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Enzymes of Entamoeba Histolytica RAWEWAN JARUMILINTA 1 & B

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Enzymes of Entamoeba Histolytica RAWEWAN JARUMILINTA 1 & B Bull. Org. mond. Sante 1969, 41, 269-273 Bull. Wid Hith Org.J Enzymes of Entamoeba histolytica RAWEWAN JARUMILINTA 1 & B. G. MAEGRAITH 2 In spite ofextensive studies of the pathogenic activities ofEntamoeba histolytica upon animal hosts by many investigators, thefactors that govern thepathogenesis oftheparasite are still largely unknown. Attempts have been made to determine the factors concerned in the pathogenicity of certain strains of E. histolytica from the aspect ofparasite itself and from the point of view of its interaction with the host. The enzyme content of the tropho- zoites of the amoebae examined has been studied in detail, both in the living organism and in its extracts. The totalpatterns ofproteolytic enzymes and ofcertain other enzymes were determined to establish whether it was possible to define the invasiveness of the various strains of E. histolytica already established as pathogenic by other workers or in our laboratories. It was found impossible to distinguish clearly between the so-called " patho- genic " and " non-pathogenic " strains ofE. histolytica by such means. EARLY INTERPRETATIONS OF INVASION substrates. In demonstrating these proteolytic activi- ties various methods have been employed. Hallman, The mechanism by which Entamoeba histolytica Michaelson & De Lamater (1950), Rees et al. (1953), gains entry into the host tissues is still not fully and Kaneko (1956) demonstrated the presence of understood. It is generally believed, however, that proteolytic enzymes in the parasite by growing the the parasite penetrates through the gut epithelium amoebae in gelatin solutions. Harinasuta & Mae- ofhumans and animals by virtue ofhistolytic enzymes graith (1954, secreted by the amoebae. This hypothesis was first 1958) showed gelatinase activity of put forward by Dobell (1919). The evidence in E. histolytica by placing the amoebae on gelatin favour of the existence of such enzymes is based on film, the activity being indicated by areas of lysis observation ofthe initial contact with the gut mucosa, of the gelatin film after intervals of incubation. and of the histological studies of lytic necrosis in Nakamura & Edwards (1959) demonstrated the the tissue at later stages, when the amoebae are ability of the amoebae to hydrolyse gelatin by visible and are often surrounded by a clear area employing the micro-test for gelatinase developed (" lytic area ") separating them from the adjacent by Thirst (1957), which consisted of a thin film of host tissue. The earliest superficial mucosal changes, gelatin on a glass slide, and also the agar-gelatin the typical bottle-neck ulcers and the honeycombing plate test developed by Smith (1957). Hydrolysis of destruction of the submucosa seen in amoebic le- gelatin on the slides or the plates was indicated by sions, indicate that lytic action is involved in the the appearance of clear areas. The existence of development of amoebic ulcers, although it is gener- caseinase activity in E. histolytica was first shown ally agreed that mechanical action may aid the by Neal (1956). amoebae in their penetration and migration. The The proteolytic activity on gelatin and casein of so-called " lytic area " has usually been ascribed to various strains of E. histolytica was demonstrated enzymatic cytolysis by the amoebae, but some of by Neal (1960) both in living amoebae and extracts. these haloes may arise as artefacts during fixation The ability of the amoebae to hydrolyse gelatin of the tissues (Torpy & Maegraith, 1957). was shown by the reduction of viscosity of a gelatin solution, whereas hydrolysis of casein solution was measured by determining the splitting of tyrosine DEMONSTRATION OF PROTEOLYTIC ENZYMES by means of the phenol reagent (Folin & Ciocalteau, Many workers have shown that E. histolytica pos- 1927). However, the results showed that the invasive sesses proteolytic activity in vitro towards various character of the amoebae did not seem to be related 1 Parasitologist, Pharmaceutical Department, CIBA Ltd., 2 Professor of Tropical Medicine, School of Tropica Basle, Switzerland. Medicine, Liverpool, England. 2368 269- -270 R. JARUMILINTA & B. G. MAEGRAITH to the proteolytic activity. Of 5 strains freshly iso- gelatin and casein but not haemoglobin, fibrin or lated from patients with amoebic dysentery, 2 exhib- epithelium. The chromatographic patterns of amino ited high proteolytic activity, whereas 3 other patho- acids produced by hydrolysis of the gut epithelium genic, freshly isolated strains, as well as 2 non- by all the strains of E. histolytica and by trypsin pathogenic ones, showed low proteolytic activity of were found to be similar. It was thus considered the same order. It was concluded that the amoebic that it might be trypsin (or a trypsin-like enzyme) proteinase resembles trypsin and that high pro- which provides E. histolytica with a potential teolytic activity may not be required for tissue pathogenic mechanism. penetration but merely acccompanies some other The use of proteins as substrates as described factor. above does not, however, allow differentiation of the different proteinases (endopeptidases) contained in a given mixture. Since Bergmann & Fruton (1941, STRAIN DIFFERENCES IN ENZYME ACTIVITY 1942) had found that it was possible by the use of The proteolytic enzyme contents of E. histolytica specific simple peptides to determine and estimate have been further studied in detail both in the living various proteinase (endopeptidase) and exopeptidase organism and in its extract by Jarumilinta & Mae- activities from an enzyme mixture, Jarumilinta & graith (1961a, 1961b). The first studies were carried Maegraith (1961b) studied the action of E. histolytica out with regard to the proteolytic activity of the on various synthetic substrates considered specific trophozoites and freeze-thaw extracts of various for proteinases, i.e., trypsin, pepsin and chymo- strains of E. histolytica (5 isolated from patients trypsin, and for exopeptidases, i.e., carboxy-, amino-, with amoebic dysentery and proved pathogenic to and dipeptidase. Trophozoites and saline extracts of rats and guinea-pigs, and 4 isolated from human the same strains of the amoebae as in the studies carriers and also proved non-pathogenic to animals) with the 5 protein substrates described above were on gelatin, casein, fibrin, haemoglobin and suspen- used. Control suspensions of associated bacteria of sions of epithelial cells from the guinea-pig caecum. the corresponding strains were included in the experi- A strain of free-living amoeba, Acanthamoeba sp., ments. The hydrolysis of these substrates after maintained in axenic culture, was also subjected to various intervals of incubation (3 h-24 h) with the similar studies. Hydrolysis of gelatin, casein, and parasite under optimum conditions was demonstrated haemoglobin was measured by determining the both qualitatively by paper chromatography and content of a-amino nitrogen liberated (by means of quantitatively by estimating the amino group liber- ninhydrin reagent, Yemm & Cocking, 1955) after ated in the reaction. The data obtained showed that various intervals of incubation (3 h-24 h) with the E. histolytica, whether pathogenic or not, possessed parasite under optimum conditions of temperature tryptic and peptic but not chymotryptic activity. and pH. Control suspensions of associated bacteria Non-pathogenic strains of E. histolytica possessed and their extracts were also set up in parallel. For carboxy-, amino-, and dipeptidase, whereas patho- the guinea-pig caecal epithelial suspensions, the genic strains did not contain carboxypeptidase. amino acids liberated after the hydrolysis by the Acanthamoeba sp. possessed only peptic activity and amoebae were determined by 1- and 2-dimensional amino- and carboxypeptidase. These results thus con- paper chromatography. Hydrolysis of the epithelial firmed those described above that all strains of E. suspensions by trypsin, pepsin and HCI was also histolytica studied contain trypsin whereas Acanth- studied in parallel. In determining the fibrinolytic amoeba sp. does not. Both E. histolytica and Acanth- activity, a modification of Permin's (1950) fibrin amoeba sp. were shown to possess pepsin. However, plate method was employed. Trypsin was used as digestion of the epithelium by pepsin is regarded as the standard for reference of the fibrinolytic activity unlikely because the parasites are inactive in vitro of the parasite. The area of dissolved fibrin which within the pH range of pepsin activity. appeared after incubation for 22 h was taken as a measure of the activity of the added trophozoites ENZYME ACTIVITY AND STRAIN PATHOGENICITY or extracts. It was demonstrated that both living organisms and extracts of all strains of E. histolytica It has thus not been possible by the study of these examined were capable of hydrolysing all the sub- proteolytic enzymes to distinguish between the patho- strates used, including the guinea-pig gut epi- genic and non-pathogenic strains of E. histolytica, thelium, whereas the free-living amoebae lysed only since all of them contain trypsin, which is capable ENZYMES OF ENTAMOEBA HISTOLYTICA 271 of digesting guinea-pig caecal epithelium within the to demonstrate activity of this enzyme in 5 strains range ofpH in which it is hydrolysed by the parasites. of E. histolytica which had been maintained in A distinction could, however, be drawn between the vitro over a long period, but did demonstrate potential activity of E. histolytica in so far as their activity in the same strains
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