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Orozdore, S.N., eds), pp 136-145, Petrozavodsk Dzika, E, and Dubas, W. (1988) W~ad Parazytol. I, 37-45 Lux, E, (1990)Angew. Parasitot, 31, 143-149 Kuperman, B.I, and Shulman, R.E. (1978) Parasitolog~'a 12, I 0 I- 107 Hanzelova, V. and Zitnan, R. (1983)Helminthologia 20, 137-150 Kamiso, H.N. and Olson, P..E. (1986)j. Parasitol. 72, 125-129

14 Gonzalez-Lanza, C. and Alvarez-Pellitero, P. (I 982)J. Helminthol, 56, 263-273 15 Koskivaara, M., Valtonen, E.T. and Prost, M, (1991) Aqua Fennica 21,47-55 16 H6glund, J. and Thulin, J. (1989)J. Helminthot. 63, 93-101 17 Pojmanska, T. and Dzika, E. (1987) Acta Parasitot. Pot. 32, 139-161 18 Koskivaara, M, and Valtonen, E,T. (1992) Parasitology 104, 263-272 19 Esch, G.W., Bush, A,O. and Aho, J.M. (1990)

Parasite Communities: Patterns and Processes, Chapman & Hall 20 Moser, M, (1991) Parasitology Today 7, 182-185 21 Poulin, R. (I 992) Parasitology Today 8, 58-61

Mari Koskivaara ps at the Department of Biology, University of Jyvdskyld, 40100 Jyvdskyl•, Finland.

Praziquantel: The Enigmatic Antiparasitic T.A. Day, J.L. Bennett and R.A. Pax Praziquantel (PZQ), a pyrazinoisoquinoline, was introduced as a novel anthelmintic in 1975. PZQ is currently the drug of choice for the treatment of a wide range of both veterinary and human trematode and cestode infections, including human schistosomiasis. Current estimates suggest that 150 million humans are infected with schistosomes, and it is expected that PZQ will play the lead role in chemotherapeutic control of those infections ~. Despite the time that has passed since its introduction and its obvious importance in global health care, it is not yet understood why PZQ is so selective and effective. The target molecules for PZQ have not been defined, nor are the sites of its effects within the parasites known. Here, Tim Day, James Bennett and Ralph Pax summarize some of the progress that has been made toward reaching these objectives in recent years. This paper focuses on the information that has provided clues as to the site and mechanism of the action of PZQ (Fig. I). Since most efforts to understand the actions of PZQ on susceptible species have used schistosomes as models, and since by far the largest clinical application of PZQ is for the treatment of schistosomiasis, this paper will also focus on the schistosome.

some metabolism, including decreases in glucose uptake, lactate release, glycogen content and ATP content 6. All of the effects of PZQ could be attributed either directly or indirectly to a single cause: an alteration of intracellular Ca 2+ homeostasis at one or more sites in the worm. With regard to tegumental disruption, increased intracellular C a 2+ is documented as the etiology of cytoskeletal disruptions and membrane blebbing in a wide variety of cell types 7. Cytochalasin B, which inhibits actin polymerization and, therefore, directly disrupts cytoskeletal elements, causes a disruption of the tegument analogous to that caused by PZQ 8. Therefore, increases in tegumental cytoplasmic Ca 2÷ could cause the observed tegumental disruption. The ability of PZQ to cause the exposure and release of schistosome antigens may be attributable to the primary effect of tegumental disruption. Clearly, the formation of host antibodies that work in synergy with the

What Does PZQ Do? The most obvious effects of PZQ on the schistosome are tegumental disruption 2'3 and the induction of a paralytic muscle contraction 4, both of which occur within seconds. PZQ also induces changes in the antigenicity of the parasite, causing the exposure or release of previously concealed parasite antigens s. PZQ also causes alterations in schisto-

Fig. I. The structure of praziquantel. (Reproduced, with permission, from Ref.

19.)

direct actions of PZQ is important in explaining how the drug allows for the elimination of schistosomes 9. The exposed antigens critical to synergy are surface antigens, in particular a 200 kDa glycosylphosphatidyl-anchored antigen localized in the tubercle '°. The exposure and release of this tubercle antigen and other antigens could be a consequence of the cytoskeletal and tegumental disruptions that result from the increased C a 2+ in the tegumental cytoplasm. PZQ-induced alterations in schistosome metabolism can also be attributed to the primary effects of tegumental disruption and paralytic contraction. The tegument is the primary surface for the absorption of glucose by the schistosome ~', which directly metabolizes this glucose obtained from the host's bloodstream '2. Disruption of tegumental function would therefore be expected to decrease glucose uptake and decrease the amount of worm ATP. Furthermore, a sustained contraction, coupled with a decrease in available glucose, would be expected to diminish the worm glycogen content. Schistosome muscle contraction can obviously be attributed to increases in intracellular Ca 2+ levels within the muscles. In fact, the tegumental effects could even be involved in muscle contraction, since the tegumental cytons are joined to the contractile fibers of the muscle by low-resistance pathways 13, Increases in tegumental cytoplasmic Ca 2+ could be directly transmitted to the muscle and so contribute to the contraction. However, since worms with the outer layer of the tegument removed still contract in response to PZQ r4, there is evidence that PZQ's effect on the muscle is not completely mediated by its action on the tegument. ~) 1992,ElsevierSciencePublishersLtd,(UK)

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Where Does PZQ Act? Although it seems clear that PZQ does alter Ca 2÷ homeostasis in the worm, it is unclear if schistosome tissues are in general sensitive 1:0 PZQ or if there are specific tissues or cell types within the worm that are PZQ susceptible. A consideration of the effects of PZQ might lead to the conclusion that the drug interacts with the parasite's tegument and muscle membranes. However, the studies elucidating these effects have all been performed with whole animals, such that observed effects might be mediated by any one of a number of different schistosome tissues. For example, observed muscle contraction may not be an effect of PZQ's interaction with the muscle, but may result from a direct effect of PZQ on schistosome motor neurons, central neurons or sensory structures. Similar arguments can be made with respect to PZQ action on the tegument. To date, there has been no measurement of any PZQ effect that can be attributed to the direct action of the drug on a specific tissue or cell type. It is also not known whether there are specific pools of Ca 2+ that are altered by PZQ and, in fact, Ca 2÷ from more than one source may be involved. The schistosome, by virtue of its anatomy, may have some unique means of Ca 2+ compartmentalization. Not only do extraworm Ca 2+ and intracellularly stored Ca 2+ need to be considered as possible sources for Ca 2+ influx into the muscles or the tegument, but also intraworm, extracellular Ca2÷ needs to be considered. The muscle layers lie beneath the tegument, which is continuous around the entire animal, such that it forms a barrier between the extraworm environment and the extracellular environment of the underlying muscles. Therefore, th,~ extracellular environment of the muscles may provide a source of Ca2+ even when extraworm Ca 2+ is low. Even when worms are incubated in zero Ca 2÷ medium for 60rain, the worms retain 70% of their total Ca 2+ (Ref. 15). It is often mentioned that the exposure of schistosomes to PZQ in medium containing ethyleneglycoltetraacetic acid (EGTA) or even in medium only nominally Ca2÷-free blocks the contraction ~s as well as tegumental disruption j6. Although this is true, the inhibition of both of these primary effects by the removal of extraworm Ca 2+ does not occur immediately, rather they are time-dependent. The partial inhibition of PZQ-induced con-

traction requires an incubation in Ca 2+free medium for 10 min and the partial inhibition of tegumental disruption requires at least 60 min for its full effects. If PZQ's induction of tegumental disruption were dependent on the influx of extraworm Ca2+ across the outer tegumental membrane, then the removal of Ca 2+ and addition of EGTA to the extracellular medium would immediately block the effect, since EGTA's chelation of free Ca2+ is immediate on this time scale. It does not do this, neither does it immediately block PZQ-induced muscle contraction 8. All of this evidence suggests that initiation of tegumental disruption and contraction are not dependent on Ca2+ fluxes from outside the worm. The time dependence of zero Ca 2÷ treatments suggests that their inhibition is more likely to be a function of the leaching of Ca 2+ from intraworm sites. Also well described are the inhibitory effects of high extraworm Mg2+ ; Ca 2+ ratios on these primary effects of PZQ 8. Mg2÷ inhibition is not immediate either, requiring a 30 min incubation period for a full block of tegumental disruption. It is likely that this time dependence is due to the time dependence of the equilibration of intraworm Mg2+: Ca 2÷ ratios with those outside of the worm. However, exposure to PZQ does cause an influx of extraworm Ca2÷ (Ref. 4) and the sustained component of both paralytic contraction ~ and maximal tegumental disruption 16 are dependent on the presence of extraworm Ca2+. So, although the expression of the full and sustained effects of PZQ requires extraworm Ca 2+, the initiation of the primary effects do not.

Praziquantel

Target Membrane protein? Cytosolic protein? Phospholipid? Other?

Location Tegument? Muscles? Nerve terminals? Central neurons? Other? -

-

ACa 2÷ Homeostasis

ACa2+ Tegument ~

ACa 2+ Muscle

Tegumental ~ Paralytic disruption ~ c o n t r a c t i o n AAntigenicity

&Metabolism

Fig. 2. All the antischistosomal effects of praziquantel may result from the drug's ability to alter Ca2+ homeostasis within the worm.

How Does PZQ Act? Several lines of evidence suggest that PZQ interacts with some non-typical molecule involved in Ca 2÷ regulation within the schistosome. One possibility is that PZQ alters important ATPases involved in Ca 2+ extrusion from the cytoplasm, but evidence has shown that PZQ does not alter total ATPase activity of schistosome homogenates 17. Another possibility is that PZQ alters the function of voltage-gated Ca 2+ channels. However, PZQ-induced Ca 2+ influxes are not inhibited by the voltagegated Ca 2÷ channel blockers such as D-600, which do block depolarizationinduced Ca2+ influxes 4. Yet another possibility is that PZQ acts by allowing Ca 2+ influx through ionotropic or ionotropically coupled worm neurotransmitter receptors. PZQ's action is not inhibited by inhibitors of putative neurotransmitters of schistosomes ~8. All of this, and other evidence, has pointed toward a non-typical interaction of PZQ with the schistosome. It has been suggested that PZQ may alter Ca 2÷ homeostasis by interacting with membrane phospholipids 19'2°. PZQ can, in the presence of Ca 2+, enhance the transition of the ionimpermeable bilayer structure of phospholipids to a hexagonally packed structure that is not a functional barrier to ion permeation ~9. PZQ can also destabilize phospholipid membranes without modifying the organization, simply by inserting itself as a spacer among the phospholipids in the membrane 2°. However, these experiments were both performed using artificial schistosome membranes and concentrations of PZQ markedly higher than therapeutically effective doses. If PZQ allows Ca 2÷ influx into the muscles by an interaction with lipids of the muscle membrane, it would change membrane resistance to ion flow. In our laboratory, we have been using patch-clamp methods on isolated schistosome muscle fibers to measure ion flow through their membranes, but concentrations of PZQ as high as I 0 I~M do not produce any detectable change in the membrane resistance of these isolated fibers. There is evidence suggesting that PZQ interacts with a specific, unique schistosome protein, rather than phospholipids. The remarkable specificity of PZQ's action on trematodes and cestodes suggests this. Thus far, there has been nothing discovered that is unique and distinguishing about the lipid content of these organisms that would

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explain the drug's selective action against them. It seems more likely that these parasites have unique proteins. The stereo-specificity of PZQ action is further indication that it exerts its effects via interaction with a protein. The EDs0 for L-PZQ eliciting contraction 2~ or for inducing tegumental disruption 22 in Schistosoma mansoni is about half that of the racemic PZQ. The action of PZQ on schistosomes is also dependent on the developmental stage of the schJstosome 22'23, with juvenile worms being much less susceptible than adult worms, This stage specificity could be well explained by stage-specific differences in protein expression. W e believe that all of this information taken together supports the hypothesis that PZQ interacts with a specific, unique protein involved in Ca 2+ homeostasis in trematodes and cestodes (Fig, 2). A better understanding of the selective action of PZQ against these parasites will require the identification of both the molecule with which PZQ interacts and the site of this interaction within the worms. Identification of the molecular targets of PZQ action, whether protein or lipid, may be accomplished through w o r k with whole

worms or worm homogenates, but it is clearly of interest exactly where in the worm these molecules are located and what physiological mechanisms are altered there. Studies involving the use of whole parasites will provide little information about the site of PZQ action; rather, this will require new approaches that use specific cells and tissues of the parasites.

References

I World Health Organization (I 989) Document WHO/SCHISTO/89.102 2 Becket, B. et al. (1980) Z. Parasitenkd. 63, 113-128 3 Shaw, M,K. and Erasmus, D,A. (1987) Parasitology 94, 243-254 4 Fetterer, R.H., Pax, R.A. and Bennett, J.L. (I 980) Eur.J. Pharmacol.64, 31-38 5 Sabah, A.A. et al. (I 985) Exp. Parasitol. 60, 348-354 6 Harder, A., Andrews, P. and Thomas, H. (1987) Parasitol.Res. 73, 245-249 7 Klaassen, C.D. and Eaton, D.L. (1991) in Casarett and Doull's Toxicology(Amdur, M.O., Doull, J. and Klaassen,C.D., eds), pp 12-49, Pergamon Press 8 Bricker, C.S. et al. (1983)Z. Parasitenkd 69, 61-71 9 Brindley, P.J. et al. (1989) Mol. Biochem. Parasitol. 34, 99-108 10 Sauma, S.Y., Tanaka, T.M. and Strand, M. ( 1991) Mol. Biochem.Parasitol.46, 73-80

I I Gomme, J. and Albrecthsen, S, (I 988) Camp. Biochem. Physiol.90, 651-657 12 Teilens,A.G.M., van den Heuvel,J.M. and van den Bergh, S.G.(1990)Mol. Biochem.Parasitol. 39, 195-202 13 Thompson, D.P., Pax, R.A. and Bennett, J.L. (I 982) Parasitology85, 163-178 14 Depenbusch,J.W. et al. ( t 983) Parasitology87, 61-73 15 Wolde Mussie, E. et al. (1982) Exp. Parositol. 53, 270-278 16 Xiao, S.H. et al. (1984)J. Parasitol. 70, 177-179 17 Nechay, B.R., Hillman, G.R. and Dotson, M.J. (1980)J. Parasitol.66, 59(~600 18 Pax, R.A., Thompson, D.P. and Bennet:, J.L. (1983) in Mechanism of Drug Action (Singer, T.P., Mansour, T.E. and Ondarza, R.N., eds), pp 187-196, Academic Press 19 Harder, A., Goossens, J. and Andrews, P. (1988) Mol. Biochem.Parasitol.29, 55-60 20 Schepers,H. et al. (I 988)Biochem. Pharmacol. 37, 1615-1623 21 Andrews, P. (1985) Pharmacol. Ther. 29, 129-156 22 Xiao, S.H. and Catto, B.A. ( 1989)j. Infect. D~s. 159, 589-592 23 Sabah, A.A. et al. (1986) Exp. Parasitol. 61,294-303 24 Shaw, M.K. (1990) Parasitology 100, 65-72 Tim Day and ]ames Bennett ore at the Department of Pharmacology and Toxicology and Ralph Pax is at the Deportment of Zoology, Michigan State University, East Lansing, MI 48824, USA.

Transformation of Caenorhabditis elegans With Genes From Parasitic Nematodes W.N. G rant Our knowledge of many aspects of the molecular biology of animal parasitic nematodes has rapidly expanded in recent years but the classical genetic analysis of this group of organisms has yet to emerge as a viable discipline. For example, it is not possible to routinely perform crosses between single males and females to examine the genetic basis of even simple phenotypes such as anthelmintic resistance. This has meant that the function of many cloned parasite genes can only be inferred from sequence comparison with genes from other organisms where the function is known, or by correlation of DNA polymorphisms linked to the gene with phenotypic differences between strains or individuals. In the absence of classical genetic techniques, a molecular solution is to transform a

suitable host with the gene of interest, but what de/fnes a suitable host? Here, Warwick Grant describes recent work that aims to provide such a host. As more molecular data on a diverse range of parasitic nematodes have accumulated, two general features have emerged. First, the function of genes that have been cloned using nonfunctional criteria (eg, immunological criteria for potential vaccine and immunodiagnostic antigen genes) has often proven elusive. T w o good examples of this are two candidate vaccine antigen genes cloned from Trichostrongylus colubriformis. One contains a domain with striking homology to the gut active peptide valosin ~ and the second contains a

globin-like domain 2. These homologies suggest possible functions for these antigens, but how can these hypothetical functions be tested? If this problem arose in yeast or Drosophila, the answer would be to first turn to the genetic system of those organisms to look for informative mutants so that the link between disruption of the gene and its normal function could be made, In the absence of the genetic means to do this, transformation of an appropriate host with the candidate gene has often been employed as a means of establishing or confirming function. For parasitic nematodes, transformation is the only option available and the choice of transformation host is an important consideration. Ideally one would like a host whose biology is as similar as ~) 1992.ElsevierSciencePublishersLtd.(UK)