Entamoeba histolytica remains an important but enzgmatic parasite. It displays ... 0rE. histolytica, and possibly in its differentiation and invasive mechanisms.
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Reducing agents and Entamoeba histolytica R.K, Mehlotra* and O. P. Shukla Entamoeba histolytica remains an important but enzgmatic parasite. It displays both nonpathogenic and invasive pathogenic types, which can be distinguished clinically and by isoenzyme markers. Yet as debated in Parasitology Today last year ~, the relationship between these two forms remains unclear. Bacterial associates and reducing agents are known to play an important role in the culture 0rE. histolytica, and possibly in its differentiation and invasive mechanisms. This article briefly reviews available information on the role of reducing agents, and explores the possibility that bacteria may play a role in reduction of toxic oxygen product- thereby promoting the virulence 0rE. histolytica. The review is not de~nitive, but should help to stimulate further research in this neglected area. Entamoeba histolytica is an oxygen tolerant organism 2 and has also been found to consume oxygen under certain conditions3-5. However, the normal environment of the trophozoites (feeding stages) of this pathogen is essentially anaerobic, and maintenance of low redox potential is obligatory for its optimal growth in vitro. To maintain a reduced potential in culture, investigators have used chemicals such as cysteine, thioglycolate or glutathione in different media6-9. Diamond used a combination of c-cysteine (0. I%) and ascorbic acid (0.02%) as the reducing agent in the axenic TP-S-I and TYI-S-33 (or BI-S-33) media t0,11.Good growth of the amoebae in Diamond's media has also been achieved using L-cysteine (0.2%) without ascorbic acid, or reduced glutathione (0.2%) 12,13, D-cysteine, ascorbic acid, Lcystine 14, or sodium thioglycolate (FLE. Reeves and B. West, unpublished). Recently we found that reduced glutathione (0.2%) supported as good a growth of E. histolytica as the combination of cysteine and ascorbic acid or L-cysteine (0.2%) in BI-S-33 medium. Moreover, 0.25% reduced glutathione supported considerably higher growth of amoebae, similar to the growth obtained with 0.3% L-cysteine t5. It is asserted in the large body of literature that E. histolytica is anaerobic. But the concentration of reducing agent required in the axenic medium (eg. 0. 1% w/v cysteine) is considerably higher than that required for the growth of anaerobic bacteria, where 0.02-0.05% cysteine is adequate and higher concentrations are usually toxic 16. This suggests certain other roles of these reducing agents for the organism, in addition to the maintenance of low redox potential to facilitate growth. Gillin and Diamond reported a specific requirement of cys* This paper is dedicated to the late Dr B.N Singh, with whom I learnt more in conversation than from the literature.
teine and ascorbic acid in shortterm survival, attachment and motility of E. histolytica in a defined MM-I medium 17.18.In TYI-S33 medium, both cysteine and cystine protected E. histolytica trophozoites from the lethal effects of increased oxygen tension 19. Neither the presence of free sulfhydryl (-SH)groups, nor the natural L-configuration seems to be essential. Reduced glutathione supports the growth of E. histolytica, but this organism lacks enzymes for glumthione metabolism 2° - a specific metabolic effect is therefore unlikely. The use of reduced glutathione has further demonstrated the importance of low redox potential for successfulcultivation of axenic E. histolytica, and the fact that reduced glutathione normally oxidizes less rapidly than cysteine might be important (FLC. Fahey, pers. commun.). Proteins containing Fe2÷ and labile sulfhydryl play a major role in the electron transport and metabolism of E. histolytica 21. Therefore the influence of various thiol-reducing agents in the survival and growth of the amoebae may also be through their effects on the synthesis and maintenance of structural integrity of these iron-sulphur proteins. Thiol proteinases have been detected and characterized in E. histolytica and may have important functions in metabolism and pathogenicity of the organism22,23. The reducing agent seems to be one of the essential factors which may induce encystment of axenic E. histolytica. According to a recent report, a strongly reducing medium (87 mM thioglycolate) can induce encystment of E. histolytica in TYI-S-33 medium 14. In minimal medium 'precyst' formation occurred even in the absence of cysteine but they were readily disintegrated 2s. Meerovitch et OI.26 have shown that the ability to invade is an index of adaptation of amoebae to grow at higher than the normal redox potentials. Thus, the loss of virulence of E. histolytica strains after prolonged periods of in vitro cultivation could be due to their adaptation
to a low redox potential, such as is usually prevalent in the culture tubes. Another point of interest is that E. histolytica can tolerate up to 5% oxygen in the gas phase, and is able to detoxify products of oxygen reduction in the medium 2. Although E. histolytica is primarily a colonic pathogen, it also invades the brain, pericardium, lung and liver. Thus neither the tissues invaded by the amoebae nor the media used for their cultivation are notably deficient in oxygen, Curiously, E. histolytica lacks both catalase5,27 and peroxidase27, but superoxide dismutase has been found in this pathogen27. Hence if the toxic superoxide anion 0 2 is formed by the partial reduction of oxygen in aerobic enzymatic oxidations, it would be removed with the formation of H20 2. The obvious variable effective in detoxifying the H20 2 is the availability of excess sulfhydryl groups. In other systems, toxicity of thiols at levels comparable to that of oxygen (i.e. about 0.2 mM) has been observed and is thought to involve peroxide-forming oxidation as follows: 2RSH + 0 2 = RSSR + H20 2 At higher thiol levels toxicity disappears presumably because the excess thiol destroys the peroxide (R.C. Fahey, pers. comm.)'. 2RSH + H20 2 -- RSSR -I- 2H20 In this sense the reducing agent may also be utilized as a detoxification mechanism in E. histolytica which, otherwise, has a limited ability to detoxify products of oxygen reduction. This idea is also supported by the observation2 that the uninoculated culture medium, containing 0. I% cysteine, exposed to 5% oxygen for 24 h, will support the normal growth of the amoebae. However, exposure for 72 h was unfavourable for the organism, possibly because toxic products of oxygen reduction accumulated in excess of the organism's ability to detoxify them. The toxicity of the medium could be reversed by adding fresh cysteine (sulfhydryl compounds can detoxify products of oxygen reduction28). In this context, it would be of interest to see if addition of catalase or peroxidase to the growth medium lowers the requirement for thiol.
D o Bacteria protect Amoebae?
In the host, it may be that tissues such as liver have localized regions with little oxy~)1988. ElsevierPubhcat,ons.Cambr,dge0169~758~88/$02.00
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gen where the organism can grow, or that bacteria detoxify the products of oxygen reduction to protect the amoebae. Bracha and Mirelman 29considered that the induction of oxidized molecules within the amoebae inhibited their virulence by consuming the cell's reducing power, which could then be restored by the ingestion of bacteria. The ingested bacteria may function as broad range scavengers for oxidized molecules and metabolites through the contribution of enzymatic systems, components, or products. In cultures of certain anaerobic bacteria, the need for reducing agents can be obviated by the use of an Escherichia coli membrane fraction that scavenges oxygen and provides a suitable reducing environment 30. It appears that reducing agents have structural and metabolic functions as well as redox functions for E. histolytica, but none of these can individually explain all the observed effects. Although no conclusive evidence is available, it would be premature to discount the possible participation of reducing agents in differentiation and pathogenicity, and in pi-otecting the trophozoites from oxygen toxicity. Further work is needed to clarify these processes and to decipher the biological and metabolic peculiarities of this enigmatic pathogen.
References
I Mirelman, D., Sargeaunt, P.G. [Debate] (I 987) Parasitology Today 3, 37-43 2 Band, R.N. and Cirrito, H. (IC'79)]. Protozool. 26, 282-286 3 Wittner, M. (I 968)]. Protozool. 15,403-406 4 Montalvo, F.E,, Reeves, R.E. ar,d Warren, L.G. (1971) Exp. Parasitol. 30, 249-256 5 Weinbach, E.C. and Diamond, L.S. (1974)Exp. Parasitol. 35,232-243 6 Hansen, E.L (1950)]. Lab. Gin A4ed. 35, 303312 7 Griffin, A.M and McCarter, W.G. (1950) ]. Parasitol. 36, 238-247 8 Shaffer, J.G., Rydon, F.W. and Frye, W.W. (I 948) Am. J. Hyg. 47, 345-350 9 Rees, C.W., Reardon, L.V.a.nd Bartgis, I.L (1950) Parasitology 40, 338-342 10 Diamond, L.S. (1968)]. Parasitol. 54, 10471056 I I Diamond, L.S., Harlow, D.R. and Cunnick, C.C. (1978) Trans. R. Sac. Trap. Med. Hyg. 72, 431432 12 Singh, B.N, Das, S.R. and Dul~, G.P. (1973) Curt. Sci. 42, 227-230 13 Singh, B.N., Dutta, G.P. and Das, S.R. (1974) Curr. Sci. 43, 71-73 14 Gillin, F.D. and Diamond, L.S. (1981) Exp. Parasitol. 5 I, 382-39 I 15 Mehlotra, R.K., Shukla, O.P. and Singh, BN. (1986) Ind.]. Parasitol. 10, 135-141 16 Hungate, R.E. (1969) Methods in Microbiology, Academic Press, London, pp 117-132 17 Gillin, F.D. and Diamond, L.S. (1980)]. Protozool. 27,220-225 18 Gillin, F.D. and Diamond, LS (1980)]. Protozoa/. 27, 474-478 19 Gillin, F.D. and Diamond, L.S. (1981) Exp. Parasitol. 52, 9-17 20 Fahey, R.C. eta/. (1984) Science 224, 70-72 ~) 1988,BsevierPublications,Cambridge0169~t758/88z$0200
21 Weinbach, E.C. ( 1981 ) Trends Biochem. Sci. 6, 254-257 22 Scholze, H. and Werries, E (1986) Mol. Biochem. Parasitol. 18, 103-112 23 Scholze, H., Otte, J. and Werries, E. ( 1986)Mol. Biochem. Parasitol. 18, 113-121 24 Rivera, P.R. and Correa-Lemus, I. (1986) Arch. Invest. Med. (Mexico) 17, 19-23 25 Mitra, S. and Krishna Murti, C.R. (1978) Proc. Ind. Acad. Sci. 87B, 9-23 26 Meerovitch, E., Eaton, R.D.P. and McLaughlin,]. (1976) In Amibiasis, (Sepulveda, B. and Diamond, L.S. eds) pp 628-635, Instituto Mexicano del Seguro Social, Mexico
27 Sykes, D.E. and Band, RN. (I 977)] Cell Biol. 75, p. 86a 28 Jocelyn, P.C. (1972) Biochemistry of the SH Group, Academic Press, New York 29 Bracha, R. and Mirelman, D. (I 984)]. Exp. Meal 160, 353-368 30 Adler, H.I. and Crow, W.D. (1981) Biotechnology and Bioengineering Symposium II, 533-540
t~K. Mehlotra and O.P. Shukla are at the Division of Biochemistry, Central Drug Research Institute, Lucknow 22600 I, India. Thisis CDRI communication no. 4162.
Trichinosis R e v i s i t e d A n o t h e r Look at Modes of Transmission W.C. Campbell The epidemiology of trichinosis is often illustrated by means of intricate diagrams with interlocking circles or squares and with enough variously pointing arrows to accommodate a battlefield of mediaeval maniacs. The cyclical aspect of the life cycle is often obscured. I suspect such diagrams contribute more to the amusement of their creators than to the enlightenment of their readers, yet it is easy to understand their widespread production. Trichinella has been reported from about 150 mammalian species, and these include animals with a great deal of diversity in their interspecies contacts and feeding proclivities, as well as diversity in the climatic zones that they occupy. Furthermore, there have been divergent views on the role of rats in transmission, long-standing doubts about the simple garbagecentred transmission picture that was drawn some half century ago, and a complementary growing awareness of the role of wildlife and the complexity of transmission when viewed in global perspective. Fortunately there are patterns, and I think these are sufficiently distinct and sufficiently few to warrant separate diagrams, each of which can be kept fairly simple and which together might give a broad, if inevitably superficial, understanding of the subject. Trichinell(] pseudospiralis Garkavi, 1972, is widely though not universally, accepted as a distinct species from the familiar T. spiralis. It does not induce capsule formation in host muscle and appears to be primarily a parasite of birds, though it will infect mammals. It is
of great biological interest but is rarely found, Since it is not at present a parasite of public health significance I will not consider it further. Because the present article is in the nature of a revisitation, references to the literature will be limited to a few recent reports, and readers are referred to my earlier article I for more extensive documentation. Trichinosis is transmitted from one animal to another through the ingestion of flesh containing Trichinelb larvae -the larvae being the progeny of adult worms that had inhabited the gut of the first animal and destined to mature to adulthood in the gut of the second. A useful distinction has been made between the 'sylvatic' or 'wildlife' cycle of Trichinelk] transmission and the 'domestic' or 'synanthropic' cycle. By 'domestic cycle' is meant the cycle of transmission involving human settlements, where man lives in association with pigs and rats. The association with such synanthropic animals may of course be very remote, but the urban banker eating his breakfast sausage is, in the present context, as surely associated with swine as the farmer who raisesthe pig. It is generally recognized that the wildlife cycle operates independently of man, although sometimes serving as a source of human infection. When it comes to the domestic cycle however, man's anthropocentric predisposition often leads him to place man metaphorically (and, in the making of diagrams, often literally) at the centre of the picture. Yet human infection should be considered incidental to both cycles, even though human activity is
