Unique roles for lipids in Schistosoma mansoni

Parasitology Today, vol. 7, no. 2, 1991

59

Unique Roles for Lipids in Schistosoma mansoni S.T. Furlong The dynamic interplay among lipids has been exploited by S. mansoni to evolve some unique processes that are vital for its long-term survival within the mammalian host. Lipids are required by the parasite not only to maintain its surface integrity and structural requirements but also for egg production and cell-cell signalling. However, S. mansoni is incapable of synthesizing essential lipids and must obtain these from its host. In this review, Stephen Furlong describes the roles and routes of acquisition oflipids by this parasite. Some of the most interesting aspects of the study of parasitology ,are the means that parasites implement to exploit the host environment. A good example is Schistosoma mansoni, which lives in the host vasculature in intimate contact with elements of the host immune system, often surviving many years. Many explanations have been proposed to account for this parasite's survival in the face of the host's immune system, including acquisition or mimicry of host antigens, sloughing of bound antibodies and parasite antigens, or an inherent tolerance to immune attack due to the physical properties of the parasite's surface membrane. Recent studies now suggest that lipids, both those synthesized by the parasite as well as those derived from the host, may play a role in the parasite's defense from host immunity. However, the importance of lipids to the parasite is not limited to its defense. In fact, some aspects of these defensive strategies may have originated as a consequence of the parasite's need to acquire lipids that it cannot synthesize. Furthermore, in several other unique respects lipids are essential for completion of this parasite's life cycle. Lipids in the Life Cycle of S. mansoni

All eukaryotic cells contain phospholipids and sterols as the major components of cellular membranes. Many cells also produce biologically active lipid metabolites. In these general respects, schistosomes are similar to host cells. However, the parasite also exploits lipids in ways distinct from those of host cells, ranging from invasion by cercariae of the ~) 199 h ElsevierScience Publishers Ltd, (UK) 0169J~707/9 I/$02.00

mammalian host to the production of eggs by the adults (Table I ). Fatty acids, like those present on the skin surface, stimulate penetration by cercariae and may also promote the transformation of cercariae to schistosomula 1'2. In general, essential fatty acids appear to be more active in this regard than nonessential fatty acids. Fatty acid metabolites, namely eicosanoids, are also produced by cercariae in vitro. Eicosanoid production by cercariae can be inhibited by ibuprofen or esculetin with a corresponding decrease in penetration. The likely explanation for these results is that the free fatty acids may be important as precursors for eicosanoids while the eicosanoids play an active role in cercarial penetration 2. Eicosanoids are also produced and released by both schistosomula and adult worms 3. While such release may have a biological effect on host cells, this has yet to be directly shown. After host penetration, lipids are important for the maturation and development of the worm. One of the most characteristic changes in the parasite that occurs following skin penetration by cercariae and during the transformation process into schistosomula is the appearance of a double membrane that envelops the entire organism. As described below, immune responses to the parasite may be influenced by the lipid content of these surface membranes. As the larval schistosomula mature, other lipid metabolites, ecdysteroids, may be important 4 since both ecdysone and 20-hydroxyecdysone

have been identified in S. mansoni and also from the sera and urine of infected hosts. (Ecdysteroids are cholesterol metabolites that have been most studied as insect moulting hormones.) In schistosomes it is presumed that these molecules promote sexual development and vitellogenesis by the worms. Finally, egg production by adults can be suppressed by mevinolin, an inhibitor of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase 5. HMG-CoA reductase is an important enzyme for de novo cholesterol synthesis in mammalian cells. However, in schistosomes the drug appears to act by inhibiting the synthesis of nonsteroidal lipids such as dolichols. Dolichols serve as intermediates for protein glycosylation. Thus, while the parasite uses lipids to satisfy structural requirements and for the production of lipid mediators typical of most eukaryotic cells, lipids are also used for functions unique to the organism. Lipid Composition and Metabolism

Work from several laboratories has defined the lipid content and major lipid synthetic pathways of the parasite (Fig. I). Studies have also been initiated to characterize the lipid content of the surface membranes and the synthesis of lipid-linked proteins. Unlike many protozoan parasites that synthesize characteristic lipids, the lipid composition of S. mansoni is similar to that of host cells. Furthermore, variations in lipid content

Table I. Important lipids in the life cycle ofSchistosomamansoni Lipid class

Function

Reference

Fatty acidsand metabolites

Penetration of skin by cercariae Secreted by schistosomulaand adults

2 3

Phospholipids and sterols

Membrane components (both cellular and tegumental membranes)

6-8

Glycolipids

Membrane components and antigens

Fatty acidsand phospholipids

Lipid anchorsfor protein antigens

9

Phospholipids, sterols and lipoproteins

Defense from host immunity

Ecdysones

Sexual development Marker of infection

4 4

Dolichols

Egg production

5

20-23 27,28,30-36

60

Parasitology Today, vol. 7, no. 2, 1991

~Glycosphingolipids Cholesterol esters t / /

t

~

|

\

"Sphing°myelin X long chain b a s e s /

Cholesterol ~" ~

from h o s t l /

/ ~/+serine

"~'Fatty acids'~'/~ / Acetate |

/ /

~ Triacylglycerols /

/ MPPC ~ ~ acylated proteins ~,

/ "-Z Eicosanoids ~ //-'-~--" Ph'Cho r~/ -Phlno -- s

t\

Dolichols

PhEth~

GI }o rote,as y p '

f ~'N~ Ph S e r a P h {

t\

\

/ /

protein linkage

ad group precursors

Fig. I. Major lipid biosynthetic pathways in S. mansoni. The parasite doesnot synthesize fatty acids or sterols de novo. Dolichols are synthesized from acetate, and acetate is also utilized to modify fatty acid chains. Fatty acids are incorporated into neutral lipids, phospholipids and protein linkages and are also precursors for synthesisof eicosanoids. Phospholipids can be synthesizedfrom head groupprecursors or by modification of existing phospholipids. Unpublished data from our laboratory has shownevidence of phosphatidylserine decarbaxylation to phosphatidylethanolamine and has also suggested a low or negligible rate of long chain basesynthesis.Many of the details of these synthetic pathways as well as regulatory factors remain to be clarified. PhCho, phosphatidylcholine; MPPC, monopalmitaylphosphatidylcholine; PhEth, phosphatidylethanolamine; PhSer, phosphatidylserine; Phlnos, phosphatidylinositaL

between different developmental stages of the parasite6 may reflect differences between mammalian and snail host lipids rather than differences in synthetic capabilities between different stages of the parasite. The predominant phospholipid in cercariae, schistosomula and adults is phosphatidylcholine ~8. Also present in smaller amounts are phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin. Only minor differences in the relative proportions of these lipids have been found between developmental stages6. In adult schistosomes only cholesterol is found, but in cercariae and schistosomula other sterols are also found, including desmosterol, campesterol, stigmasterol and ~-sitosterol 6. As these sterols are also present in uninfected Biomphalaria glabrata, it is likely that these lipids are transferred from the snail host rather than metabolized by the parasite6. The predominant fatty acids in all stages of

the parasite are palmitic, stearic and oleic acids with slightly different proportions of each appearing between parasite developmental stages. However, a relatively high proportion of the fatty acids is eicosenoic acid, which is particularly enriched in phosphatidylethanolamine and phosphatidylserine8. Eicosenoic acid is also present in the hepatopancreas of the snail8 but is not typically found in appreciable quantities in mammalian cells. In addition, antigenic glycolipids have been identified from adult male worms, cercariae and eggs9. Morphological studies have shown differences between the inner and outer tegumental membranes t°, the double bilayer that constitutes the parasite's surface. Freeze-fracture electron microscopy has shown a paucity of intramembranous protein particles in the outer tegumental membrane ~°, indicating that it may be particularly lipidrich. Because the outer tegumental

membrane is the interface between parasite and host, its lipid composition may be significant. However, separation of outer and inner tegumental membranes is difficult and complete lipid analyses have only been conducted on preparations containing both 8. Relative to whole worm homogenates, tegumental membranes isolated by any of three methods had a higher sphingomyelin and eicosenoic acid content but were otherwise similar to the whole worm 8. Although it is not known what alterations occur in the lipid composition of the worm or of its tegumental membranes during transformation and maturation, it has been shown that changes in membrane fluidity accompany the transformation process ~r. Furthermore, measurement of the lateral diffusion of fluorescent lipid probes on the parasite's surface suggests asymmetric distribution of lipids between the tegumental membranes. While the lateral mobility of lipid analogs in the outer monolayer from adult worms is typical of most biological membranes, there is restricted mobility in the inner leaflets of these membranes ~2 S. mansoni cannot make sterols or fatty acids de novo 13.14.However, these organisms are able to modify fatty acid chain length 13'Is and to synthesize fatty acid metabolites 2'3. Furthermore, fatty acids are incorporated into triacylglycerols, cholesterol esters and phospholipids, glycerol is incorporated into triglycerides and phospholipids, and phospholipid head group precursors into phospholipidsl 3,~s-19.The synthetic rate for lipids may vary between stages. When corrected for mass, I I-day-old schistosomula have been found to have a much higher rate of phospholipid synthesis than either newly transformed schistosomula or adults ~8. There is very little information on how the parasite regulates its lipid synthesis or content, although such mechanisms are undoubtedly important. Another important class of molecules that are influenced by the lipid metabolism of the organism are proteins with lipid anchors, including proteins covalently linked to fatty acidsaswell asthose linked to glycophosphatidylinositol (GPI). Biosynthetic labeling experiments have shown that the organism incorporates fatty acids both into proteins containing GPI20 and those without 2 I . Such linkages may be particularly important for antigens on the surface of the parasite (Fig. 2) (Refs 22 and 23) since, for example, GPl-linked antigens can be released from the parasite by treatment with phospholipase C and some of these antigens may be shed from the parasite

Parasitology Today, vol. 7, no. 2, 199 /

surface in a membrane-bound form 23. There also may be some stagedependent differences in the expression of GPl-linked surface antigens23, Means of Lipid Acquisition from the Host The inability of the parasite to synthesize essential lipids means that it has to acquire exogenous lipids if it is to survive in the host. Because lipids such as fatty acids and sterols are essentially insoluble in aqueous solution, the acquisition of lipids from free solution in serum is unlikely. However, serum proteins such as albumin commonly bind lipids, particularly free fatty acids, and this is a potential source of lipids for the parasite. Both schistosomula and adult schistosomes incorporate fatty acids into phospholipids and neutral lipids from culture media containing free fatty acids and bovine serum albumin Ls'18,24. A second mechanism by which the parasite may acquire host lipids is serum lipoproteins. It was first suggested by Rumjanek and colleagues:z5 that a protein expressed on the surface of schistosomula may serve as a receptor for low density lipoprotein (Fig. 2). Such a protein has recently been identified and purified from adult S. japonicum 26. Furthermore, several protozoa have been shown to possess lipoprotein receptors and this may be a common mechanism for lipid uptake by parasite organisms 27. However, although lipoprotein receptors have been identified in these organisms, there is at present little direct

61 evidence showing lipid transfer between host serum lipoproteins and parasites. A third possible mechanism by which S. mansoni may obtain host lipids is from host cell membranes. Host cells both lyse on and fuse with the surface of the parasite and this may be due to the production of monopalmitoylphosphatidylcholine by the parasite (Fig. 2) (Refs 15 and 28). Furthermore, treatment of red blood cell ghosts with an exogenous source of this phospholipid such as that produced by the parasite causes release of cholesterol from red blood cell membranes29. Thus, monopalmitoylphosphatidylcholine may free cholesterol for the parasite. Although the currently available evidence suggests that the parasite may obtain lipids from all three sources, more studies must be done to demonstrate this transfer directly and to determine the relative contribution from each mechanism. Lipids and I m m u n e Responses Both humoral and cellular immune response to S. mansoni are modified by lipids. Schistosomula cultured in an exogenous source of phosphatidylcholine, sphingomyelin and phosphatidylethanolamine are less susceptible to complement-dependent antibodymediated killing but phosphatidylserine and phosphatidic acid had no effect 3°. Other work has shown that phosphatidylethanolamine methylation by schistosomula may influence complementmediated worm killing3~. Both biochemical and morphological evidence

Shed Membrane Eicosanoid Release

Lipid-linked Proteins

~

Host Cell

e U enta outer inner

\/ Membrane Proteins Fig. 2. Lipid-related event,.; at the surface of the parasite. PC, phosphatidylcholine, MPPC, monopalmitoylphosphatidylcholine; PI, phosphatidylinositol; Idl, low-density lipoprotein.

suggest that when components of the complement cascade bind they stimulate membrane biogenesis by the worm 32'33. Activation of the alternative complement pathway is also prevented by the parasite's acquisition of a lipid-anchored host protein called decay-accelerating factor 34. In addition to complementmediated killing of the worm, antibody binding to the parasite also may be influenced by tipoproteins. While lowdensity lipoprotein reduced the binding of antischistosomal antibodies to schistosomula3s, lipoproteins appeared to increase antibody-binding to adults36. Because monopalmitoylphosphatidylcholine is a phospholipid with detergent properties, the acquisition of host antigens such as decay-accelerating factor may be facilitated by the parasite's production of this phospholipid and its subsequent interaction with host cells. Furthermore, immune cells that are fused to the parasite's surface or lysed are certainly compromised in their ability to damage the parasite 28. As described above, eicosanoids are also released by schistosomula and adult worms, but how their release may affect host cell immune function remains to be determined. Similarly, lipids as well as protein antigens are shed by cultured worms Is, but neither the size nor the lipid content of these antigen complexes, nor how tipids associate with these antigens and influence processing has yet been determined. In summary, recent studies have shown that both host and parasite lipids are important in the life cycle ofS. mansoni. Additional studies are needed to characterize the lipid content of the outer tegumental membrane during transformation and maturation of the worm, to clarify the mechanisms the parasite uses to acquire lipids, to determine the factors that regulate lipid synthesis and to provide more information as to how lipids influence immune responses to the parasite. Such studies may provide not only a better understanding of how schistosomes adapt to the host environment but also an opportunity to exploit this knowledge for vaccine and drug development. Acknowledgements The author gratefully acknowledgesthe contribution of John Caulfleld to the studies discussed from our laboratory. This work was supported by grant no. AI24570 from the NIH. References

I Haas,W. (1984) Exp. Parasitol. 58, 215-222 2 Fusco, A.C. et al. (1988)J. Parasitol. 74, 253-26 I 3 Salafsky, B. and Fusco, A.C. (1987) Exp. Para-

62 sitol. 64, 361-367 4 Nirde, P. et al. (1984) FEBSLett. 168,235-240 5 Vande Waa, E.A. et al. (I 989) Am. J. Physiol. 257, R618-R625 6 Furlong, S.T. and Caulfield, J.P. (1988) Exp. Parasitol. 65,222-23 I 7 Young, B.W. and Podesta,R.B. ( 1984)J. Parasitol. 70,447-448 8 Allan, D., Payares,G. and Evans,W.H. (I 987) Mol. Biochem. ParasitoL 23, 123-128 9 Weiss, J.B., Magnani,J.L. and Strand, M. (I 986) J. Immunol. 136,4275-4282 10 Hockley, D.J. et al. (1975) Tissue Cell 7, 485-496 II Foley, M., Kusel,J.R. and Garland, P.B. (1988) Parasitology 96, 85-97 12 Foley,M. etal. (I 986)]. CellBiol. 103,807-818 13 Meyer, F., Meyer, H. and Bueding, E. (1970) Biochim. Biophys. Acta 210, 257-266 14 Smith, T.M., Brooks, T.J.,Jr and Lockard, V.G. (1970) Lipids 5, 854-856 15 Furlong, S.T. and Caulfield, J.P. (1989) Exp. Parasitol. 69, 65-77 16 Goldring, O.L., Kusel, J.R. and Smithers, S.R.

Urinary Schistosomiasis and the Coming of Age in Nigeria Studies continue to suggest that cultural and behavioural habits in many Nigerian societies play a greater role in parasite transmission than do most other parameters hitherto identified ~'2.What is worrying is that the health planners seem to share these folk beliefs. Otherwise, how does one explain the apparent disregard of schistosomiasis in Nigeria, especially when one considers the emphasis that is placed on river blindness, sleeping sickness and malaria control? Yet, schistosomiasis is the second most prevalent parasitic disease in Nigeria3. Perhaps the neglect of schistosomiasis is due to the marked haematuria which is the major symptom of heavy infections of schistosomiasis haematobium. Amongthe Song people of Gongola, where schistosomiasis prevalence is estimated to be 45%, the occurrence of haematuria has become incorporated into local custom and is considered to signify the coming of age. It is therefore usual for the family of a bride to inquire discreetly if her suitor has attained manhood as indicated by the passing of blood. This pre-marital requirement has superceded the ability of the suitor to pay the additional brideprice. Unlike the Song, where the prospective father-in-law would not be prepared to give his daughter away to someone who has not metthe requirements of adulthood, the Luguda people are not so rigid. Instead, the brideprice is made at least fourfold greater for those who have not'come of age' than for those who have. In the old days, amongthe Luguda, a boy who sawthe first signsof adulthood brought it to the attention of his older brothers who in turn informed the uncle. Consequently, the uncle began to address him as 'little man'

Parasitology Today, vol. 7, no. 2, 1991

(I 977)Exp.ParasitoL 43, 82-93 17 Young, B.W. and Podesta, R.B. (1985) Mol. Biochem. Parasitol. 15, 105- I 14 18 Vial, H.J. et al. (1985) Mol. Biochem. Parasitol. 17, 203-218 19 Young, B.W. and Podesta, R.B. (1982) MoL Biochem. ParasitoL 5, 165-172 20 Sauma, S.Y. and Strand, M. (1990) Mol. Biochem. ParasitoL 38, 199-209 21 Wiest, P.M. et al. (1988) Biochem. J. 254, 419-426 22 Espinoza,B. et al. (1988)Mol. Biochem. Parasitol. 29, 171- 179 23 Pearce,E.J.and Sher,A. (I 989)J. Immunol. 142, 979-984 24 Rumjanek, F.D. and Simpson, A.J. (I 980) Mol. Biochem. Parasitol. I, 31-44 25 Rumjanek, F.D., McLaren, D.J. and Smithers, S.R.( 1983)MoLBiochem. ParasitoL 9, 337-350 26 Rogers,M.V. et aL (1990) Mol. Biochem. Parasitol. 4 I, 93- 100 27 Furtong,S.T.(I 989)Exp. ParasitoL 68,482-485 28 Golan, D.E. et al. (1986)J. Cell Biol. 103, 819-828

instead of'boy'. This appellation drew the attention of the entire village, and his peer group, who with envy may have watched him pass 'manly urine' rather than 'ordinary water' during the usual afternoon swimming sessions, usually helped to spread the word. Many old people express regret that this practice is dying out owing to the influence of other ethnic groups, education and communication. The Margi also had a custom dating back hundreds of years in which the urination of blood was a prerequisite for initiation into certain cults, the most notable being the war-scouts (who may be regarded as modern day cadets). War-scouts could touch war arsenals like arrows and spears, which were forbidden to women and those who had not had blood in their urine. Interestingly, these customs have not developed in southern Nigeria. Schistosomiasis may be more prevalent in the north owing to the close ties of these people with Egypt, from where many Nigerian ethnic groups claim to originate. In Egypt schistosomiasis haematobium is as old as the pyramids and found the length of the river Nile. It is possible that this disease was introduced into what is now Nigeria during the movement of people westwards.

29 Golan, D.E. et al. (1988) Biochemistry 27, 2661-2667 30 Billecocq,A. (1987) Mol. Biochem. Parasitol. 25, 133-142 31 Parra,J.F. et 01. (1986) Mol. Biochem. Parasitol. 21,151-159 32 Zhou, Y. and Podesta, R.B. (1989)J. Parasitol. 75,333-343 33 Young, B.W. and Podesta,R.B. (1986)J. Parasitol. 72, 802-803 34 Pearce, E.J., Hall, B.F. and Sher, A. (1990) J. ImmunoL 144,2751-2756 35 Chiang, C.P. and Caulfield, J.P. (1989)Am. J. Pathol. 134, 1007- I 018 36 Hartnett, W. and Kusel,R. (1986) Exp. Parasitol. 61,146-150 Stephen T. Furlong is at the Department of Medicine, Harvard Medical School, and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA0 2 1 1 5 , USA.

Moreover, the traffic of goods and slaves along the trans-Saharan trade routes from North Africa and the Middle East, and the ravaging of the entire central region of Nigeria (where schistosomiasis is highly endemic) during the kwararafa campaigns and later during the Kanem Bornu expansion of the last century, may all have played a significant role in the introduction and spread of the parasite within Nigeria. With schistosomiasis deeply rooted in our culture and traditions and the disease afflicting at least one third of the Nigerian population, why is it that our public health planners are still unable to recognize it as a serious parasitic disease? Perhaps they share in the belief that to be a man one has to pass something more than water.

Oladele B. Akogun ParasiteStudyGroup Box 10121 University PostOffice Jos, Nigeria

References I Akogun,O.B. (1989) Parasitology Today 5, 39 2 Akogun, O.B. (1989)J. Trop. Meal. Hyg. 92, 193-196 3 Ramsay,G.W. (1934) W. Afr. MedJ. 8, 2

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