Cytolytic Activity Released from Tetrahymena 1

J. Protozool., 32(4), 1985, pp. 657-660 0 1985 by the Society of Protozoologists

Cytolytic Activity Released from Tetrahymenal J. FLORIN-CHRISTENSEN,2 M. FLORIN-CHRISTENSEN,*J. KNUDSEN, and L. RASMUSSEN Institute of Anatomy and Cytology and Institute of Biochemistry, Odense University, 5230 Odense M, Denmark

ABSTRACT. Using a hemolytic assay system, we have detected cytolytic activities in extracellular medium from Tetrahymena thermophila and T. pyriformis. In addition, we have identified two phospholipase activities (types A and C) from the same source by thin layer chromatography analysis of the breakdown products of a phospholipid preparation. The hemolytic activity peaks at low pH values. It is inhibited by egg lecithin, supporting the view that a phospholipase activity is involved in the cytolytic processes. Cytolytic activities may play important roles in Tetrahymena, both in nutrition, especially in parasitic and scavenger forms, and in defense against predators. Tetrahymena is probably partly protected from its own released cytolytic phospholipase by having a high proportion of phosphonolipids on its surface membrane. stirring. Initial population density was 5000 cells/ml and the final cell density was 450,000 cells/ml, reached after 48 h. Except for some flattening due to hypertonic conditions, all cells showed normal shape and motility. Hemolytic assays were camed out using sheep erythrocytes. The blood was obtained aseptically and stored in sterile Alsever solution at 4°C. Prior to use, the red cells were washed thrice in 20 mM Tris-HCV150 mM NaCl (pH 7.0) and resuspended in the same buffer to a final cell concentration that resulted in an OD,,,n, of 0.3 upon lysis by 10-fold dilution with distilled MATERIALS AND METHODS water. This concentration is about 1% of red cells by volume. Proteose peptone and yeast extract were purchased from Difco Serial dilutions ofthe growth medium in 20 mM sodium acetate/ 150 mM NaCl (pH 5.5) were tested for hemolytic activity by Laboratories (Detroit, MI, USA); soy bean lecithin and p-nitrophenylphosphate from Sigma Chemical Co. (St. Louis, MO, addition of 0.1 ml of the red cell suspension to 0.9 ml of each USA); asolectin from American Lecithin Co. (Woodside, NY, dilution. The tubes were incubated at 37°C for 1 h and then USA); silica gel from Merck (Darmstadt, FRG), and p-nitro- centrifuged. The absorbancy of the released hemoglobin in the phenyl-N-acetyl-P-D-glucosaminidase from Serva (Heidelberg, supernatant was read at 540 nm. The dilution of the medium FRG). Sheep blood was kindly provided by Dr. Per Svendsen, in which 50% of the hemoglobin was released was determined by linear interpolation. By definition, this dilution contains 1 Biomedical Laboratory, Odense, Denmark. Cells. Tetrahymena thermophila (12) strains DIII, NP 1 (1 3), hemolytic unit (HU)/ml. The reciprocal of the dilution factor and BIII were grown axenically at 37°C while T. pyriformis (12) therefore expresses the number of HU present in 1 ml of the was grown at 28°C. The nutrient medium consisted of 0.7S0/o undiluted medium. The kinetics of hemolysis at 37°C was camed out by recording (w/v) proteose peptone, 0.25% (w/v) yeast extract, and salts (14). This medium is designated PPYS. Release of enzymes to growth the change in absorption at 500 nm using a Pye Unicam SP 100 medium was studied in cells grown in test tubes with 4 ml of spectrophotometer equipped with thermostatized cuvette holdmedium, inoculated at an initial density of 5000 cells per ml. ers. The red cell concentration was adjusted to give an apparent OD,,, , of 0.9 when read against medium without red cells. Duplicate parallel cultures were used for each determination. Detection of lipolytic activity in the medium. Mixtures conInteraction between erythrocytes and Tetrahymena was studied using cells adapted to grow in PPYS medium supplemented sisting of 0.32 ml of BIII or DIII growth medium, 0.04 ml of with 150 mM NaCI. Adapted cells show a doubling in generation 0.5 M sodium acetate (pH 5.0), and 0.04 ml of a 30 mum1 time to 5 h and reach half of the final density with respect to suspension of either asolectin or purified soy bean lecithin were cells grown under the same conditions in non-supplemented incubated for 1 h at 37°C. These substrates were dispersed by medium; however, the pH values in the adapted cultures remain sonication in a Branson sonifier (50 W; 20 min; room temperaround neutral in contrast to those of the non-adapted ones, ature). After the incubation period, the lipids were extracted where high pH values have detrimental effects on the cells. according to Bligh & Dyer (2) and chromatographed on Silica Therefore, the NaCl-supplemented cultures can be kept without Gel HR 60 thin layer plates, developed with chloroform/methcell death or breakage for at least twice as long as the non- anovacetic acid/water (50:25:7:3) for separation of phosphoadapted ones (4-5 days against 2-3 days, respectively), allowing lipids or with hexane/diethylether/methanoVaceticacid (90:20: accumulation of higher amounts of cytolytic activity. The NaCl- 3:2) for separation of neutral lipids. Spots were made visible by supplemented cultures of DIII cells were used as a source of exposure to iodine vapor. Assay of hydrolases. Acid phosphatase and N-acetyl-8-D-gluextracellular medium to study the kinetics of hemolysis and its variation with pH. These cultures were grown in 450-ml Fern- cosaminidase activities were determined by the method debach flasks containing 75 ml ofmedium at 37°C for 72 h without scribed by Miiller (1 1). The assays were linear with respect to time and enzyme concentration. One unit (1 U) is defined as the amount of enzyme that releases 1 rmol of p-nitrophenol per minute at 37°C. I We thank the Danish Ministry of Foreign Affairs, the Carlsberg Foundation, the Danish Natural Science Research Council, and the Danish-ArgentineSociety for support. We also thank Dr. Hans 0. HanRESULTS sen, Institute of Biochemistry, for fruitful discussions and Tove Nielsen Growth media from Tetrahymena cultures have hemolytic for preparing the manuscript. Present address: Laboratorio de Invertebrados I, Facultad de Cien- activity against sheep erythrocytes. Figure 1 shows the time course of hemolysis. A lag period without change in apparent cias Exactas y Naturales, Ciudad Universitaria 4" P I1 42,1428-Buenos absorbency, the prolytic period, is followed by a period of rapid Aires, Argentina.

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ANY enzyme activities have been reported from the extracellular fluids of Tetrahymena (1 1). So far, there have been no studies of lipases from this source. Previous results, however, have indicated that cell homogenates have strong lipolytic activity (20). Using a hemolytic assay system, we show here that Tetrahymena growth and starvation media display cytolytic activity. The possibility of this activity being caused by a phospholipase and its physiological significance are discussed.

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Fig. 1. Kinetics of hemolysis of erythrocytes exposed to extracellular fluid in which Tetrahymena thermophila BIII cells have grown for 72 h at 37°C. Apparent OD,,,, of a sheep red cell suspension read against medium without red cells as a function of time. The lines labeled p and s show the duration of the prolytic period and slope of the curve at the 50% level of the change in OD, respectively. Note that the OD is decreasing with time, indicating that the suspension becomes more transparent as the cells are lysing. The cells were grown in PPYS-medium supplemented with 150 mM NaCI.

changes, the lytic period. Similar results were obtained with human red cells. We have compared hemolytic activity and release of lysosoma1 enzymes in Tetrahymena media from various strains. Table I shows both types of activities 24 and 48 h after inoculation. The BIII cells produce much less hemolytic activity than DIII cells and they may also produce less lysosomal enzymes. Tetrahymena pyriformis is less active than BIII in the release of hemolytic activity. Cultures of DIII cells adapted to NaCl show a considerable increase in hemolytic activity after they entered stationary growth phase. At 48 h of growth, the TABLE I. Hemolytic and lysosomal enzyme activities in growth medium of Tetrahymena thermophila. Hemolytid activity (HU/ml)

N-acetyl-8Acid phos- B-glucosphatase aminidase Cell density (mU/ml) (mU/ml) (celldml) ~

24 h 48 h

BIII DIII BIII DIII