Download - Lipid Metabolism Mammalian Protozoan Evolutionary Hypothesis · 298 ORMERODANDVENKATESAN like, andrather sluggish form;duringthe course ofthis transformation, cell division occurs

MICROBIOLOGICAL REVIEWS, Sept. 1982, p. 296-307 Vol. 46, No. 30146-0749/82/030296-12$02.00/0Copyright © 1982, American Society for Microbiology

Similarities of Lipid Metabolism in Mammalian andProtozoan Cells: an Evolutionary Hypothesis for the

Prevalence of AtheromaW. E. ORMERODl* AND S. VENKATESAN2

London School ofHygiene and Tropical Medicine, London WCI E 7HT,1 and Medical Research CouncilLipid Metabolism Unit, Hammersmith Hospital, London W12,2 England

INTRODUCTION ...................................................... 296CHOLESTEROL ESTER ACCUMULATION....................................... 297

Aortic Smooth Muscle Cells..................................................... 297Trypanosomes ...................................................... 297

LETHAL FACTORS IN PLASMA................................................ 298Trypanockid Activity of Plasma Against Trypanosoma brucei........................ 298Possible Nature of Trypanocidal Factors.......................................... 299Relationship Between Cholesterol Uptake and Trypanocidal Activity

of High-Density Lipoprotein .................................................. 299Factors Lethal to Other Species of Trypanosoma ................................... 299

INHIBITORY FACTORS IN iILASMA ..................... ....................... 300Action of "Supplements" on T. brucei and Trypanosoma vivax....................... 300Action,of "Supplements" on Trypanosoma kwisi................................... 300

LIPID DIETARY FACTORS ACTING ON PROTOZOA ............................. 300Effect of Polyunsaturated Fat in the Diet on Parasitic Protozoa ....... ............... 300Inhibitory Effect of Milk on Parasitic Protoza ........... .......................... 300

DIRECT ABSORPTION OF LIPIDS BY PROTOZOA ......... ...................... 301Trypanosomes ...................................................... 301Leishmania ..................................................... 301Entamoeba..................................................... 301

INTERACTIONS OF THE MALARIA PARASITE .................................. 301Absorption of Lipoprotein ...................................................... 301Liberation of Hydrolytic Enzymes................................................ 302Further Metabolism of Liberated Lipid........................................... 302Shimlarities of Malaria and Trypanosome Infections ................................ 302

CONCLUSIONS ...................................................... 302Possible Relationship Between Aortic and Protozoal Disease ......................... 302Hypothesis of Evolutionary Development of a Protective Lipoprotein ...... ........... 303Lack of Selective Pressure to Eliminate Toxic Factor ......... ...................... 304Evolution of Parasitic Protozoa.................................................. 304Reasons for Pursuing Studies in Lipid Metabolism of Protozoa ....... ............... 305

SUMMARY .............. ....................................... 305LITERATURE CITED...................................................... 305

INTRODUCTION

It is today widely accepted that cholesterolesters of fatty acids, derived from the diet, arecarried in plasma low-density lipoprotein (LDL)and that high levels of plasma cholesterol areassociated with the deposition of plaques, con-sisting mainly of cholesterol ester and crystals offree cholesterol, in the aortic smooth muscle cell(ASMC).

There is a similar association, less widelyrecognized, between the absorption of choles-

terol esters of fatty acids and the behavior ofcertain parasitic protozoa which are found in theblood. This relationship is most clearly seen inTrypanosoma brucei, in which cholesterol fattyacid esters enter the trypanosome cell and it isdestroyed in a manner strikingly similar to theprocess with ASMC. Related mechanisms oflipid absorption and sometimes of cytolysis arealso found in other pathogenic protozoa, butthey are less clearly defined and their similarityto the destruction of the ASMC is less obvious.The purpose of this review is to compare and

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MAMMALIAN AND PROTOZOAN LIPID METABOLISM 297

contrast the mechanisms of uptake of cholester-ol and fatty acids and their storage and lethalproperties for both mammalian and variousmodel protozoan cells; we will also put forwardhypotheses to explain the facts. Discussion ofthe process in the ASMC need only be brief,amounting to a setting of the scene, since thework is generally well known and has been ablydescribed in a number of recent reviews (3, 9,46). More detail is necessary for the protozoancells, since the relevant work has not hithertobeen reviewed. The fact that there are importantgaps in our knowledge of the lipid metabolism ofpathogenic protozoa does not seem a reason forpostponing such a review; rather, such a reviewshould serve as a means of emphasizing theurgency of filling the gaps.

CHOLESTEROL ESTER ACCUMULATION

Aortic Smooth Muscle CeilsAll eucaryotic cells require cholesterol and

fatty acids for the synthesis of their membranes,and mammalian cells obtain them in two ways:by direct synthesis within the cell (29, 88) and bytransport via the plasma from sites of synthesiselsewhere in the body or from the diet. Aboutthree-quarters of the plasma cholesterol is car-ried in the 3-lipoprotein, or LDL, and most ofthe remainder of the a-lipoprotein, or high-density lipoprotein (HDL); a small fraction oc-curs in the pre-6-, or very low-density, lipopro-tein (78). The cholesterol esters of fatty acids,which are by themselves insoluble in plasma, arelocated in the LDL as a nonpolar core surround-ed with a polar shell of phospholipids, apopro-tein, and unesterified cholesterol, thus ensuringsolubilization and transport. Normally the syn-thesis of cholesterol and its conversion to theester is regulated in the cell by a homeostaticmechanism based on the amount ofLDL choles-terol presented to the outside of the cell (8).Various mechanical and toxic factors have beensuggested as means by which the homeostaticmechanism can be deranged, but probably themost important clinical factor in increasing thestorage of cholesterol, at least in ASMC, ap-pears to be an increase in the dietary consump-tion of cholesterol and of saturated fatty acids,which have the specific effect of increasing theamount of cholesterol incorporated in the lipo-protein and therefore available to be taken upfrom the blood. The result is an enhanced uptakeand storage of cholesterol esters, which appearas globules within the cell, giving it the charac-teristic appearance of a "foam cell" (42). ASMCseem to be particularly at risk, presumably be-cause of the structural role that they play in thebody and because they cannot easily be re-

placed; they are transformed into the atheroma-tous plaques of the arterial wall which Virchow(91) recognized as consisting mainly of lipid.Besides humans, a number of different speciesof animals also develop atheroma under theinfluence of a high-cholesterol diet; an experi-mental model has been developed in rabbits,which, when fed on a diet high in cholesterol,develop, as do other species, typical foam cellsin the aorta. In this model (63), large globules ofcholesterol ester accumulate and are associatedwith the release of hydrolytic enzymes, includ-ing those usually associated with the activationof lysosomes, and these enzymes gradually de-stroy the cell, leaving behind a deposit of choles-terol and cholesterol ester.Although LDL is recogniied as the main

carrier of cholesterol, attention has recentlybeen focused on the action of HDL, which mayreverse the process of cholesterol accumulationin ASMC (11, 66). Experiments with fibroblastscultured in vitro have demonstrated the exis-tence of receptors for LDL to which HDL mayalso become attached, and it is thought that thisattachment of HDL may be responsible forremoval of excess cholesterol from the cell (53).An alternative hypothesis, however, suggeststhat HDL may compete with LDL for the samereceptors, and, since it is removed from thesereceptors to the interior of the cell less rapidlythan is LDL, it may block, or at least slowdown, the rate of accumulation of cholesterolcarried by LDL into the cell (10, 80). The degreeof blocking may be regulated by the constitutionof the HDL; for instance, it has been shown thata diet very rich in cholesterol produces a form ofHDL which, although only a small fraction ofthe whole plasma HDL, becomes fixed toASMC in vitro at an increased rate (47).

Centrifugal analysis of lipoprotein suggestssome heterogeneity (44), three fractions beingfound in the LDL and three or four being foundin the HDL; although it is possible that some areartifacts of centrifugation, others, at least, havedistinct apoprotein structures. Modification ofthese structures (48) increases the diversity oftheir physiological actions, which suggests thatactivity may be determined more by apoproteinthan by lipid structure. The diverse reactions oflipoprotein fractions on protozoa and the lack ofspecificity of action of the individual lipid com-ponents (see Trypanosomes) suggests that inthis field also, diversity of action may be afunction of apoprotein structure.

TrypanosomesPart of the life cycle of T. brucei occurs in the

blood and involves the transformation of a long,narrow, and highly active trypomastigote, whichemerges from the tissues, into a stumpy, leaf-

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298 ORMEROD AND VENKATESAN

like, and rather sluggish form; during the courseof this transformation, cell division occurs andthe residual stumpy form, in which division hasceased, accumulates globules of lipid (61). Atthe same time, the stumpy form acquires anexcess of cholesterol, mostly as the ester whichis presumably contained in these globules (90).The stumpy forms are then destroyed, liberatingtheir lipid, some of which is taken up by macro-phages (M. Guy, unpublished data); the rest isprobably mobilized (see Interactions of the Ma-laria Parasite). The destruction of parasites inthe blood seems to be due to the liberation ofintracellular hydrolytic enzymes which are se-creted in response to the accumulation of lipidand begin to dissolve the cytoplasm of the cell;these events in the trypanosome (89) closelyresemble those that occur in the ASMC of therabbit aorta model (63).The hydrolytic enzymes so far identified in

trypanosomes undergoing dissolution are ca-thepsin D, acid phosphatase (89), and phospholi-pase A1 (85). Phospholipase A1 has been identi-fied in both Trypanosoma congolense and T.brucei (87), and its action on phospholipids givesrise to a hemolytic mixture of free fatty acids,mainly unsaturated: C14:0 (2.8%), C16:0 (20%),C18:0 (24%), C18:1 (7%), C18:2 (22.5%), and C18:3(2.8%); C18:2 (linoleic acid) is said to be the mostactive as a hemolytic agent (85). Similar work onmalaria parasites is described in Interactions ofthe Malaria Parasite.The stages of T. brucei which occur in the

blood do not synthesize sterols, and in thisrespect they differ markedly from mammaliancells; moreover, culture forms, which are pre-sumably related to the stages found in the insectvector, the tsetse fly, do synthesize sterols, butthese turn out to be phytosterols, such as ergos-terol, and not, apparently, cholesterol (17). Itseems likely, therefore, that the cholesterol andcholesterol esters accumulated in the stumpytrypanosomes are derived entirely from theblood and are therefore predominantly of dietaryorigin.

Free cholesterol and free fatty acids do notoccur normally in the blood; they are incorporat-ed into the lipoprotein, which is then absorbedby the trypanosome. Confirmatory evidence ofthis mechanism of uptake is provided by dietaryexperiments in which rats were fed on a dietdeficient in fat; on this abnormal diet, an abnor-mal fatty acid (eicosatrienoic acid, C20:3,9) ap-peared in the blood, replacing the normal C20:2and C20:3,6 acids. The abnormal fatty acid was,in these experiments, absorbed unchanged bythe stumpy trypanosomes, and this providesadditional evidence that absorption occurs viathe lipoprotein, since in these experiments cho-lesterol and fatty acids were mainly found asso-ciated together with the phospholipids that

would .be expected as normal constituents ofplasma lipoprotein (90). Although it seems likelythat cholesterol is carried into the trypanosomeby LDL, as it is carried into the ASMC inhumans, it is not possible to state this with anydegree of assurance, since the experiments werecarried out in rats. In rat blood, LDL is presentin low concentration, so in this species HDLbecomes the main carrier of cholesterol (79);however, accumulation of lipid occurs in thetrypanosomes infecting a wide range of otherspecies, including humans (57), so it is likely thatthe main carrier of cholesterol esters (be it HDLor LDL) is involved in the accumulation ofcholesterol esters in the trypanosome, whateverits host species.

LETHAL FACTORS IN PLASMA

Trypanocidal Activity of Plasma AgainstTrypanosoma brucei

It has been known for many years that a factorpresent in human plasma is lethal to T. brucei(41). This factor is also found in the bloods ofhigher apes and monkeys and seems to havebeen developed as a protective mechanismagainst trypanosomes. However, some strainsof trypanosomes have become resistant to thetrypanocidal factor, so that although humans areprotected from most of the strains that infectcattle, antelopes, and carnivores, they remainsusceptible to certain strains of T. brucei (which,to distinguish them from strains that do notinfect humans, are usually placed in the subspe-cies T. brucei gambiense and T. brucei rhode-siense). The "gambian" and "rhodesian"strains of T. brucei are identical to the strains ofT. brucei usually found in animals except in theirability to overcome the trypanocidal factor;these are the causative organisms of "Africansleeping sickness" in humans (67). (Thomas andBreinl [84a] showed that T. brucei gambiense,inoculated intraperitoneally, could infect ba-boons, but gave a benign infection with very lowparasitemia.) In contrast, the baboon, which hasa particularly high level of trypanocidal activityin its plasma, cannot be infected with any strainof T. brucei unless the trypanosomes are inject-ed directly into its cerebrospinal fluid, where thetoxic lipoprotein factor occurs in low concentra-tion (65). Early work appeared to suggest thatthe trypanocidal factor was related to antibody,although it was noted at the time that the plas-mas of some individuals-particularly those whosuffered from liver diseases, such as cirrhosis,infective hepatitis, or obstructive jaundice-haddiminished trypanocidal activities (72). Morerecent work has also appeared to confirm therelationship of the toxic factor with antibody byassociating its trypanocidal action with the mac-roglobulin fraction of plasma when this had been

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fractionated by ion-exchange and gel filtrationchromatography (34). However, it was also not-ed that lipoproteins were associated with thesame chromatographic fraction of plasma, so thetrypanocidal factor could not then be identifiedwith certainty. The matter has not been settledconclusively by Rifkin (68, 69), who has shownnot only that the trypanocidal activity is associ-ated specifically with the HDL fraction but thatits activity is greatly reduced in the plasma of acase of Tangier disease, a genetic abnormality inwhich HDL is virtually absent. However, Rifkindid not suggest that all of the HDL fraction wastrypanocidal; indeed, she showed that the try-panocidal activity occurred in only part of theelution curve of HDL.

Possible Nature of Trypanocidal FactorsTwo types of trypanocidal factor-active in

vitro and in vivo, respectively-occur in theplasmas of humans and baboons, the propor-tions of these activities varying among differentindividuals; moreover, the stabilities of the twoactivities are not the same in any one plasma(32), and it is therefore likely that two sub-stances are involved. Of these two, the in vitro-acting substance seems to be a fraction of HDL;the identity of the in vivo-acting substance isless certain, although Hawking et al. (34) suggestthat it may be of a different nature, perhaps asubstance which is self-activated in the recipi-ent's body. One candidate for the role of activa-tor or activated agent is the enzyme lecithin:cho-lesterol acyltransferase (EC 2.3.1.43), which ispresent in high concentrations in the bloods ofhumans, baboons, and other higher primates butis reduced in cases of liver disease (20), as areboth trypanocidal factors. The action of lecithin:cholesterol acyltransferase is to incorporatecholesterol into lipoprotein, forming the choles-terol ester and displacing lecithin; HDL is mostreadily affected and can be changed in this waywhen the enzyme is transfused from one personto another (21). In addition, lipoprotein from onespecies can be transformed after transfusion intoanother species (7); it is therefore reasonable tosuppose that the lecithin:cholesterol acyltrans-ferase of one species might act on the lipoproteinof another and that this might occur in the invivo test. One must, however, recognize thatsuch evidence is indirect and that there is as yetno direct evidence in support of the hypothesisthat the in vivo factor can be identified aslecithin:cholesterol acyltransferase.

Relationship Between Cholesterol Uptake andTrypanocidal Activity of High-Density

LipoproteinWhat, then, is the relationship between the

formation of lipid globules due to the uptake of

lipoprotein by the stumpy form of T. brucei andthe trypanocidal activity of HDL? Are they thesame phenomenon seen from different perspec-tives, are they closely related phenomena, or arethey totally independent of one another?The evidence so far seems to suggest that the

formation of lipid globules and the action oftrypanocidal HDL are not the same phenome-non, since the dynamics of the two processesdiffer considerably: the formation of globules is,on the one hand, a slow process which takes 3 to5 days to reach a maximum, after which thetrypanosomes are gradually destroyed over aperiod of about 24 h by liberation of hydrolyticenzymes, as described above (61); trypanocidalHDL, on the other hand, acts rapidly and willproduce complete lysis on incubation in vitro for15 min.Not all strains of T. brucei accumulate glob-

ules of lipid in their cytoplasms. There is, in-deed, great variability in the amounts of lipidtaken up by different strains, ranging fromstrains found in southern Africa, where globulesare large and the autolysis correspondinglygreat, through the more virulent strains foundnorth of Lake Victoria, which accumulate onlysmall globules, to strains without globules thathave acquired an extra virulence by rapid pas-sage between laboratory animals (57, 58). Al-though some strains visibly absorb substantialamounts of lipoprotein and others appear toabsorb none, all strains are fully susceptible tothe action of trypanocidal HDL unless they haveacquired a specific resistance to it (see Trypano-cidal Activity of Plasma Against Trypanosomabrucei). Even when such a specific resistance ispresent, only a few individual trypanosomes areable to resist the trypanocidal activity longenough to establish an infection; the majority ofthe inoculum is destroyed. It seems likely, al-though this cannot yet be proved, that the for-mation of lipid globules and the action of trypan-ocidal HDL are distinct yet related phenomena,but it is not yet clear in what way they arerelated.Factors Lethal to Other Species of TrypanosomaAlthough most work has been done on the

effects of serum on T. brucei, other species ofTrypanosoma are similarly affected; thus, thefailure of other trypanosomes (such as T. congo-lense and Trypanosoma vivax) affecting cattleand game animals to infect humans may or maynot be due to similar substances in human plas-ma. For instance, Hawking has shown that somestrains of T. congolense and T. vivax may behighly resistant to human serum, indicating thatthe resistance of humans to these trypanosomesmust be due to some other mechanism (31).Conversely, studies have been made on nonhu-man host species that produce trypanocidal sub-

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stances. One such species is the cotton rat,Sigmodon hispidus, which has in its serum apowerful trypanocidal substance lethal to T.vivax (83). The active substance is again found inthe part of the chromatogram where lipoproteinsand macroglobulins are eluted, and it was at firstidentified tentatively as a macroglobulin (83);the nature of the factor in cotton rat blood whichkills T. vivax therefore should be reassessed.

INHIBITORY FACTORS IN PLASMAAction of "Supplements" on T. brucei and

Trypanosoma vivaxEarly studies on the lethal actions of human

and baboon sera on T. brucei demonstrated thattheir trypanocidal actions could be inhibited byserum from another species of animal if it wasgiven concurrently. Sheep or rabbit serum wasusually, but by no means always, effective,whereas mouse serum was never effective (94).This work has not yet been repeated, but thefocus of interest has shifted to the inhibitoryeffect of serum on the lethal factor (or factors)which kills T. vivax when it is inoculated intorats. T. vivax will not normally infect laboratoryrats unless it is injected together with the serumof a susceptible animal, such as a cow, a sheep,or an antelope, and, although it is sometimespossible for a strain that has been passagedmany times in the presence of supplements to"take" eventually without supplementation, re-peated supplements normally have to be given inorder to maintain the infection (14-16).Action of "Supplements" on Trypanosoma kwisiA similar phenomenon relates to the infection

of mice with the rat trypanosome Trypanosomalewisi. This infection will only occur if the miceare given rat serum supplements at intervals.The nature of the supplement has been studiedin greater detail in the T. lewisi-mouse modelthan in the T. vivax-rat model, and it has beenpossible to exclude the participation of immuno-globulins M and G (y-2). However, other poten-tially active substances remain in the fraction,and these include siderophilin, immunoglobulinG (-y-1) (27, 28). In these experiments the lipidcontent of the fraction was found to be below0.5%, and although the possibility that a specificlipoprotein was present cannot be eliminated, itis clear that at so low a concentration it wouldhave to be exceptionally active.

LIPID DIETARY FACTORS ACTING ONPROTOZOA

Effect of Polyunsaturated Fat in the Diet onParasitic Protozoa

Infections with a number of protozoal para-sites are modified or even suppressed by

changes in the lipid concentration of the diet.Godfrey (22, 23) has shown that T. congolenseinfections are markedly depressed by feedingthe host on a diet containing cod-liver oil. T.vivax infection maintained in rats with a sheepserum supplement was completely suppressedby cod-liver oil. No suppression was noted ininfections with Trypanosoma cruzi or with T.brucei, although it is possible that the latter mayhave been one of the highly virulent strains(noted above) which do not visibly absorb lipid.Babesia rodhani and Plasmodium berghei weresimilarly inhibited by cod-liver oil. On furtherexamination, Godfrey (24) and Taylor (82) wereable to demonstrate that the toxic factors in cod-liver oil were the polyunsaturated fatty acidsC20:4, C20:5, C22:5, and C22:6, of the family oflinolenic acid (C18:3), to which they are degradedin vivo (36).The action of these polyunsaturated fatty ac-

ids in inhibiting parasitemia could be preventedby increasing the dietary intake of a-tocopherol(vitamin E) or other in vivo antioxidants whichhave the effect of preventing the formation ofepoxides and free radicals that are formed frompolyunsaturated fatty acids and are highly toxicto cells.There has been some argument as to whether

similar substances may be toxic to mammaliancells, and there is evidence that derivatives,possibly carcinogenic, of the peroxidized fattyacids may be stored in tissues when such acidsare fed to mammals; however, no toxicity orstorage occurs when dietary intake is also ac-companied by an increase in a-tocopherol, be-cause presumably the peroxidized acids becomereduced before intracellular absorption occurs(62, 64, 81). Free fatty acids as such do notnormally occur in the blood; they are in facthighly toxic, especially the common saturatedfatty acids (13, 35), but the extensive uptake oflipid by parasites, which Godfrey's experimentsimply, is most likely to occur via the absorptionof lipoproteins, and their activity, which is somuch greater on the parasites than on the cells ofthe host, is probably due to preferential uptakeof lipoprotein by the parasites.

Inhibitory Effect of Milk on Parasitic ProtozoaMice fed on a diet of unextracted casein

cannot support infection with T. congolense.This might be due to the presence either of lipidor of some other toxic factor, but is usually heldto be due to the absence from milk of somedietary factor essential for the growth of T.congolense (37). A similar explanation is usuallygiven for the inhibitory effect of a milk diet on P.berghei (30, 45) infection, in which the effect isusually assumed to be due to the absence of 4-aminobenzoic acid, an essential dietary factor

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for the malaria parasite, which mammalian cellscan mostly synthesize. However, in a number ofexperiments the expected inhibition did not oc-cur (33), and it is possible that additional factorsare operating. In one set of experiments (26), theinhibitory effect of a milk diet did not occur ifskim milk was used, and this suggested that thepresence of lipid might also be important andcontribute to the inhibitory effects caused by theabsence of 4-aminobenzoic acid; a similar ex-periment (74) was less conclusive in its result.Milk fats are 65% saturated (C14, C16, and C18),but they also include a substantial (31%) amountof monounsaturated fats (C16:1 and C18 l, mainlythe latter); only a small amount of polyunsatu-rated (3.4%) linoleic acid (C18g2) is present (36).Since polyunsaturated fatty acid forms only asmall proportion of the lipids of milk, it seemsunlikely that the action of the lipid is due tooxidation or that it would be reversed by a-tocopherol. Recent repetition of these experi-ments under more carefully controlled condi-tions (H. F. El Bashir, E. B. Fern, and W. E.Ormerod, unpublished data) showed that theinhibitory effect of skim milk was not primarilydue to deficiency of lipid so much as to distur-bance of appetite. Inhibition of malaria was thuscaused by protein deficiency (18), and it is notrelated to the uptake of lipid.

DIRECT ABSORPTION OF LIPIDS BYPROTOZOA

TrypanosomesAlthough free lipids do not normally occur in

the blood, parasitic protozoa do apparently havethe ability to absorb them. Thus, T. lewisi andTrypanosoma equiperdum (now usually consid-ered to be a subspecies of T. brucei) have beenreported to absorb globules of fat if these areadded to the blood in which they are suspended(92, 93).

LeishmanaLeishmania species appear also to absorb

micellar globules of lipid, although this processis probably more a function of the macrophagein which the amastigote of this parasite is har-bored. The process has been used to presenttoxic chemotherapeutic substances to the para-site, since macrophage and parasite absorb theglobules in greater concentration than do theother cells (4).

EntamoebaEntamoeba histolytica, which is not a blood

parasite, has the property of absorbing nativecholesterol when this is added to the culturemedium; under these conditions its behavior is

changed from that of a nonpathogenic species,which develops in the lumen of the gut withoutcausing lesions, to a pathogenic species thatinvades the gut wall and produces ulcers (5, 76).Although this behavior appears to be well sub-stantiated, it is in apparent conflict with themore recently accepted concept that the patho-genicity of E. histolytica is a genetically con-trolled factor that can be identified by isoen-zyme markers (73); however, it is interesting todraw the analogy between the accumulation ofcholesterol in T. brucei, with subsequent releaseof hydrolytic enzymes, and the possible require-ment of cholesterol by E. histolytica in orderthat it shall produce the hydrolytic enzymeswhich are a necessary factor in its pathogenicity(55, 56).

INTERACTIONS OF THE MALARIAPARASITE

Absorption of LipoproteinNote has been taken above of the accumula-

tion of lipid in the cells of African trypanosomesvia the lipoprotein and of the trypanocidal actionof HDL. It has also been noted that polyunsatu-rated fats and possibly other types of fat takenby mouth can inhibit the growth of malariaparasites. It is therefore pertinent to reviewevidence which might indicate that the malariaparasite also interacts with plasma lipoprotein ina manner similar to that of the trypanosomes.

Clofibrate, a synthetic drug which lowers thelevel of plasma lipoprotein, has been shown todecrease the parasitemia in experimental malar-ia caused by P. berghei (52); this activity sug-gests that lipoprotein may carry essential lipidmetabolites which act as limiting factors on thegrowth of the malaria parasites, but the possibili-ty that clofibrate acts directly on the parasite asan inhibitory agent cannot be excluded.

Experimental malaria (49-51) in mice appearsto be associated with an increase in LDL andvery low-density lipoprotein, whereas in hu-mans infection with P. vivax has induced acondition resembling endogenous hyperlipemia(Frederickson type IV), a transient increase inLDL reversible by chloroquine therapy; howev-er, in another study of patients infected with P.vivax (38), the opposite appears to obtain, withtransient disappearance of plasma HDL and itsreappearance after the parasitemia has beenremoved by chloroquine treatment. The authorsof the latter study suggest that the HDL mayhave been taken up by the parasites and point tothe very high concentrations of phosphatidyl-choline in both the parasites and the HDL.

Extracts of P. berghei have been shown tocontain sterol esters amounting to 65% of the

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neutral fat lipids present, and, although thesterols involved were not identified in this in-stance, there is no evidence that any sterol otherthan cholesterol occurs in the malaria parasite(6). Since malaria parasites do not synthesize denovo either cholesterol or fatty acids (12), itseems likely that the sterol ester is derivedentirely from absorbed plasma lipoprotein.Although the evidence for absorption of lipo-

protein by, and accumulation of cholesterol es-ter in, the malaria parasite is preliminary andsomewhat inconclusive, it is reasonably consis-tent in supporting the hypothesis that the level ofplasma lipoprotein is an important factor in thegrowth of the malaria parasite in the blood, butthere are as yet no data on the mechanism ofabsorption, i.e., whether it occurs through thestructure of the erythrocyte or directly from theplasma.

Liberation of Hydrolytic EnzymesThe uptake of lipoprotein by the malaria para-

site, as in trypanosomes, is followed by libera-tion of hydrolytic enzymes, and this suggeststhat a process similar to that described in Cho-lesterol Ester Accumulation for ASMC and try-panosomes also occurs in the malaria parasite.Evidence of a possible connection between ab-sorption of lipid and the liberation of hydrolyticsubstances has arisen from the study of a so-called lytic factor isolated from several speciesof Plasmodium. This lytic factor, stated to lyseparasitized and normal corpuscles, has been saidto consist of monounsaturated C18:1 fatty acids(39), and the main activity has been ascribed tovaccinic acid (Ci8:Iw7), rather than to the moreusual oleic acid (C,8:1, ) (40). Recent work,however, has failed to identify vaccinic acid inisolates, but has disclosed a mixture similar tothe hemolytic mixture of fatty acids obtainedfrom trypanosomes (see Cholesterol Ester Ac-cumulation) consisting of C16:0 (33%), C18:I(36%), and C18:2 (18%) with the minor constitu-ents C18:0, C18:3, and C19:0 (together, 13%).Boiling for 5 min changes the proportion of theseacids, and the lytic activity is reduced (6); al-though the authors state that vaccinic acid wasnot present, it is not clear from the methodsdescribed in the paper whether they were ade-quate to distinguish C181w7 from its isomer,C18:lw9.

Although the mixture of fatty acids appears tohave a specific hemolytic effect, whether pro-duced from trypanosomes, malaria parasites, orfrom the cells of the host, it is important toemphasize that the liberation is likely to occur asa result of the production of hydrolytic enzymesby the parasite as it matures. Cathepsin D andacid phosphatase (as in trypanosomes) havebeen identified as being produced by several

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species of malaria parasite (43) as they maturetowards schizogony, and much of the activityremains in the erythrocyte ghost after the daugh-ter parasites (merozoites) have been liberatedinto the blood. It is almost certain that lipids areliberated by a lipolytic enzyme similar to thephospholipase of trypanosomes. Such a processof liberating the lipid has been postulated byMaurois et al. (49).

Further Metabolism of Liberated LipidMaurois and his colleagues further suggest

that the enzymic liberation of lipid may give riseto an increased mobilization of lipid, whichcauses an increase in very low-density lipopro-tein into which the lipids are rapidly incorporat-ed; this process is followed, in turn, by a rise inLDL. Such a sequence of events suggests thatthe route of catabolism of lipids liberated byparasites in the blood may be similar to the routeof those absorbed from the gut and mobilizedaccording to the endogenous and exogenouscycles of Brown et al. (9).Thus, an increase in lipoprotein in the blood

may be caused by increased lipid mobilizationby the parasite, but a decrease may occur as aresult of the parasite consuming lipoprotein. Thepreliminary results of Lambrecht et al. (38) maybe an example of this, but it is a difficult conceptto establish because the increase in blood vol-ume, which frequently occurs in pathogenicinfections, would tend to cause an apparent fallin one factor or another.

Similarities of Malaria and TrypanosomeInfections

In malaria and trypanosome infections, a simi-lar sequence of three events takes place: (i)absorption of considerable amounts of choles-terol esters offatty acids, (ii) intracellular libera-tion of hydrolytic enzyme, and (iii) dissolution ofthe cell. The differences, however, are first thatthe trypanosome cell itself is destroyed, whereasthe malarial merozoites escape from the dis-solved erythrocyte, and second that it is only inthe trypanosome that there is a clear causalrelationship between the different stages of thelytic process; nevertheless, in both parasites thelytic process appears to be an integral part of thelife cycle.

CONCLUSIONS

Possible Evolutionary Relationship BetweenAortic and Protozoal Disease

The object of this review has been to point outthat there are analogies in the uptake of lipopro-tein and the damage that it can cause to the cellthat receives it, between, on the one hand,


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mammalian body cells (notably, ASMC) and, onthe other, various parasitic protozoa that arefound in the blood. The study of lipid physiologyin mammalian cells has, because of its impor-tance to human arterial disease, become anadvanced and sophisticated branch of science;similar studies on protozoa are in their infancy,but the diversity of effect, both in the directaction of lipoprotein factors which are toxic andin the indirect blocking action of others (or sothe preliminary evidence seems to suggest), indi-cates that the study of lipoprotein-cell interac-tion in protozoa might illuminate significantlythe study of mammalian lipid physiology (2).Although it is not, perhaps, surprising that the

study of parasites should reveal biochemicalmechanisms that are similar to those of the hostcells that they mimic, it is possible that therelationship between the actions of lipoproteinin mammals and in their protozoan parasitesmay be closer and more subtle than a meresimilarity of biochemical mechanisms.

Hypothesis of Evolutionary Development of aProtective Lipoprotein

The overall function of lipoprotein is to carryessential lipid metabolites for the cells of thehost, but it also carries lipid to blood parasites.Since parasites in the blood reproduce fasterthan the cells of the host, any effects, toxic orbeneficial, that the lipoprotein produces are like-ly to be greater on the parasite than on the host.We suggest that in the course of evolution,modifications have occurred in the structures ofcertain lipoproteins in the blood that have madethem toxic to parasitic protozoa. Such changesin structure are likely to give a net advantage tothe host, but such an advantage may also beoffset by certain disadvantages when the pro-duction of lipoprotein begins to assume a dualfunction, not only of transmitting lipid to bodycells that require it but also acting as a protectivemechanism. Through the period of evolution ofthe higher mammals, protozoa must have beenimportant competitors and pathogens; but formodem humans, especially in the northernhemisphere where protozoal disease has largelybeen eradicated, their constraining influence hasgreatly diminished. Atherogenic diets, however,are a relatively modem phenomenon and areunlikely to have affected significantly the evolu-tion of Homo sapiens.

Protozoal infection is an ancient phenomenonwhich may have antedated the evolution ofvertebrates or even of an immune system as weknow it today. Hosts, throughout the ages, haveevolved a variety of strategems for ridding them-selves of guests that have outstayed their wel-come, and the specific lipoprotein factors whichkill trypanosomes, and perhaps other parasites

as well, may be the survivors of some suchprimitive device. There may, indeed, be otherdiverse mechanisms which ensure a home onlyto the parasite that has specialized closely incolonizing a particular host species and whichlimit the aggression of other, merely opportunis-tic invaders. The highly specialized life cycles ofthe trypanosomes, malaria parasites, piro-plasms, and other hematozoa are an indicationof the extent to which mutual adaptation hasoccurred between parasite and host during thecourse of evolution.The usefulness of possessing a lipoprotein

factor which is lethal to an invading parasite hasnot been outdated in mammals by the possessionof a fully developed system of humoral and cell-mediated immunity; the continued usefulness isshown in the higher primates by their possessionof a highly effective trypanocidal factor in theHDL. Since trypanosomes are able to changetheir main antigenic structure with great speed(25, 70, 71), thereby evading the immune re-sponse, this additional protective mechanism isclearly of great value. Although sleeping sick-ness trypanosomes have in their turn been ableto adapt themselves in such a way as to evadethe lethal effects of HDL and establish an infec-tion, this property should not obscure the factthat Homo sapiens and its near relatives contin-ue to be protected from a range of trypanosomespecies widely prevalent in Africa with whichthe ruminants, which have no such effectivefactor, have had to come to terms by othermeans.Humans, the higher apes, monkeys and, par-

ticularly, baboons have in their bloods the mosteffective trypanocidal factor possessed by anyspecies, but they also seem to be the mostsusceptible species to the development of ather-oma (1); the susceptibility of these speciesseems to support the hypothesis that the devel-opment of a factor toxic to trypanosomes alsoimplies toxicity to the hosts' own cells. But thepicture is a complicated one: there are at leasttwo lipoprotein factors active against trypano-somes, a slow-acting factor (present in HDL inrats and possibly LDL in other species) and afast-acting factor, particularly active in the HDLof humans and of baboons. There also appear tobe inhibitory factors (usually referred to as se-rum supplements) in the bloods of a number ofmammals; these substances may (as has beensuggested above) also be located in the HDL.The inhibitory factors seem to act by competi-tive absorption into the trypanosome cell, there-by preventing the toxic factor (also probably inHDL) from being absorbed. The existence oftoxic factors active against protozoa and inhibi-tory factors competing against them suggests aneven greater degree of functional heterogeneity

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ofHDL than the interactions between mammali-an cells and lipoprotein (discussed at the begin-ning of this review) seem to display, and theunderstanding of this heterogeneity in the studyof protozoal lipid metabolism may contributesignificantly to the study of mammalian physiol-ogy.

In mammalian cells, cholesterol ester is takenup mainly from the LDL, and this providescholesterol and fatty acid additional to what isproduced by intracellular synthesis, a processwhich is lacking in protozoal cells. It is generallybelieved that there is a homeostatic mechanismto balance absorption with synthesis, preventingexcess absorption; when this mechanism breaksdown, as it appears to do under the influence ofcertain toxic factors, excessive accumulation ofcholesterol takes place. It is possible that one ofthese toxic processes may be the result of thesame trypanocidal factor in the HDL which,having been developed as a defense against aforeign invader, may yet have some residualaction against the host's own cells. Conversely,factors which inhibit the toxicity against humancells are believed also to occur in human HDL.Such factors appear to act by competing for thesame loci on the cell that normally take up theLDL, and we suggest that they may be related tothe inhibitory factors (serum supplements)which allow protozoal infection to occur inabnormal hosts. Excessive absorption or syn-thesis of lipoprotein might be removed frommammalian cells by a process of "cellular defe-cation" making use of the lysosome system.Although this process occurs in a wide variety ofmammalian cells, the removal of excessive lipo-protein components has not been specificallyidentified; however, it does appear to occur intrypanosomes (89). Perhaps they need such aprocess more than do mammalian cells, sincetrypanosomes do not have any homeostaticmechanism to control the amount of lipoproteinthat they absorb. If damage does occur, mostmammalian cells can be replaced, like trypano-somes in blood; but this may not apply to cellswhich have a structural role, such as ASMC; itmay be the irreplaceability of ASMC whichmakes them the point of least resistance in thehost's defences against excessive lipid accumu-lation.

Lack of Selective Pressure to Elininate ToxicFactor

It may be argued that had humans acquired atoxic factor which acted against their own cells,genetic pressure to reduce the prevalence of theallele would have increased as soon as subjec-tion to parasitic protozoa had ceased to be animportant feature of their environment; argu-

ments which suggest that such selective pres-sure is likely to be unimportant are as follows:first, humans have only recently (say, in the pastcentury) lived in such a favored environment,relatively free from parasites and with an athero-genic diet available to them, and, second, al-though the mortality from atheroscelerosis ishigh, it occurs mainly, if not exclusively, afterthe age of reproduction, and mortality fromatherosclerosis is therefore unlikely to causeselective pressure. Although wild animals keptin captivity frequently develop atheroma (19),only the higher primates-together with the Af-rican elephant (77), which is so affected onlyunder conditions of environmental stress-de-velop atheroma in the wild. During the periodthat humans or their forebears were developingantiprotozoal mechanisms, they were unlikely tohave lived long enough or to have been so well-nourished as to have developed atheroma to theextent that this would have created any evolu-tionary disadvantage to outweigh the great ad-vantage of possessing an effective antiprotozoalmechanism.

Evolution of Parasitic ProtozoaNot only have humans and their mammalian

relatives undergone evolution, but so too havethe protozoan parasites that infect them; in sodoing they have even taken advantage of someof the protective mechanisms that the host hasused against them, and the development ofresistance in some strains of T. brucei to toxicHDL has been noted in Lethal Factors in Plas-ma. However, in addition T. brucei has evolveda life cycle in its mammalian host which actuallyrequires the uptake of excess lipoprotein; oneeffect of this uptake is the accumulation ofcholesterol esters, the intracellular liberation ofhydrolytic enzymes, and the destruction of largenumbers of circulating trypanosomes. One ad-vantage of this process is in ensuring that thevascular system of the host does not becomeoverloaded with vast numbers of trypanosomesand thereby killed; another is in producing theconditions described by Ormerod (59, 60) inwhich multiple division forms can be produced,for it is these forms, when they are intracellular,that provide optimal conditions for the survivalof both host and parasite, the blood forms andother extracellular forms which endanger thehost being merely the agents for the parasite'sinsect transmission. Malaria parasites may alsohave evolved to take advantage of the absorp-tion of lipoprotein; we know that they accumu-late cholesterol esters and that this is followed insequence by intracellular secretion of hydrolyticenzymes and by destruction of the carrying cell.The sequence of events has a more obvious

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causal connection in the trypanosome, but in themalaria parasite their causal connection remainshypothetical. Similarly, other protozoal para-sites (Babesia, Leishmania, and Entamoeba) areknown to absorb lipids, and some (Entamoebaand the trichomonads [54, 75]) are known tosecrete hydrolytic enzymes. These hydrolyticenzymes have an importance in the pathogenesisof protozoal disease which is probably greatlyunderestimated; however, this aspect is not suf-ficiently close to the present topic to be pursuedfurther in this review.

Reasons for Pursuing Studies in LipidMetabolism of Protozoa

Much more is known about the mechanisms ofabsorption of lipid into mammalian than aboutprotozoal cell mechanisms; indeed, the lack ofprecise knowledge of the latter is often frustrat-ing. However, it is possible that a more detailedknowledge of the lipid metabolism of protozoamight be of value in understanding mammalianmechanisms, and hence in understanding thepathogenesis of atheroma; for instance, if theinhibiting substances (serum supplements)which antagonize the ability of a particular hostto resist a protozoal infection can be shown to belipoprotein in nature, then the range of diversityof active lipoprotein factors, possibly activeagainst mammalian cells, will have been extend-ed and tools will have been provided for thestudy of LDL and HDL receptors and the waysin which they can be activated or blocked. Theinfant science of protozoal lipid metabolism maystill have little to offer the wider field ofmamma-lian lipid physiology in terms of concrete fact,but it does offer much in terms of illuminatinghypothesis, usually the catalyst for further ex-perimentation.

SUMMARYASMC and sleeping sickness trypanosomes

(T. brucei) both store globules of excess fattyacids esterified with cholesterol that are derivedfrom the plasma lipoproteins; in both instancesthe globules are associated with intracellularliberation of hydrolytic enzymes that destroy thecells. The malaria parasite, Plasmodium, alsoabsorbs lipoproteins, and hydrolytic enzymesare also liberated and the carrier cell (the eryth-rocyte) is destroyed, but connection betweenthese processes (in Plasmodium and in severalother blood protozoa) has not been establishedas clearly as in T. brucei and ASMC.There is a diversity of serum factors-some

lipoprotein, others as yet uncharacterized-which either assist the host in destroying itstrypanosomal parasites or antagonize this ac-tion; analogous factors also affect cytolysis of

ASMC. Species with powerful trypanocidal fac-tors seem particularly prone to develop athero-ma. We suggest that lipoprotein factors havebeen evolved as antiprotozoal mechanisms butthat they have residual toxicity against hostcells, ASMC being particularly susceptible.

Genetic pressure on the parasite to adapt tosuch mechanisms has been strong, and popula-tions of trypanosomes (e.g., T. brucei rhode-siense) have developed resistance to trypanoci-dal lipoprotein; however, genetic pressure onhumans to eliminate alleles for toxic actionagainst their ASMC is weak, because the illeffects of atheroma occur mainly after the age ofreproduction and because atherogenic diets andenvironments free from protozoal pathogens arerecent phenomena.

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