was employed for the removal of lysozyme. The method used for absorption was in principle that of Myrvik and Weiser (1955). Four mg. bentonite washed in Hanks' solution were employed for the absorption of 1 mg. lysozyme.

EXPERIMENTS The penetration by toxoplasma in the presence of lysozyMe.-Lysozyme was added to suspensions of toxoplasma parasites which were tested in cell cultures. It was found that in the presence of lysozyme the number of parasites which penetrated the host cells was considerably increased (Fig. 1). The use of 1 mg. 100 _

80 -

60 -4

40 -

20 - 00

1 3 6 Hours FIG. 1. The effect of lysozyme on penetration by toxoplasma into HeLa cells. The relative number of infective units, (RNIU) is plotted against the incubation time after inoculation of the cell cultures with parasites. The stippled curve refers to the results obtained with cultures to which lysozyme (1 mg./ml.) was added. The fully drawn curve shows the results obtained with cultures without lysozyme. lysozyme per ml. of the parasite suspending fluid resulted in 20-60 per cent larger RNIU values than were obtained with suspensions without lysozyme. This effect was demonstrable whether or not the suspensions contained serum in addition to lysozyme (Table I). TABLE I.-Penetration by Toxoplasma of HeLa Cells in the Presence of Lysozyme (1 mrg./ml.) Parasite suspending fluid RNIU Hanks' solution . 55* 2 Hanks' solution + lysozyme . 75 * 7 Normal human serum . . 62- 7 Normal human serum + lysozyme 98- 4 PENETRATION OF TOXOPLASMA INTO CULTURED CELLS 191

The relation of the penetration-promoting effect to the concentration of lysozyme used is illustrated by Fig. 2. The concentrations of lysozyme tested ranged from 0.001-10 mg. per ml. of the parasite suspension. In the figure, the concentrations of lysozyme are plotted against the mean values of the RNIU in 4 experiments. Concentrations of 0.01 mg. per ml. or less caused no effect on the penetra-

0 01 1 0 10.0 mg. ml. FIlo. 2. The effect of different concentrations of lysozymne on penetration by toxoplasma into HeLa cells. The concentration of lysozyme (mg./ml. of inoculated parasite suspension) is l)lotted against the relative number of infective units (RNIU). tion, but when amounts larger than 0*01 mg. were tested the RNIU increased proportionately to the concentration of lysozyme. Absorption with bentonite inhibited the penetration-promoting effect of lysozyme (Table II).

TABLE II.-Inhibition of the Penetration-promoting Effect of Lysozyme by Absorption with Bentonite Parasite suspending fluid RNIU Cell culture medium . . 37 * 9 Cell culture medium + lysozyme 54 8 Cell culture medium + lysozyme, absorbed with bentonite . 39 :2

The penetration-promoting effect of lysozyme supplemented with protamine sulphate, EDTA or polymyxin.-Like lysozyme, protamine sulphate is a basic protein and has in certain cases a similar effect (Pontieri, Imperato and Cavallo (1962). Sodium ethylenediamine tetra-acetate (EDTA) and polymyxin are known to influence the action of lysozyme on the cell walls of bacteria (Repaske, 1956, 1958; Warren, Gray and Yurchenco, 1954). In a series of experiments the effect of these compounds on the penetration-promoting activity oflysozyme was studied. 192 E. LYCKE, EBBA LUND AND 0. STRANNEGARD

Lysozyme, protamine sulphate and polymyxin were each used in a concentration of 1-0 mg. per ml. of the parasite suspension inoculated into the cultures. EDTA was employed in a concentration of 0.001 M. A serum-free suspension of parasites was used. The results obtained are listed in Table III.

TABLE III.-The Effect on the Penetration by Toxoplasma of HeLa Cells when Adding Lysozyme, Protamine Sulphate, EDTA or Polymyxin Compounds tested RNIU Protamine sulphate 51*9 Protamine sulphate + lysozyme 61-0 Lysozyme ...... 80- 2 Control. No additive 51 * 6 EDTA . 54-1 EDTA + lysozyme . 62 * 8 Lysozyme . 69- 4 Control. No additive . . 51 2 Polymyxin. a5 1 Polymyxin + lysozyme 68- 7 Lysozyme 66- 7 Control. No additive . 538

None of the three compounds tested seemed per se to affect the penetration of toxoplasma or to enhance the penetration-promoting effect of lysozyme. On the other hand the use of protamine sulphate together with lysozyme seemed to inhibit the effect of the enzyme. The effect of hyaluronidase on the penetration of toxoplasma parasites.-Like lysozyme hyaluronidase was found to promote the penetration of toxoplasma parasites into HeLa cells. The RNIU values found when 1 mg. hyaluronidase per ml. was added to the suspending media were usually about the double of those obtained in tests without the enzyme. The effect of 0.01 mg. hyaluronidase corresponded to that of about 1 mg. lysozyme. When lysozyme and hyaluronidase were used together, a synergistic effect was noted (Table IV). Thus with the combination of 0 05 mg. of lysozyme and

TABLE IV.-The Effect of Hyaluronidase, Used Alone or in Combination with Lysozyme, on the Penetration of Toxoplassma into HeLa Cells Compounds tested RNIU Lysozyme (0 5 mg./ml.) . . . . 71*9 Hyaluronidase (05 mg. /ml.). . . 102 - 6 Lysozyme (05 mg./ml.) + Hyaluronidase (0 005 mg./ml.) . 73-3 Lysozyme (U 05 mg./ml.) + Hyaluronidase (0 05 mg./ml.) 125-7 Lysozyme (0 005 mg./ml.) + Hyaluronidase (0 05 mg./ml.) 87 * 9 Control. (No additive) . 49 9 hyaluronidase respectively, a considerably larger number of parasites were found to penetrate the host cells than if each enzyme had been used separately in a concentration 10 times higher. Table V lists the results of an experiment where hyaluronic acid was added to the parasite suspension at a concentration of 1 mg./ml. ofthe inoculated suspension. The acid was also used mixed with hyaluronidase, 0-1 mg./ml., or lysozyme, 1 mg./ml. Hyaluronic acid inhibited the effect of hyaluronidase as well as that of PENETRATION OF TOXOPLASMA INTO CULTURED CELLS 193 TABLE V.-Inhibition by Hyaluronic Acid of the Penetration-promoting Effect of Lysozyme and Hyaluronidase Compounds tested RNIU Hyaluronidase (O 1 mg./ml.) . . . . . 98* 6 Hyaluronidase (0 1 mg.n/ml.) + hyaluronic acid (1 mg.//ml.) . 614 Lysozyme (1 mg./ml.) ...... 70 0 Lysozyme (1 mg./ml.) + hyaluronic acid (1 mg./ml.) . 58-5 Hyaluronic acid (1 mg./ml.) . . . . . 54*2 Control. No additive ...... 53.2 lysozyme, although the latter enzyme less effectively. However, in the concentration used the hyaluronic acid itself did not affect the penetration of the parasites. The effect of treatment of host cells or parasites with lysozyme or hyaluronidase prior to the inoculation.-A number of cell cultures were exposed to lysozyme or hyaluronidase at 370 for 2 hr. and then washed in culture medium before being inoculated with parasites. A concentration of 1 mg. of the enzyme tested per ml. of the cell culture medium was used. The results listed in Table VI show that washing of the cells after exposure to the enzymes eliminated the enhancement of penetration.

TABLE VI.-The Effect of Treatment of HeLa Cell Cultures with Lysozyme or Hyaluronidase Prior to Inoculation of Toxoplasma Parasites Treatment of host cells RNIU Lysozyme ...... 69- 8 Lysozyme and washing 1 x ...... 59.7 Untreated control ...... 57.5 Hyaluronidase ...... 79 3 Hyaluronidase and washing 1 x . . . . 75-1 Hyaluronidase and washing 3 x . . . . . 54.5 Untreated control ...... 53*0

When the parasites instead of the host cells were pretreated with enzyme, similar results were obtained. Table VII shows the results of an experiment in which parasites were suspended in a medium containing 1 mg. of lysozyme and then incubated at 370 for 1 min. or 1 hr. After the incubation the parasites were washed twice. Each time they were centrifuged at 440 g. for 20 min. and resus- pended in medium. Suspensions of parasites to which no lysozyme had been added were treated in the same way as those supplied with lysozyme and served as controls. The parasites were counted before inoculation ofthe suspensions and differences in the concentration of parasites were adjusted by addition of small amounts of medium. Cell cultures were then inoculated with the suspensions and the RNIU was determined. The same number ofparasites which gave an RNIU value of 55- 1 with parasites washed after exposure to lysozyme for 1 hr. gave when inoculated without any preceding attempts to remove the lysozyme an RNIU value of 69*8. Table VII shows that the values obtained with parasites washed after exposure to lysozyme were higher than the values of the corresponding control tests. Most of the lysozyme effect disappeared however if the parasites were washed after treatment with the enzyme. 194 E. LYCKE, EBBA LUND AND 0. STRANNEGARD TABLE VII.-The Effect of Treatment of Toxoplasma Parasites with Lysozyme Prior to Inoculation into HeLa Cell Cultures Time of incubation in min. Lysozyme RNIU 1 . + . 53-5 1 . - . 47-3 60 + . 55-1 60 . - . 46 6 The penetration in the presence of lysozyme or hyaluronidase by parasites treated with ultraviolet light or formalin.-Three ml. of a parasite suspension were exposed to UTV-light (30 W Philips "Sterilamp") at a distance of 30 cm. for one hour. As a result the concentration of parasites was reduced from 9 0-4-8 x 106 organisms per ml. The parasite suspension to which 1 mg. of lysozyme per ml. was added was inoculated into cell cultures. The RNIU as well as the number of parasites formed in the cultures were estimated. Tests using non-irradiated parasites instead of the same number of irradiated parasites were included as controls in the experiment. The RNIU values obtained are listed in Table VIII. TABLE VIII.-The Effect of Lysozyme on the Penetration of Ultraviolet-irradiated Toxoplasma Parasites into HeLa Cells Inoculum Lysozymne (1 mg./ml.) RNIU Irradiated parasites . . + 47.4 32-5 Untreated parasites . . + 53-3 35.4 On an average, 22 per cent of the intracellular non-irradiated parasites were found to have divided during the observation period, whereas the corresponding figure for the irradiated parasites was 5-3 per cent. Lysozyme enhanced the penetration of the irradiated parasites almost as well as it did that of the parasites not treated with ultraviolet light (Table VIII). In additional experiments parasites were treated with 10 per cent formalin for 2 min. at room temperature and were washed 3 times in cell culture medium. They were then tested in cell cultures in the presence of 1 mg. of lysozyme or hyaluronidase per ml. of inoculum. In the same way as in the preceding experiment controls with untreated parasites were included. To check that formalin remaining after the washings should not have destroyed activity of the enzymes, controls with formalin-treated human erythrocytes were carried out as well. For these control tests the erythrocytes were treated with formalin and washed in the same way as the parasites. The treated erythrocytes were mixed with untreated parasites and inoculated into cell cultures. The same number of parasites, whether formalin-treated or not, were inoculated in all the tests. The results of the RNIU determinations are found in Table IX. The test with untreated parasites to which no enzyme had been added showed that 16 per cent of the intracellular parasites had divided during the incubation period. Of the intracellular, formalin-treated parasites, only 0*8 per cent were found to have multiplied, and the RNIU values were reduced from 33-7-15*2 by the formalin-treatment. The addition of the enzymes also had an evident effect on the penetration of the formalin-treated parasites. Thus the RNIU values increased from 15-2-31-8 and 33-6 in the presence of lysozyme or hyaluronidase respectively. PENETRATION OF ToXOPLASMIA IN-TO CtULTUIREI) CE LLS 195v

'A BLE IX. The Effect of Lysozyjme or Hyaluronidaise on the Penetration I,y Formiain -tracted Toxoplasma Par(rsites into HeLa (ells 1 110()1111110l A(1(litix ' IIN-II Fl r1alllinl-treatdptarelIlasites 3 1 IUntreated PlxasiteS 1'S(o'-Ille 50-4 ForImalini-treated pa)llaSites t1tIuIIrolli(dIase .33 t; Unltreatedl P)aIasitesi *HH-lulItironidase 152- FoIIllnl' -treated1 pra)i1Sites *-No ad1lditive No ad(d(litive 33 * 7 Utr1eate(dPalrasites 51 -3 FoirIIalin-t reatedl cythll(oytes IltIiteate(t pIarasites H-Ivaluroniidase F()1rlllinl-trleateld (erytllTr(otes llultreated( lpalasites No aI(ldditive 33-4 Attem)ipts to detect at penetrtation-prom.otin(y elect of preparations of lysed toxo- plaQsatw para.sites.-.Some 2-4 , 106 *washed toxoplasnia palasites w-ere lsed in distilled w-ater aid tflien tnltrasoinicated for 1 5 mimi. at full pow-er in a 200 W.. 10 kc. magynetostictive Raytheon oscillator. The pre)aratiolls tltins hlomogeniize(l. 2-4 ml. eaclh, w\ere cen-trifuigedI at :2500 g. for 20nmiii. The supernatants obtainiedl w-ere ad(led aloireo1 towether-ith l%sozvme to suspensions of parasites tested in cell cnltures. TABLE X. The, Effect oJ al Preparation of Lysedl Toxoplasmiil,a Parasites, Used Alone, or in (Comiibination with Lysozymte. on the Penetration by Toxoplasm%iia, into HeLa (1ells A(ldit ive LysedI paaI sites. 1 1 59-6 d 1: 5 .) Lxy( p)arasites, ' LV,-sed p1aIasites, I: -1, lysozymne (1 Ilng. /Inl.) 82 -*, LVSO3Z7JVIie (1 Ing./1111.) 74 0 (Cotrol. -No additive 5)4- 8

Table X siummarizes the results of oiie experimenit. As cani be seeni in the table, the results may ind(licate the presence of a penietrationi-promotiing activity in prepalatioiis of lysed p)arasites. The enhl-ancemenit of the penietrationi w-as more evidenit -when the lysates -were used together witlh lysozyme. The mtultiplication of toxoplasmia in the presence of lysozymwe. Table XI anid

TI'ABLE XI. Penetration a nd Reprod(ltetion of Toxoplasmata in the Presence or Absence of Lysozyisie T(ttal I10. of -No of peletratecl illt iaeelluilal' I,-VSoZv111e Tiicie iII l1iou0 s RNI U p)arasites < 1 ()-5 paratsites ;Y 10-5 *6 . 1L78 - oi5 2 26 71 - 6 2 35 14 70-6 2 49 19 71 -7 48 4() - 7 3 *6 0) 60 0 -62 - 1(I 50 -4 () 60 0-64 14 48 -9 () ()5 0- 79 19 50 8 0 61 1 *23 196 E. LYCKE, EBBA LUND AND 0. STRANNEGARD Fig. 3 demonstrate the growth rate of toxoplasma in the presence or absence of 1 mg. lysozyme per ml. of the inoculated parasite suspension. Each culture was inoculated with 3-38 x 105 parasites. The greatest number of parasites which penetrated the host cells in the presence of lysozyme was calculated (Lycke and Lund, 1964b) to be 2-26 x 105 and, in the cultures which had been inoculated with the parasite suspension without lysozyme, to be 61 x 104. Table XI also shows the estimated total number of initracellular parasites at each time of observation.

1*0

0-4 02 / /~~~~~~~

0-2

5 10 15 20 Hours FI(. 3.-- -Relpro()duction of toxolplasma ill tlhe p)Irese(ice or abseInce of l--sozY.inie. The nimber of (laughter p)arasites produced jer )arasite found to liave p)enetrate(l the HeLa cells is p)lotted againist the observation time after the inioculatioIn of the cell cultuIres. The filledi ci-cles ii(lidcate restilts obtained in the presernce of lysozyme. The open circles show the results obtained w-ith. cultuires without lvsozvmIe.

As iindicated by the R_NJIU values ill the table, in the absence of lvsozvme all the parasites in the inioculum able to penietrate the host cells appeared initracel- lularlv at the 6 hr. reading. Thus from then on thie RNIU values did niot clhaiige significantly during the rest of the observationi period. In the presenice of l-so- zyme the penietration phase did not seem to be completed at 6 hr., but from 10 hr. nio signiificant change in the RNIU values was noted. To study the growth rate of toxoplasma in the presence or absence of lysozyme, the number of parasites found to have been produced in the cultures was divided by the inumber of parasites whiclh had penetrated the cells. In Fig. 3 the results obtained are plotted against the observation time. As seen from the figure, there is nio evidence for an increased rate of multiplication in the presence of lvsozyme. The relative number of parasites produced in cultures with lysozyme was lower than the number produced in the untreated cultures. This may onlv reflect that PENETRATION OF TOXOPLASMA INTO CULTURED CELLS 197 in the presence of lysozyme more non-reproductive parasites gained entrance into the cells. Tests with other enzyMes.-Besides lysozyme and hyaluronidase three other enzymes were tested. Neuraminidase (RDE), ,-amylase and phospholipase A were each tested alone or together with hyaluronidase, 0 05 mg., or lysozyme 0.1 mg. per ml. of the inoculated parasite suspension. Concentrations of 1 mg. RDE, 0.1 mg. amylase and 0 01 mg. phospholipase per ml. of parasite suspension were employed. No effect on the penetration by the parasites was demonstrable and when used together with hyaluronidase or lysozyme there seemed to be no additional enhancement of the penetration.

DISCUSSION Little is known about the mechanism by which the toxoplasma parasite pene- trates its host cell. Active invasion and passive ingestion have both been considered as possible ways in which the parasites can infect susceptible cells (Pulvertaft, Valentine and Lane, 1954). Parasites liberated by rupture of cells in an infected cell culture are actively motile and able to invade new cells (Pulvertaft et al., 1954; Guimaraes and Meyer, 1942; Lund, Lycke and Sourander, 1961a). Within a few minutes after the liberation of parasites by the rupture of an infected cell the active motility of the parasites seems to disappear. Parasites showing no evidence of active motility nevertheless gain entrance into cells. The present report shows that two enzymes, lysozyme and hyaluronidase, have a promoting effect on the penetration by Toxoplasma gondii into HeLa cells. This effect seems to be due to specific enzymatic actions. This assumption is supported by the following observations. The basic amino acids in the lysozyme molecule seem to be responsible for its antiphage effect (Ribble and Hook, 1959), but protamine sulphate, being a basic protein like lysozyme, was not shown to have any effect on the penetration of the toxoplasma parasites. The action of lysozyme on Gram-negative bacteria is considered to be mediated by agents supposed to disorganize protein and lipoprotein structures in the bacterial cell wall (Wardlaw, 1962). In the present investigation two such agents, EDTA and polymyxin, were examined but were not found to increase the penetration-enhancing action of lysozyme. The presence of complement is considered important for obtaining the bacteriolytic activity of lysozyme in normal serum (Wardlaw, 1962). The experiments presently described do not indicate however that complement factors or other serum components are of any importance for the penetration-promoting effect of lysozyme. Lysozyme, and hyaluronidase are both enzymes acting on mucopolysaccharide- mucoprotein complexes by attacking the glycosidic linkages of amino sugars. Their effect on the penetration of the parasites may therefore be due to specific actions on mucopolysaccharide containing substrates in the walls of the host cells or between the cells. Amino acid-amino sugar complexes are considered to be of importance for the incorporation of material into cells. Thus, Burgos (1960), studying the cells in the proximal convolutions of the rat kidney, has suggested that the mucopolysaccharide coat of the membranes of these cells contains the binding sites for material entering the cells. It has been suggested that, as a primary stage in the pinocytosis of proteins in the amoeba, the binding of protein 198 E. LYCKE, EBBA LUND AND 0. STRANNEGARD to the cell surface depends on precipitation of mucoproteins in the mucus coat (Chapman-Andresen, 1962). The experiments reported show that, when cells or parasites are washed after treatment with the enzymes, the penetration-promoting effect fails to appear or is markedly reduced. To obtain the penetration-promoting effect it seems therefore necessary for the enzymes to be present at the contact between the parasite and its host cell. Moreover, if the effect on the penetration is due to enzymatic reactions with cell wall components, the alterations caused must be easily revers- ible. Addition of hyaluronic acid inhibited the effect of both the enzymes, although less effectively that of lysozyme. This does not mean that the enzymes must have been working on the same substrate. The synergistic effect found when lysozyme and hyaluronidase were used together rather indicates that different reaction sites in mucopolysacharide complexes are affected. The synergistic effect might be due to the degradation by hyaluronidase of material containing hyaluronic acid, thus making reaction sites available to the action of lysozyme. Lysozyme did not seem to affect the multiplication of the parasites. The penetration by irradiated parasites with reduced capacity for multiplication was promoted by lysozyme, as was that of the non-irradiated parasites. Previously reported experiments have shown that high doses of irradiation which inhibited the multiplication of toxoplasma did not influence the penetration by the parasites (Lund et al., 1961b). Reproduction and ability to penetrate seem thus to be two independent activities. When no lysozyme was present the penetration phase was completed in 6 hr. In the presence of lysozyme the penetration phase continued for 10-14 hr. This observation and the results of the tests with parasite suspensions treated with formalin indicate that the addition of lysozyme or hyaluronidase allows parasites, presumably unable to take any active part in the process of penetration, to gain entrance into cells. It seems reasonable to assume that an enzymatic activity of toxoplasma may play a role in the process of penetration. Attempts have therefore been made to trace a penetration-enhancing activity in preparations of lysed parasites. The results obtained suggest that such an activity may exist. Further studies are however necessary before any statement concerning this matter can be made.

SUMMARY Lysozyme and hyaluronidase were found to have a promoting effect on penetration by Toxoplasmca gondii into HeLa cells. This effect seemed to be due to specific enzymatic actions of the enzymes. To obtain the effect it seemed necessary for the enzyme used to be present at the contact between parasite and host cell. A synergistic action of lysozyme and hyaluronidase was observed. Enhancement of the penetration was also exerted when the enzymes were added to parasites unable to take any active part in the process of penetration. Some results suggested that a penetration-promoting activity was present in preparations of lysed toxoplasma parasites. We should like to thank Miss Mona Thorstensson and Mrs. Marie Louise Persson for their skilled technical assistance. PENETRATION OF TOXOPLASMA INTO CULTURED CELLS 199 REFERENCES BURGOS, M.-(1960) Anat. Rec., 137, 171. CHAPMAN-ANDRESEN, C.-(1962) C. r. Trav. Lab. Carlsberg, 33, No. 3. GUIMARXES, F. N. AND MEYER, H.-(1942) Rev. bras. Biol. 2, 123. LUND, EBBA, LYCKE, E. AND SOURANDER, P.-(1961a) Brit. J. Exp. Path., 42, 357.- (1961b) Ibid., 42, 404. LYCKE, E. AND LUND, E.-(1964a) Acta path. microbiol. scand., 60, 209.-(1964b) Ibid., 60, 221. Jidem, STRANNEGAIRD, 0. AND FALSEN, E.-(1965) Ibid., in press. MYRVIK, Q. N. AND WEISER, R. S.-(1955) J. Immunol., 74, 9. PONTIERI, G., IMPERATO, S. AND CAVALLO, G.-(1962) Giorn. Microbiol., 10, 93. PULVERTAFT, R. J. V., VALENTINE, J. C. AND LANE, W. F.-(1954) Parasitol., 44, 478. REPASKE, R.-(1956) Biochim. biophys. Acta, 22, 189.-(1958) Ibid., 30, 225. RIBBLE, J. C. AND HOOK, E. W.-(1959) Fed. Proc., 18, 2332. WARDLAW, A. C.-(1962) J. exp. Med., 115, 1231. WARREN, G. H., GRAY, J. AND YURCHENCO, J. A.-(1954) Med. J. Osaka Univ. 4, 401.