of Entamoeba histolytica Hemolysins. Salvador Said-Fernfindez* and Rub6n Ldpez-Revilla**. Departamento de Biologia Celular, Centro de Investigaci6n del ...
Z Parasitenkd (1983) 69 : 435-438
Parasitenkunde Parasito[ogy Research
9 Springer-Verlag 1983
Latency and Heterogeneity of Entamoeba histolytica Hemolysins Salvador Said-Fernfindez* and Rub6n Ldpez-Revilla** Departamento de Biologia Celular, Centro de Investigaci6n del IPN, Ap. Postal 14-740, 07000 Mbxico, D. F., M6xico
Abstract. We have found that the hemolytic potency of P 30 - a subcellular fraction that contains most of the hemolytic activity of Entamoeba histolytica trophozoites - increased up to 100 times by preincubation for 36 h at 36 ~ C. This remarkable increase in potency was due to the in vitro generation of heat-labile as well as heat-stable hemolytic components and could be prevented if P 30 was preincubated at 4 ~ C or subjected to either heating at 90~ or to repeated freeze-thawing cycles before preincubation at 36 ~ C. The major hemolytic activity ofE. histolytica thus appears to depend on latent, heat-labile and heat-stable hemolysins that are probably unmasked through the action of endogenous amebal enzymes.
The major hemolytic activity of E. histolytica homogenates, which may be involved in the in vivo production of the necrotic lesions of amebiasis, is strain-specific (L6pez-Revilla and Said-Fernfindez 1980), depends on pH and calcium concentration, and is located in a subcellular vesicular fraction called P 30 (Said-Fernfindez and L6pez-Revilla 1982). In this paper we show that the hemolytic activity of E. histolytica homogenates (1) is mostly latent in recently isolated P30, (2) appears to require endogenous amebal enzyme action to become fully patent, and (3) is due to heterogeneous hemolytic components. Materials and Methods Parasites. Trophozoites of the E. histolytica strain HK9 were axenically cultured in TPS-1
medium (Diamond 1968). * Present Address." Unidad de Investigaci6n Biom6dica del Noreste (IMSS), Apartado Postal
020-E, 64720 Monterrey, N. L., M6xico ** Offprint requests to. R. L6pez-Revilla
S. Said-Fernfindez and R. L6pez-Revilla
Subcellular Fractionation. Subcellular fractions and their protein content were obtained as described before (Said-Ferndmdez and L6pez-Revilla 5982). Briefly, trophozoites from cultures in late logarithmic phase of growth were washed twice by centrifugation in ice-cold phosphatebuffered saline (PBS), suspended in 2 vols ice-cold balanced salt solution (BSS) containing 3 mM sodium azide, and disrupted with a Potter-Elvehjem homogenizer. From total homogehates the 'nuclear fraction' was obtained as the sediment formed after 5 rain centrifugation at 535g and 4~ C; the supernatant was then centrifuged for 15 rain at 30,000g and 4~ C, and the resulting pellet (vesicular fraction 'P30') containing most of the hemolytic activity was immediately assayed upon rat erythrocytes. Hemolytic Assays. The assays were performed as described (Said-Fernfindez and L6pez-Revilla 1982) by colorimetric measurement of the amount of hemoglobin released after 1 h incubation. One hemolytic unit (HU) was defined as the amount of P 30 required to release an amount of hemoglobin equivalent to that contained in 3 x 1 0 6 rat erythrocytes and having an optical density of 0.05 at 455 nm. Effect of Preincubation, Heating, and Freeze-Thawing on the Hemolytic Activity of P 30. Fresh samples of P 30 were subjected to one of the following treatments before assaying their hemolytic activity: (1) preincubation at 4~ C or 36~ C, (2) immersion in boiling water (90~ C in Mexico City), or (3) repeated cycles of freezing (at - 20~ C) and thawing at room temperature. All analyses were performed in triplicate samples containing 2 or 4 mg P30 proteins per ml. Chemicals. Reagent grade inorganic salts were purchased from J.T. Baker (Mexico). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Results Increase in Hemolytic Potency by Preincubation at 36 ~ C. H e m o l y t i c activity increased exponentially for up to 12 h in P 30 samples p r e i n c u b a t e d at 36 ~ C, whereas it r e m a i n e d a l m o s t u n c h a n g e d in samples p r e i n c u b a t e d at 4 ~ (Fig. 1). Prevention of the Increase of Hemolytic Potency. T h e rise in h e m o l y t i c p o t e n c y was p r e v e n t e d as an exponential function o f the time o f heating at 90 ~ C (33% loss per m i n o f exposure) or o f the n u m b e r o f successive freeze-thawing cycles (30% loss per cycle) to which P 30 was subjected before p r e i n c u b a t i o n at 36 ~ C (Fig. 2). T h e increase in h e m o l y t i c p o t e n c y was c o m pletely p r e v e n t e d after 9 rain at 90 ~ C (Fig. 2). Generation of Heat-Stable and Heat-Labile Hemolytic Components During Preincubation at 36 ~ C. T h e h e m o l y t i c p o t e n c y increased to a m a x i m u m (100 times higher t h a n in fresh P30) after 36 h p r e i n c u b a t i o n at 36 ~ C, a n d then decreased (Fig. 3). H e a t i n g at 90 ~ C d e s t r o y e d some o f the h e m o l y t i c activity generated in P 30 samples o b t a i n e d at different periods o f p r e i n c u b a tion at 36 ~ C (Fig. 3). R e i n c u b a t i o n o f such samples did not result in a further increase o f the h e m o l y t i c potency. The r e m a i n i n g (i.e., heat-stable) h e m o l y t i c activity was f o u n d to increase a s y m p t o t i c a l l y with time to r e a c h a m a x i m u m at a r o u n d 24 h p r e i n c u b a t i o n ; by substracting heat-stable f r o m total h e m o l y t i c activity we noticed t h a t the heat-labile h e m o l y t i c activity p r e d o m i n a t e d initially, attained a m a x i m u m after 36 h p r e i n c u b a t i o n , a n d then rapidly decreased.
E. histolytica Hemolysins
f, Fig. 1. Increase in the hemolytic potency of P30 during its preincubation at 36 ~ C. Specific hemolytic activity was determined on P 30 samples containing 2 mg protein/ml and preincubated for different periods of time either at 4 ~ C (o) or at 36 ~ C (o)
o_ u__ o to
Fig. 2. Prevention of the rise in hemolytic potency by heating or freeze-thawing P30 before its preincubation at 36 ~ C. Specific hemolytic activity was determined on P 30 samples containing 2 mg protein/ml and preincubated for 12 h at 36 ~ C, after being immersed in water at 90 ~ C (o), or subjected to successive cycles of freezing at - 2 0 ~ C and thawing at room temperature (e). The increase in potency was calculated by substracting the activity from the non-preincubated controls to that of the preincubated samples
4 8 12 HOURS OF PREINCUBATION AT 56~
6 9 12 OF HEATING AT
Fig. 3. Total and heat-stable hemolytic activities generated during preincubation at 36 ~ C. Aliquots from a P 30 sample containing 4 mg protein/ml were preincubated at 36 ~ C for different periods of time and assayed immediately (o), or after heating for 9 min at 90 ~ C
4000 2 000
o,~ , J , , 0 12 24 36 48 HOURS OF PREINCUBATION AT 36 ~
S. Said-FernAndez and R. L6pez-Revilla
The major hemolytic activity of E. histolytica trophozoite homogenates (Lbpez-Revilla and Said-Fernfindez 1980) is located in a subcellular vesicular fraction called P 30 (Said-Fernfindez and Lbpez-Revilla 1982). We are interested in this activity because of its potential involvement in the in vivo production of the necrotic lesions characteristic of amebiasis. The increase in hemolytic potency obtained by preincubating P30 at 36 ~ C but practically not at 4 ~ C (Fig. 1) and the destruction of this potentiality by heating or freeze-thawing P 30 (Fig. 2) are compatible with an enzyme-mediated mechanism. The increase in hemolytic potency could itself depend either on de novo synthesis or activation of hemolytic factors already present in P 30. The major lytic effect of E. histolytica homogenates is significant and increases considerably with preincubation. We calculated before (Said-Fernfindez and L6pez-Revilla 1982) that the amount of P30 recently obtained from a single trophozoite lyses around 20 erythrocytes; the same amount of maximally active P 30 after 36 h preincubation at 36 ~ C could therefore lyse around 2,000 erythrocytes. The hemolytic components generated during preincubation of P30 at 36 ~ C are heat-labile as well as heat-stable (Fig. 3) and thus heterogeneous. Strict quantitation of the hemolytic activity applies perhaps only to the heat-stable component, because it occurred once the heat-labile component had been inactivated. Quantitation of the heat-labile activity is only tentative because it cannot be measured directly but calculated by substraction, and might be significantly affected by interactions between the hemolytic components. Based on the properties of known cytolysins (Thelestam and M611by 1979) we are tempted to speculate that the heat-labile hemolytic component may be an enzyme, and that the heat-stable component may be non-enzymatic. Once the molecular nature of the major E. histolytica hemolysins is known, it might be possible to understand their mechanism of action and determine their contribution to amebic pathogenicity. Acknowledgments. This work was supported in part by grant PCSANAL 790272 and a predoctoral fellowship to S.S.-F. awarded by CONACYT (Mexico).
References Diamond LS (1968) Techniques of axenic cultivation of Entamoeba histolytica, Schaudinn 1903 and E, histoIytica-like amebae. J Parasitol 54:1047-1056 L6pez-Revilla R, Said-Fernfindez S (1980) Cytopathogenicity of Entamoeba histolytica: hemolytic activity of trophozoite homogenates. Am J Trop Med Hyg 29:209-212 Said-Fern/mdez S, L6pez-Revilla R (1982) Subcellular distribution and stability of the major hemolytic activity of Entamoeba histolytica trophozoites. Z Parasitenkd 67 : 249-254 Thelestam M, M611by R (1979) Classification of microbial, plant and animal cytolysins based on their membrane-damaging effects on human fibroblasts. Biochim Biophys Acta 557:156-169 Accepted March 8, 1983