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 hyaluro- nidase. MATERIAL AND METHOD The parasite suspensions. The RH strain of Toxoplasma gondii was used. The prepara- tion 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 lyso- zyme 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 concen- trations of lysozyme are plotted against the mean values of the RNIU in 4 experi- ments. 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 sul- phate, 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.) .