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25 Lindsay, D.S. and Dubey, J.P. (1989)J. Parasitol. 75, 772-779 26 Dubey, J.p. and de Lahunta, A. ]. AppL Parasitol. (in press) 27 Mayhew, I.G. et al. (1991)J. Small Anita. Pract. 32, 609-612 28 Yamane, R.D. et al. Am. J. Vet. Res. (in press) 29 Barta,J.R. and Dubey, J.P. (1992)Parasitol. Res. 78, 689-694 30 Brindley, P.J. et al. (1993)Am. J. Trop. Med. Httg. 48, 447-456 31 Cole, R.A. et al. (1993)J. Vet. Diagn. Invest. 5, 579-584 32 Lindsay, D.S. and Dubey, J.p. (1989)J. Parasitol. 75, 990-992 33 Lindsay, D.S. and Dubey, J.P. (1992)J. ParasitoL 76, 177-179 34 Hay, W.H. et al. (1990)J. Am. Vet. Med. Assoc. 197, 87-89 35 Lindsay, D.S. et al. (1990)J. Hehninth. Soc. Wash. 57, 86-88 36 Trees, A.J. et al. (1993) Vet. Rec. 132, 125-126 37 Greene, C.E., Cook, J.R. and Mahaffey, E.A. (1985) J. Am. Vet. Med. Assoc. 187, 631-634 38 Dubey, J.P. et al. (1990)J. Vet. Diagn. Invest. 2, 6~69
39 Thorton, R.N., Thompson, E.J. and Dubey, J.P. (1991) NZ Vet. J. 39, 129-133 40 Ogino, H. et al. (1992)J. Comp. Pathol. 107, 231-237 41 Wouda, W. et al. (1992)Tijdschr. Diergeneeskd 117, 599-602 42 Abbitt, B. et al. (1993)J. Am. Vet. Med. Assoc. 203, 444-448 43 Bryan, L.A. et al. Canad. Vet. J. (in press) 44 Jardine, J.E. and Last, R.D. (1993)J. S. Afr. Vet. Assoc. 64, 101-102 45 Barr, B.C. et al. (1991)J. Vet. Diagn. Invest. 3, 39M6 46 Nietfeld, J.C. et al. (1992)J. Vet. Diagn. Invest. 4, 223-226 47 Barr, B.C. et al. (1993)J. Am. Vet. Med. Assoc. 202, 113-117 48 Rogers, D.G. et al. Agric. Practice (in press) 49 Lindsay, D.S. et al. (1993) Comp. Cont. Educ. Pract. Vet. 15, 882-889 50 Conrad, P.A. et al. (1993)Parasitology 106, 239-249 51 Conrad, P.A. et al. (1993)]. Vet. Diagn. Invest. 5, 572-578
The Role of Parasites in Generating Evolutionary Novelty D, Bermudes and K.A. Joiner in this review, David Bermudes and Keith Joiner discuss the interrelationship between parasitism and m u t u a l i s m and examine the parallel mechanisms used by parasites and mutualists to access and persist w i t h i n the intracellular environment. By drawing analogies with mutualistic associations, they suggest mechanisms by which some parasites m a y ultimately benefit their hosts. They ~¢rther speculate that some hosts m a y even become dependent upon their parasites.
Parasitism is a well-recognized evolutionary forceL The co-evolution of hosts with their parasites accounts for numerous physiological modifications of the host (eg. immunological defenses 2) as well as driving the p h y l o g e n y of the parasites, limiting host geographic distribution 3-5 and potentially contributing towards the adaptive value of sexual reproduction 6,7. However, less appreciated is the relationship of parasitism to mutualism and the potential for parasitism ultimately to lead towards novel host adaptations. The physiological advantages to the host accrued from mutualisms involving specialized functions such as nitrogen fixation or photosynthesis appear obvious. Such relationships have even led to the origin of specific organelles, including plastids and mitochondria 8. Mutualistic symbioses function as a mechanism of evolution by genetically introducing significant new traits to their host 9. By providing for host needs or introducing new capabilities, symbiosis can greatly increase the host's ability to inhabit certain environments.
ism, with parasitism considered a separate and unrelated phenomenon. The original usage reflects De Bary's finding of a propensity for organisms to form relationships, while also recognizing that the immediate and long-term effects of interaction are not always known. Furthermore, there is an interrelationship of the two types of interaction. From the perspective of the host, the effects of symbiotic interactions on fitness can be placed along a continuum, with mutualism (advantageous relationships) at one end and parasitism (disadvantageous relationships) at the other (Fig. 1). Practical indicators of host fitness c o m m o n l y include nutritional status and growth rate, which m a y be significantly modified by the interaction. Although some associations appear relatively fixed in their outcome, m o v e m e n t along the c o n t i n u u m occurs in both directions, as often observed in parasites ranging in virulence. The effect of an organism on the host as measured by practical indicators can be variable and m a y be d e p e n d e n t u p o n factors including environmental parameters, host population dynamics and genetically based characters (resistance of the host, virulence of the parasite). Not only are small shifts observed in the relative effects of parasites on their host, but large shifts from
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Interrelationships of mutualism and parasitism The term 'symbiosis' (the living together of two or more dissimilar organisms) was originally coined by De Bary 1° to describe both parasitic and mutualistic associations. Symbiosis is often equated with mutual-
David Ben-nudes and Keith Joiner are at the Yale University School of Medicine, Department of Internal Medicine, Infectious Diseases Section, New Haven, CT 06510, USA.
Host
Fitness
negativeeffect
Parasitism
no effect
Commensalism
positive effect
Mutualism
Fig. I. Ranges of effects of symbionts (parasites or mutualists) upon their hosts occur as a continuum. Overall positive or negative e~ects as measured by practical indicators define the relationship as parasitic, commensalistic or mutualistic. ~ 9 9 3 . Flse'4(~ S(icr c Pllbhsl-(I ~ i t 1 I i F
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one end of this continuum to the other are also known. Mutualistic associations may, under given conditions, have no detectable effect (commensalism), while, under other conditions, they will become parasitic. For example, mycorrhizal symbioses (the association of fungi with the roots of vascular plants) are usually advantageous to onions for obtaining phosphate 11. Phosphate, more efficiently obtained by the fungal mycelium, is translocated to the plant and results in a measurable increase in plant growth, despite concomitant translocation of sugars from the plant to the fungus. However, this advantage is absent at higher population densities. Under crowded conditions, all available phosphate is taken up by the plant roots and there is no additional uptake associated with the mycorrhizae. Further, under excess phosphate concentrations, the fungus now acts as a parasite, reducing host vigour 12. Apparently, the sugars translocated to the fungus are sufficient to diminish plant growth relative to uninfected plants, with no compensation by fungal-mediated phosphate uptake. Resource availability has been shown to determine the nature of interaction in other associations. The ciliate Lambornella clarki is a fatal pathogen to its mosquito host (Aedes sierrensis) when the food supply is abundant, but can result in greater production of progeny when food is limiting 13. Many mutualisms, such as the association of Rhizobium with legumes, are believed to have evolved into their present form of co-operation from parasitic associations 14. In a recent study 15, it was found that a fungal plant pathogen could be converted to a mutualist as a result of mutation of a single locus. The evolution of virulence has recently been reviewed by Read and Schrag 16. Natural selection for co-operation is believed to be promoted when the chief mode of transmission is vertical (infecting the offspring of the host), thereby not only limiting potential hosts, but linking present and future parasite reproduction to the continuance of that host and its progeny 17. Conversely, it has been shown that increased opportunity for horizontal transmission (not infecting the host offspring) of parasites promotes evolution towards increased virulence 18. In the study by Herre 18, waspparasitic nematode species using more than one host have a greater affect on wasp reproduction. In such populations, virulence increases parasite reproductive success, with future parasite success independent of one particular host and its progeny.
Transition from parasite to obligate mutualist The only directly observed example of transition from lethal pathogen to obligate symbiont has been studied by Jeon and co-workers19, 20. The parasitism first occurred when a normal (D) strain of Amoeba proteus became infected with an unknown species of obligately parasitic Gram-negative rod (x-bacteria). Most of the amoebae were killed but, by separating individual amoebae, infected survivors (xD amoebae) were obtained. At this stage the bacteria harvested from resistant amoebae were still lethal to most amoebae. Subsequently, selection apparently occurred for bacteria with reduced virulence, as the bacterial strains from later stages were quickly able to establish a nonlethal association with naive amoebae. After the original xD amoebae had become stable, the amoebae
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could be cured with trimethoprim (a dihydrofolatereductase inhibitor), chloramphenicol or slightly elevated temperature. The association could be reestablished by microinjection with cytoplasm containing the bacteria of an infected amoebae or through phagocytosis from a x-bacterial suspension. However, after maintenance through 200 generations (18 months), most xD amoebae could not survive curative treatment unless rescued by re-introduction of the bacteria. Furthermore, in nuclear transplantation experiments, the nucleus from an infected cell was inviable when placed within the cytoplasm of an enucleated uninfected cell. These hybrid amoebae could also survive only if the bacteria were introduced into that cell. These observations have led to the conclusion that the association had become obligate for the amoebae. The formation of the x-bacteria and amoeba's association appears to have followed the aforementioned rules for virulence. Initially, virulence was high when unlimited hosts (large cultures) were present: parasite reproduction was not directly linked to survival of the immediate host. Reduced virulence was observed after amoebae were separated out into many individual cultures: virulent bacteria were selfeliminated by killing of their only available host. Generally, mutual interdependence occurs as a form of complementation (eg. through providing a needed metabolite or gene product). Metabolic requirements could be pre-existing or induced after initiation of the association through several different means. A host or symbiont could lose a gene function through mutation simply because there is no longer selective pressure to maintain that function. Gene transfer (which is known to have occurred for both plastids and mitochondria 8) could also account for an obligate relationship. Mechanisms actively causing impairment, such as genetic transposition or the selective action of a toxin, could also generate interdependence.
Mechanisms of intracellular persistence In examining parasite and mutualist methods for accessing and persisting within their hosts, we find that significant parallels exist, although insufficient data are available to determine if the actual mechanisms are the same. Similarities could be due either to common ancestry or to convergent evolution. However, these data are intriguing because they demonstrate that evasion of intracellular defense alone does not necessarily create virulence. A limited number of mechanisms for organisms accessing and persisting within eukaryotic cells have been described. The most common modes of entry are either by phagocytosis or active membrane penetration 21-24, although other mechanisms occur. For example, lysosome recruitment has been shown to be a mechanism for stimulating internalization of Trypanosoma cruzi 25. Inside the cell, resistance to or avoidance of digestive or defensive measures facilitates intracellular persistence. These metabolic mechanisms of the host, which are broadly lethal to ingested organisms, can include lysosomal hydrolases, acidification and reactive oxygen and nitrogen intermediates. Utilization of specific receptors. Parasite internalization into macrophages via specific receptors to avoid
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Ted Pardy Fig. 2. Hydra gastrodermal cells prepared by maceration show the presence of endobiotic Chlorella. The Chlorella are engulfed on the gut surface and transported to the basal region (arrows), but are not digested. Nu, host cell nucleus. Scale bar = 30 t~m.
a specific metabolic response is well documented and has been discussed in a previous review 24. Nearly all pathogens restricted to macrophages are internalized via complement receptors, which are not linked to the generation of reactive oxygen or nitrogen intermediates. Altering the receptors mediating entry is thought to trigger specific microbicidal cascades and result in killing of the invading organism. Evidence also suggests that intracellular mutualists must first bind to the appropriate ligand or face destruction subsequent to internalization. Hydra viridis normally contains symbiotic strains of the green alga Chlorella (these Chlorella secrete photosynthate in the form of maltose or trehalose that contribute to Htldra nutrition and absorb excess nitrogenous compounds from the animal). Normally, Chlorella are phagocytozed but not digested (Fig. 2). Antibody-coated and lectin-coated Chlorella or Chlorella treated with trypsin are not well internalized 26-2s. Moreover, Chlorella treated with ferric chloride are phagocytozed and digested by Hydra 29 and the morphology of the uptake mechanism is observably altered 3° (Fig. 3). These data suggest that recognition phenomena
Paras~tolo,~yToday,~ol 9. ~o i), 1993
involving surface charge occur at the time of phagocytosis. Initiation of the interaction between Hydra and Chlorella may be analogous to infection by Leishmania, where internalization via the complement receptors CR1 and CR3 bypasses a destructive intracellular response (for review see Hall and Joiner24). Inhibition of lysosomal fusion. Phagocytosis of bacteria is usually followed by fusion of the phagosome with lysosomes, exposing the contents of the endosomal compartment to acid pH and hydrolases. This cell function is blocked in several parasitisms, including those involving Mycobacterium tuberculosis, Chlamydia psittaci and Legionella pneumophila. The amoebae Hartmannella is also blocked in this function when infected by Legionella31. Although the mechanism for this inhibition is not well understood for any pathogen, recent studies with Legionella have identified loci involved in fusion inhibition 32-~3. Stable sequestering of mutualistic endosymbionts appears to rely extensively upon endocytosis without subsequent fusion of lysosomes. Examples include both the interaction of Chlorella with Hydra and later stages of the x-bacteria and A. proteus association. In both Amoeba 19,2° and Hydra 29, live symbionts are required to inhibit fusion and destruction of the symbiont. In the example of Hydra, uptake of heat-killed Chlorella or inhibition of photosynthesis with 3-(3,4dichlorophenyl)-l,l-dimethyl urea, followed by dark incubation after endocytosis, results in lysosomal fusion and destruction of most Chlorella29. Apparently, continued contribution from the symbiont is somehow linked to the mechanism of inhibition. Resistance to lysosomal degradation. Another generally employed mechanism for survival within phagolysosomes is resistance to the enzymatic activities presented. Leishmania major, Salmonella typhimurium and Mycobacterium avium are presumed to be resistant to the effects of lysosomal hydrolases. These organisms express large amounts of surface glycolipids (lipophosphoglycans in Leishmania, lipopolysaccharides (LPS) in Sahnonella and glycopeptidolipids in Mycobacterium) that may account for resistance. The loss of these glycolipids is generally correlated with loss of virulence, although this is not inevitably the case 34.
Fig. 3. Different modes of internalizing foreign particles by Hydra. A microvillar form (A) is used to engulf symbiotic Chlorella (arrows indicate microvillae) and broader, funnel-shaped extensions (B) are observed when latex spheres are internalized (arrows indicate unknown material deposited on the particles before internalization). Scale bars = 5.0 t~m. (Reproduced, with permission, from Ref. 30.)
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Mutualists also employ resistance to hydrolysis. When the x-bacterial symbionts of A. proteus were subjected to lysosomal fusion at early stages of the association, the isolated bacteria showed extensive recalcitrance to lysozyme, lipase, proteases and detergents2~k Later stages of the association showed no signs of lysosomal fusion. Resistance appears to have been a pre-adaptation of these bacteria, later followed by the ability to prevent lysosomal fusion. Vacuolar escape. Once inside their intravacuolar compartment, some parasites escape into the cytoplasm through rupturing the membrane. Examples including T. cruzi (Fig. 4), Listeria and Rickettsia have been reviewed by Andrews and Webster 3s. Where the process is understood, disruption of the vacuole membrane is associated with microbial secretion of pore-forming proteins, phospholipases and neuraminidases. Commensalistic or mutualistic symbionts observed in ciliates are also known to escape the phagosome 3". Intranuclear bacteria initially taken up by phagocytosis are generally free of surrounding membranes when found in either the macronucleus or micronucleus. The ciliates Spirostomum ambiguum and Neobrusaridium gigas are consistently observed to contain macronuclear-bacteria. The widespread occurrence, permanence and apparently nondestructive nature of these associations appears to indicate a mutualistic relationship. Other intranuclear symbionts of ciliates even include eukaryotes such as Leptomonas karyophilus. These trypanosomatids rupture the phagocytotic vacuole of Paramecium trichium, escaping into the cytoplasm before migrating to the macronucleus. However, there are no known selective advantages to the Paramecium of possessing these relatives of T. cruzi and they persist only when food is abundant. The mode of direct access to the cytoplasm by symbionts is often poorly understood. In addition to symbiont-mediated escape, the possibility exists that rupture of a vacuole could also be due to host activities. Some organisms may bypass endosomes entirely and enter through direct penetration, analogous to some parasites including microsporidians. Zygomycete fungi of the genus Glomus possess both bacteria and bacteria-like organelles free in the cytoplasm 37 (Fig. 5). Active invasion rather than loss of a vacuolar membrane may have been involved in establishing this association since there is no known phagocytosis in these fungi. Vacuolar modification. Residing within a membrane barrier creates a potential dilemma for nutrient procurement by the parasite. Although, in principal, such procurement can occur either via diffusion, specific transport of nutrients across the membrane, or as a consequence of vesicular traffic into the vacuole, the actual mechanisms are poorly understood. The parasitophorous vacuole membrane (PVM) of Toxoplasma gondii has recently been shown to contain a pore of unknown origin with a size exclusion of 1.3-1.9 kDa (J.C. Schwab, C.J.M. Beckers and K.A. Joiner, unpublished). The parasitophorous vacuole (PV) of T. gondii is known to be modified by dense granule proteins 3s~1 (Fig. 6), although their function has not been established. Following invasion, and increasingly over the next 16 h, dense granule proteins are present both in the vacuolar space and on the PVM 42. In
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Fig. 4. Escape ofTrypanosoma cruzi from the vacuole. One hour after infection, discontinuities appear in the vacuolar membrane (arrows). Scale bar = 0.5 t~m. (Reproduced, with permission, from Ref 58.)
addition, rhoptry proteins are associated with the PVM 42 some of which are exposed to its cytoplasmic face (C.J.M. Beckers, J-F. Dubremetz and K.A. Joiner, unpublished). One role of these parasite-induced modifications may be to contribute to pore formation. In Plasmodium, although nutrient acquisition may involve a channel across the PVM 43 or even a duct connecting the PV space with the extracellular space 44,45, this issue remains to be clarified. The x-bacteria-containing vacuoles of A. proteus are also known to be modified. The membrane of the xbacteria-containing vacuole contains a 96 kDa protein of unknown origin 46 and LPS that occurs on both
Fig. 5. (A) Bacterium in the mycelium of an unidentified fungus colonizing Vitis vinifera roots. Outside the plasma membrane (pl) of the bacterium, the cell wall (cw) shows loose fibrillar material. Electron-dense granules (dg) and a possible nucleoid material (nu) are present in the protoplasm. (13) A bacteria-like organelle in the
fungus Glomus fasciculatum colonizing Ginko biloba roots. This organelle appears to be completing division, with the daughter cells attached via a small piece of coat (arrow). Scale bars = 0.5 p.m. (Reproduced, with permission, from Ref. 37.)
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Fig. 6. Vacuolar modification by Toxoplasma gondii showing immunogold localization on intracellular T. gondii tachyzoites with an antibody to the dense granule protein, GRA3. Staining o c c u r s primarily on dense granules (large arrows), both inside the parasite and after exocytosis, and on the parasitophorous vacuole membrane (small arrows). Nu, nucleus. Scale bar -- I ~m. (Reproduced, with permission, from Ref. 42.)
sides of the membrane and presumably originates from the bacteria 47. Although it is speculated that the LPS may play a role in the prevention of lysosomal fusion, its function has not been established. Chlorella-containing vacuoles of Paramecium bursaria also appear to have protein modifications4S, 49. The number of intramembranous particles is higher in algal-containing vacuoles compared with food vacuoles, although whether the origin of modification is host or symbiont is not known. Treatment of Chlorella with proteases, antibodies or lectins prior to uptake by the Paramecium inhibits this structural modification. Positive effects of parasites upon their hosts? As we have outlined, there are several well-recognized lines of evidence suggesting an interrelationship between parasitism and mutualism. If mutualisms can begin as parasitisms, then parasitism can be the initiation of a complex interaction that eventually provides new metabolic capabilities to the host. Mitochondria may have had a pathogenic origin, similar to invading bacteria such as Daptobacter50, which gains direct contact with the cytoplasm of its bacterial hosts. Recently discovered bacterial analogs of mitochondria are also free in the cytoplasm sl. The presence of porins on the outer membrane of mitochondria structurally similar to bacterial porins 52 also suggest that the bacterial ancestors of mitochondria had direct contact with the host cytoplasm. Thus, they may have been derived either through direct invasion, escape from a phagosome or host-mediated loss of the phagosomal membrane. Although a pathogenic origin is implicated, the association ultimately resulted in bringing respiration to the host. The origin of chloroplasts may have been analogous to the association of Chlorella with Hydra ~3, where the host is the aggressive partner. Intervention
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in lysosomal fusion would probably have been necessary for survival of the endosymbionts that evolved into plastids. Often lacking highly specialized metabolism capabilities such as photosynthesis or nitrogen fixation, parasites may seem to possess little that might have positive evolutionary consequences for their host. Yet parasites carry an array of metabolic capabilities and unique gene products that might exert a positive effect on their host under certain conditions. It is instructive to recognize that, in addition to photosynthesis and respiration, plastids and mitochondria both carried with them other important metabolic functions. Mitochondria provide [3-oxidation of fatty acids, urea cycling, pyrimidine biosynthesis and intermediates in amino acid biosynthesis >4. Likewise, plastids perform other essential functions that could explain their sometimes cryptic persistance, including the presence of plastid-like DNA 5s (the 35 kb circle) in coccidian parasites. With the exception of Euglena, which can be cured of their plastids with streptomycin, possessing plastids appears to be obligatory for plastid-containing hosts. Although achlorophyllous plants do not to rely on their plastids for carbon assimilation, remnants of their plastids are still maintained. Similarly, Chlamydomonas can be maintained in the dark on acetate, but treatments to eliminate plastids are lethal. The reasons for this are not proven but probably lie in the other metabolic functions of plastids, including the reduction of sulfate and nitrite and involvement in biosynthesis of amino acids, vitamin B1, starch, pyrimidines, fatty acids, tetrapyrroles and isoprenoidsS6; representative of more rudimentary functions with which many parasites might complement their hosts. Parasites not only possess essential biosynthetic pathways but may also carry the capacity to synthesize unique secondary compounds that might also be deployed for the benefit rather than the demise of the host. Killer Paramecium carry symbiotic bacteria (eg. Caedibacter), which both secrete a toxin and confer resistance to that toxin s7. When a non-symbiotic Paramecium comes in contact with a Paramecium carrying Caedibacter, the non-symbiotic one is killed. Possessing a toxin to which the Paramecium is susceptible, it may be speculated that these bacteria were once pathogenic, yet the toxin now acts as a selective advantage to the Paramecium carrying it. Likewise, one selective advantage of the x-bacteria and A. proteus association at the initial stages of parasitism may lie in the ability of the bacteria to kill non-resistant amoebae. Conclusions The modes of interaction between host and parasite are not rigid, and can evolve to become either more or less virulent. The interrelationship of mutualism and parasitism is reflected in the parallels used for accessing and persisting within host cells, demonstrating that intracellular persistence does not imply virulence. In those cases where pathogens become less virulent, association can represent a potential opportunity for the host to gain metabolic functions. This perspective suggests that questions concerning widespread persistent parasites should include possible contributions of the parasite towards host adaptation. We also speculate that, perhaps like A. proteus
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and its x-bacterial symbionts, it may even be found that some host cells become dependent upon their parasites. We would expect to observe this as a complication of curative therapy, in which the destruction of the parasite also results in the loss of the infected host cell, although w e know of no examples at this time.
Acknowledgements We wish to thank Betsey Dyer, Genevi@ve Milon and Isabelle Tardieux for helpful d~scussions, and Ted Pat-dy for supplying the photograph for Fi,~. 2. References
l Kevmer, A.E. and Read, A.F. (1990) Parasitology Today 6, 2-3 2 Mitchell, G.F. (1991) in Immum~parasitoh~gy Today (Ash, C. and Gallagher, R.B., eds), pp A2-A5, Elsevier Trends Journals, Cambridge 3 Humphrey-Smith, I. (1989) Parasitology Today 5, 385-387 4 ]anovy, l., Jr, Clopton, R.E. and Percival, T.J. (1992) ]. Parasitol. 78, 630-640 5 Klassen, G.J. (1992) ]. Parasitol. 78, 573-587 6 Hamilton, W.D., Alelrod, R. and Tanese, R. (1990) Proc. Natl Acad. Sci. USA 87, 3566-3573 7 Ladle, R.]. (1992) Tremts Ecol. Evol. 7, 405408 8 Margulis, L. (1993) Symbiosis in Cell Evaluti(m (2nd edn), W.H. Freeman 9 Bermudes, D. and Margulis, L. (1987) Symbiosis 4, 185-198 10 De Bary, A. (1879) Die Erscheinung der Symbiosc. Vers. Deut. Naturtbrscher t4nd Aerztc :u Cassel, Tagebl. 51,121 11 Hayman, D.S. (1983) Cml. ]. Bot. 61,944-963 12 Hayman, D.S. (1987) in VA Mycorrhizal Plm~ts (Safir, G.R., ed.), pp 171-192, CRC Press 13 Washburn, J.O., Mercer, D.R. and Anderson, J.R. (1991) Science 253, 185-188 14 Sharifi, E. (1984) BioSystems 16, 269-289 15 Freeman, S. and Rodriguez, R.J. (I993) Science 260, 75-78 16 Bull, J.J., Molineux, l.J. and Rice, W.R. (1991) Evolution 45, 875-882 17 Read, A.F. and Schrag, S.J. (1901) Parasitoloxy Today 7, 296-297 18 Herre, E.A. (1993) Science 259, 1442-1445 I9 Jeon, K.W. (1987) Ann. New York Acad. Sci. 503, 359-371 20 Jeon, K.W. (1991) in Symbiosis as a Source off Evolutionmy htnoration (Margulis, L. and Fester, R., eds), pp 118-131, MIT Press 21 Moulder, J.W. (1985) Micmbiol. Rev. 49, 298-337 22 Reisser, W. ctal. (1985) J. Protozool. 32, 383-390 23 Muscatine, L. and McNeil, P.L. (1989) Am. Zool. 29, 371-389
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R A P D in the Genus Trichinella We agree with Zadenga and Nun-ell' when they urge researche,-s to type isolates of Tnchinella before publishing experimental data. Host pubhshed studies of this parasite do not mention th~s essential informatlon and recent wod< has shown that antigenic differences between ~olates can result in diffecent host responses. D N A bandqng patterns, [DNA probes t~ and isoenzyme analysis' have p,-o'ven their ability to type isolates or identif/species of TtfchJne/Id. but these methods are time consuming and often t-equate large amounts of D N A and the use of radioactive probes. We and others have used a conventional polymerase chain reacbon (PCR) to identify genom< sequences of T spnaks -~: but, with the pnmers used, vve wece unable to
differentiate the other four- species'. Recently, PlacPhecson and Gajadhad reported the value of random amplified polymo~?hic D N A (P,APD) (arbitra~7 primed PCR) foc fingerprinting parasitic organisms. We used RAPD to type 18 isolates of TnchJnello from various oc@ns, most of them kindly provided by Pozio (Istituto Superiore di SanitY, Rome, Italy), ,who had identified them as T. sprahs. T nelson. 7 pseudospirr3hs. 7 ndt~vd. T bnto,.~ or FnchJnella type S D N A was extracted by a conventional method from muscle lapvae and RAPD assays were caned out accordin~ to Wilhams e; r;I Several arbitrarily chosen pnme~°s were used. With some primers, PCR amplification (45 cycles at 94°C for I rain. 36°C for I rain and 72°C for 2 rain) yielded several D N A fragments r'anglng fl-om 0.2 to 3 kb that were polymorphic between the sl~ groups of'solatestFl~ I).lnaddltion,
isolates were compared by calculating similarity coeffcients based on both the TI T5 T2 T2 T2 T2 T3 T3 T3 T3 T3 T3 T4 T7 mw
Fig. I. Fragments amplified using primer TGGTCGCGGC on DNA extracted from the following isolates: T I (Trichinella spiralis), 1"5 ('rrichinella type 5), T2 (T. nativa), T3 fT. britovi), T4 (T. pseudospiralis), T7 fT. nelsoni). PCR products were electrophoresed on a 1.4% agarose gel and stained with ethidium bromide. Mw, molecular weight marker III from Boehringer, ranging from 0.56 to 21.2 kb.
ParasJ~olo~,yToday. ~.ol 9, no. I ~, ~ 1993
25 Lindsay, D.S. and Dubey, J.P. (1989)J. Parasitol. 75, 772-779 26 Dubey, J.p. and de Lahunta, A. ]. AppL Parasitol. (in press) 27 Mayhew, I.G. et al. (1991)J. Small Anita. Pract. 32, 609-612 28 Yamane, R.D. et al. Am. J. Vet. Res. (in press) 29 Barta,J.R. and Dubey, J.P. (1992)Parasitol. Res. 78, 689-694 30 Brindley, P.J. et al. (1993)Am. J. Trop. Med. Httg. 48, 447-456 31 Cole, R.A. et al. (1993)J. Vet. Diagn. Invest. 5, 579-584 32 Lindsay, D.S. and Dubey, J.p. (1989)J. Parasitol. 75, 990-992 33 Lindsay, D.S. and Dubey, J.P. (1992)J. ParasitoL 76, 177-179 34 Hay, W.H. et al. (1990)J. Am. Vet. Med. Assoc. 197, 87-89 35 Lindsay, D.S. et al. (1990)J. Hehninth. Soc. Wash. 57, 86-88 36 Trees, A.J. et al. (1993) Vet. Rec. 132, 125-126 37 Greene, C.E., Cook, J.R. and Mahaffey, E.A. (1985) J. Am. Vet. Med. Assoc. 187, 631-634 38 Dubey, J.P. et al. (1990)J. Vet. Diagn. Invest. 2, 6~69
39 Thorton, R.N., Thompson, E.J. and Dubey, J.P. (1991) NZ Vet. J. 39, 129-133 40 Ogino, H. et al. (1992)J. Comp. Pathol. 107, 231-237 41 Wouda, W. et al. (1992)Tijdschr. Diergeneeskd 117, 599-602 42 Abbitt, B. et al. (1993)J. Am. Vet. Med. Assoc. 203, 444-448 43 Bryan, L.A. et al. Canad. Vet. J. (in press) 44 Jardine, J.E. and Last, R.D. (1993)J. S. Afr. Vet. Assoc. 64, 101-102 45 Barr, B.C. et al. (1991)J. Vet. Diagn. Invest. 3, 39M6 46 Nietfeld, J.C. et al. (1992)J. Vet. Diagn. Invest. 4, 223-226 47 Barr, B.C. et al. (1993)J. Am. Vet. Med. Assoc. 202, 113-117 48 Rogers, D.G. et al. Agric. Practice (in press) 49 Lindsay, D.S. et al. (1993) Comp. Cont. Educ. Pract. Vet. 15, 882-889 50 Conrad, P.A. et al. (1993)Parasitology 106, 239-249 51 Conrad, P.A. et al. (1993)]. Vet. Diagn. Invest. 5, 572-578
The Role of Parasites in Generating Evolutionary Novelty D, Bermudes and K.A. Joiner in this review, David Bermudes and Keith Joiner discuss the interrelationship between parasitism and m u t u a l i s m and examine the parallel mechanisms used by parasites and mutualists to access and persist w i t h i n the intracellular environment. By drawing analogies with mutualistic associations, they suggest mechanisms by which some parasites m a y ultimately benefit their hosts. They ~¢rther speculate that some hosts m a y even become dependent upon their parasites.
Parasitism is a well-recognized evolutionary forceL The co-evolution of hosts with their parasites accounts for numerous physiological modifications of the host (eg. immunological defenses 2) as well as driving the p h y l o g e n y of the parasites, limiting host geographic distribution 3-5 and potentially contributing towards the adaptive value of sexual reproduction 6,7. However, less appreciated is the relationship of parasitism to mutualism and the potential for parasitism ultimately to lead towards novel host adaptations. The physiological advantages to the host accrued from mutualisms involving specialized functions such as nitrogen fixation or photosynthesis appear obvious. Such relationships have even led to the origin of specific organelles, including plastids and mitochondria 8. Mutualistic symbioses function as a mechanism of evolution by genetically introducing significant new traits to their host 9. By providing for host needs or introducing new capabilities, symbiosis can greatly increase the host's ability to inhabit certain environments.
ism, with parasitism considered a separate and unrelated phenomenon. The original usage reflects De Bary's finding of a propensity for organisms to form relationships, while also recognizing that the immediate and long-term effects of interaction are not always known. Furthermore, there is an interrelationship of the two types of interaction. From the perspective of the host, the effects of symbiotic interactions on fitness can be placed along a continuum, with mutualism (advantageous relationships) at one end and parasitism (disadvantageous relationships) at the other (Fig. 1). Practical indicators of host fitness c o m m o n l y include nutritional status and growth rate, which m a y be significantly modified by the interaction. Although some associations appear relatively fixed in their outcome, m o v e m e n t along the c o n t i n u u m occurs in both directions, as often observed in parasites ranging in virulence. The effect of an organism on the host as measured by practical indicators can be variable and m a y be d e p e n d e n t u p o n factors including environmental parameters, host population dynamics and genetically based characters (resistance of the host, virulence of the parasite). Not only are small shifts observed in the relative effects of parasites on their host, but large shifts from
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Interrelationships of mutualism and parasitism The term 'symbiosis' (the living together of two or more dissimilar organisms) was originally coined by De Bary 1° to describe both parasitic and mutualistic associations. Symbiosis is often equated with mutual-
David Ben-nudes and Keith Joiner are at the Yale University School of Medicine, Department of Internal Medicine, Infectious Diseases Section, New Haven, CT 06510, USA.
Host
Fitness
negativeeffect
Parasitism
no effect
Commensalism
positive effect
Mutualism
Fig. I. Ranges of effects of symbionts (parasites or mutualists) upon their hosts occur as a continuum. Overall positive or negative e~ects as measured by practical indicators define the relationship as parasitic, commensalistic or mutualistic. ~ 9 9 3 . Flse'4(~ S(icr c Pllbhsl-(I ~ i t 1 I i F
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one end of this continuum to the other are also known. Mutualistic associations may, under given conditions, have no detectable effect (commensalism), while, under other conditions, they will become parasitic. For example, mycorrhizal symbioses (the association of fungi with the roots of vascular plants) are usually advantageous to onions for obtaining phosphate 11. Phosphate, more efficiently obtained by the fungal mycelium, is translocated to the plant and results in a measurable increase in plant growth, despite concomitant translocation of sugars from the plant to the fungus. However, this advantage is absent at higher population densities. Under crowded conditions, all available phosphate is taken up by the plant roots and there is no additional uptake associated with the mycorrhizae. Further, under excess phosphate concentrations, the fungus now acts as a parasite, reducing host vigour 12. Apparently, the sugars translocated to the fungus are sufficient to diminish plant growth relative to uninfected plants, with no compensation by fungal-mediated phosphate uptake. Resource availability has been shown to determine the nature of interaction in other associations. The ciliate Lambornella clarki is a fatal pathogen to its mosquito host (Aedes sierrensis) when the food supply is abundant, but can result in greater production of progeny when food is limiting 13. Many mutualisms, such as the association of Rhizobium with legumes, are believed to have evolved into their present form of co-operation from parasitic associations 14. In a recent study 15, it was found that a fungal plant pathogen could be converted to a mutualist as a result of mutation of a single locus. The evolution of virulence has recently been reviewed by Read and Schrag 16. Natural selection for co-operation is believed to be promoted when the chief mode of transmission is vertical (infecting the offspring of the host), thereby not only limiting potential hosts, but linking present and future parasite reproduction to the continuance of that host and its progeny 17. Conversely, it has been shown that increased opportunity for horizontal transmission (not infecting the host offspring) of parasites promotes evolution towards increased virulence 18. In the study by Herre 18, waspparasitic nematode species using more than one host have a greater affect on wasp reproduction. In such populations, virulence increases parasite reproductive success, with future parasite success independent of one particular host and its progeny.
Transition from parasite to obligate mutualist The only directly observed example of transition from lethal pathogen to obligate symbiont has been studied by Jeon and co-workers19, 20. The parasitism first occurred when a normal (D) strain of Amoeba proteus became infected with an unknown species of obligately parasitic Gram-negative rod (x-bacteria). Most of the amoebae were killed but, by separating individual amoebae, infected survivors (xD amoebae) were obtained. At this stage the bacteria harvested from resistant amoebae were still lethal to most amoebae. Subsequently, selection apparently occurred for bacteria with reduced virulence, as the bacterial strains from later stages were quickly able to establish a nonlethal association with naive amoebae. After the original xD amoebae had become stable, the amoebae
459
could be cured with trimethoprim (a dihydrofolatereductase inhibitor), chloramphenicol or slightly elevated temperature. The association could be reestablished by microinjection with cytoplasm containing the bacteria of an infected amoebae or through phagocytosis from a x-bacterial suspension. However, after maintenance through 200 generations (18 months), most xD amoebae could not survive curative treatment unless rescued by re-introduction of the bacteria. Furthermore, in nuclear transplantation experiments, the nucleus from an infected cell was inviable when placed within the cytoplasm of an enucleated uninfected cell. These hybrid amoebae could also survive only if the bacteria were introduced into that cell. These observations have led to the conclusion that the association had become obligate for the amoebae. The formation of the x-bacteria and amoeba's association appears to have followed the aforementioned rules for virulence. Initially, virulence was high when unlimited hosts (large cultures) were present: parasite reproduction was not directly linked to survival of the immediate host. Reduced virulence was observed after amoebae were separated out into many individual cultures: virulent bacteria were selfeliminated by killing of their only available host. Generally, mutual interdependence occurs as a form of complementation (eg. through providing a needed metabolite or gene product). Metabolic requirements could be pre-existing or induced after initiation of the association through several different means. A host or symbiont could lose a gene function through mutation simply because there is no longer selective pressure to maintain that function. Gene transfer (which is known to have occurred for both plastids and mitochondria 8) could also account for an obligate relationship. Mechanisms actively causing impairment, such as genetic transposition or the selective action of a toxin, could also generate interdependence.
Mechanisms of intracellular persistence In examining parasite and mutualist methods for accessing and persisting within their hosts, we find that significant parallels exist, although insufficient data are available to determine if the actual mechanisms are the same. Similarities could be due either to common ancestry or to convergent evolution. However, these data are intriguing because they demonstrate that evasion of intracellular defense alone does not necessarily create virulence. A limited number of mechanisms for organisms accessing and persisting within eukaryotic cells have been described. The most common modes of entry are either by phagocytosis or active membrane penetration 21-24, although other mechanisms occur. For example, lysosome recruitment has been shown to be a mechanism for stimulating internalization of Trypanosoma cruzi 25. Inside the cell, resistance to or avoidance of digestive or defensive measures facilitates intracellular persistence. These metabolic mechanisms of the host, which are broadly lethal to ingested organisms, can include lysosomal hydrolases, acidification and reactive oxygen and nitrogen intermediates. Utilization of specific receptors. Parasite internalization into macrophages via specific receptors to avoid
460
Ted Pardy Fig. 2. Hydra gastrodermal cells prepared by maceration show the presence of endobiotic Chlorella. The Chlorella are engulfed on the gut surface and transported to the basal region (arrows), but are not digested. Nu, host cell nucleus. Scale bar = 30 t~m.
a specific metabolic response is well documented and has been discussed in a previous review 24. Nearly all pathogens restricted to macrophages are internalized via complement receptors, which are not linked to the generation of reactive oxygen or nitrogen intermediates. Altering the receptors mediating entry is thought to trigger specific microbicidal cascades and result in killing of the invading organism. Evidence also suggests that intracellular mutualists must first bind to the appropriate ligand or face destruction subsequent to internalization. Hydra viridis normally contains symbiotic strains of the green alga Chlorella (these Chlorella secrete photosynthate in the form of maltose or trehalose that contribute to Htldra nutrition and absorb excess nitrogenous compounds from the animal). Normally, Chlorella are phagocytozed but not digested (Fig. 2). Antibody-coated and lectin-coated Chlorella or Chlorella treated with trypsin are not well internalized 26-2s. Moreover, Chlorella treated with ferric chloride are phagocytozed and digested by Hydra 29 and the morphology of the uptake mechanism is observably altered 3° (Fig. 3). These data suggest that recognition phenomena
Paras~tolo,~yToday,~ol 9. ~o i), 1993
involving surface charge occur at the time of phagocytosis. Initiation of the interaction between Hydra and Chlorella may be analogous to infection by Leishmania, where internalization via the complement receptors CR1 and CR3 bypasses a destructive intracellular response (for review see Hall and Joiner24). Inhibition of lysosomal fusion. Phagocytosis of bacteria is usually followed by fusion of the phagosome with lysosomes, exposing the contents of the endosomal compartment to acid pH and hydrolases. This cell function is blocked in several parasitisms, including those involving Mycobacterium tuberculosis, Chlamydia psittaci and Legionella pneumophila. The amoebae Hartmannella is also blocked in this function when infected by Legionella31. Although the mechanism for this inhibition is not well understood for any pathogen, recent studies with Legionella have identified loci involved in fusion inhibition 32-~3. Stable sequestering of mutualistic endosymbionts appears to rely extensively upon endocytosis without subsequent fusion of lysosomes. Examples include both the interaction of Chlorella with Hydra and later stages of the x-bacteria and A. proteus association. In both Amoeba 19,2° and Hydra 29, live symbionts are required to inhibit fusion and destruction of the symbiont. In the example of Hydra, uptake of heat-killed Chlorella or inhibition of photosynthesis with 3-(3,4dichlorophenyl)-l,l-dimethyl urea, followed by dark incubation after endocytosis, results in lysosomal fusion and destruction of most Chlorella29. Apparently, continued contribution from the symbiont is somehow linked to the mechanism of inhibition. Resistance to lysosomal degradation. Another generally employed mechanism for survival within phagolysosomes is resistance to the enzymatic activities presented. Leishmania major, Salmonella typhimurium and Mycobacterium avium are presumed to be resistant to the effects of lysosomal hydrolases. These organisms express large amounts of surface glycolipids (lipophosphoglycans in Leishmania, lipopolysaccharides (LPS) in Sahnonella and glycopeptidolipids in Mycobacterium) that may account for resistance. The loss of these glycolipids is generally correlated with loss of virulence, although this is not inevitably the case 34.
Fig. 3. Different modes of internalizing foreign particles by Hydra. A microvillar form (A) is used to engulf symbiotic Chlorella (arrows indicate microvillae) and broader, funnel-shaped extensions (B) are observed when latex spheres are internalized (arrows indicate unknown material deposited on the particles before internalization). Scale bars = 5.0 t~m. (Reproduced, with permission, from Ref. 30.)
Pomsltoio£y Toddy vol 9 ~,) i 2 /99]'
Mutualists also employ resistance to hydrolysis. When the x-bacterial symbionts of A. proteus were subjected to lysosomal fusion at early stages of the association, the isolated bacteria showed extensive recalcitrance to lysozyme, lipase, proteases and detergents2~k Later stages of the association showed no signs of lysosomal fusion. Resistance appears to have been a pre-adaptation of these bacteria, later followed by the ability to prevent lysosomal fusion. Vacuolar escape. Once inside their intravacuolar compartment, some parasites escape into the cytoplasm through rupturing the membrane. Examples including T. cruzi (Fig. 4), Listeria and Rickettsia have been reviewed by Andrews and Webster 3s. Where the process is understood, disruption of the vacuole membrane is associated with microbial secretion of pore-forming proteins, phospholipases and neuraminidases. Commensalistic or mutualistic symbionts observed in ciliates are also known to escape the phagosome 3". Intranuclear bacteria initially taken up by phagocytosis are generally free of surrounding membranes when found in either the macronucleus or micronucleus. The ciliates Spirostomum ambiguum and Neobrusaridium gigas are consistently observed to contain macronuclear-bacteria. The widespread occurrence, permanence and apparently nondestructive nature of these associations appears to indicate a mutualistic relationship. Other intranuclear symbionts of ciliates even include eukaryotes such as Leptomonas karyophilus. These trypanosomatids rupture the phagocytotic vacuole of Paramecium trichium, escaping into the cytoplasm before migrating to the macronucleus. However, there are no known selective advantages to the Paramecium of possessing these relatives of T. cruzi and they persist only when food is abundant. The mode of direct access to the cytoplasm by symbionts is often poorly understood. In addition to symbiont-mediated escape, the possibility exists that rupture of a vacuole could also be due to host activities. Some organisms may bypass endosomes entirely and enter through direct penetration, analogous to some parasites including microsporidians. Zygomycete fungi of the genus Glomus possess both bacteria and bacteria-like organelles free in the cytoplasm 37 (Fig. 5). Active invasion rather than loss of a vacuolar membrane may have been involved in establishing this association since there is no known phagocytosis in these fungi. Vacuolar modification. Residing within a membrane barrier creates a potential dilemma for nutrient procurement by the parasite. Although, in principal, such procurement can occur either via diffusion, specific transport of nutrients across the membrane, or as a consequence of vesicular traffic into the vacuole, the actual mechanisms are poorly understood. The parasitophorous vacuole membrane (PVM) of Toxoplasma gondii has recently been shown to contain a pore of unknown origin with a size exclusion of 1.3-1.9 kDa (J.C. Schwab, C.J.M. Beckers and K.A. Joiner, unpublished). The parasitophorous vacuole (PV) of T. gondii is known to be modified by dense granule proteins 3s~1 (Fig. 6), although their function has not been established. Following invasion, and increasingly over the next 16 h, dense granule proteins are present both in the vacuolar space and on the PVM 42. In
461
Fig. 4. Escape ofTrypanosoma cruzi from the vacuole. One hour after infection, discontinuities appear in the vacuolar membrane (arrows). Scale bar = 0.5 t~m. (Reproduced, with permission, from Ref 58.)
addition, rhoptry proteins are associated with the PVM 42 some of which are exposed to its cytoplasmic face (C.J.M. Beckers, J-F. Dubremetz and K.A. Joiner, unpublished). One role of these parasite-induced modifications may be to contribute to pore formation. In Plasmodium, although nutrient acquisition may involve a channel across the PVM 43 or even a duct connecting the PV space with the extracellular space 44,45, this issue remains to be clarified. The x-bacteria-containing vacuoles of A. proteus are also known to be modified. The membrane of the xbacteria-containing vacuole contains a 96 kDa protein of unknown origin 46 and LPS that occurs on both
Fig. 5. (A) Bacterium in the mycelium of an unidentified fungus colonizing Vitis vinifera roots. Outside the plasma membrane (pl) of the bacterium, the cell wall (cw) shows loose fibrillar material. Electron-dense granules (dg) and a possible nucleoid material (nu) are present in the protoplasm. (13) A bacteria-like organelle in the
fungus Glomus fasciculatum colonizing Ginko biloba roots. This organelle appears to be completing division, with the daughter cells attached via a small piece of coat (arrow). Scale bars = 0.5 p.m. (Reproduced, with permission, from Ref. 37.)
462
Fig. 6. Vacuolar modification by Toxoplasma gondii showing immunogold localization on intracellular T. gondii tachyzoites with an antibody to the dense granule protein, GRA3. Staining o c c u r s primarily on dense granules (large arrows), both inside the parasite and after exocytosis, and on the parasitophorous vacuole membrane (small arrows). Nu, nucleus. Scale bar -- I ~m. (Reproduced, with permission, from Ref. 42.)
sides of the membrane and presumably originates from the bacteria 47. Although it is speculated that the LPS may play a role in the prevention of lysosomal fusion, its function has not been established. Chlorella-containing vacuoles of Paramecium bursaria also appear to have protein modifications4S, 49. The number of intramembranous particles is higher in algal-containing vacuoles compared with food vacuoles, although whether the origin of modification is host or symbiont is not known. Treatment of Chlorella with proteases, antibodies or lectins prior to uptake by the Paramecium inhibits this structural modification. Positive effects of parasites upon their hosts? As we have outlined, there are several well-recognized lines of evidence suggesting an interrelationship between parasitism and mutualism. If mutualisms can begin as parasitisms, then parasitism can be the initiation of a complex interaction that eventually provides new metabolic capabilities to the host. Mitochondria may have had a pathogenic origin, similar to invading bacteria such as Daptobacter50, which gains direct contact with the cytoplasm of its bacterial hosts. Recently discovered bacterial analogs of mitochondria are also free in the cytoplasm sl. The presence of porins on the outer membrane of mitochondria structurally similar to bacterial porins 52 also suggest that the bacterial ancestors of mitochondria had direct contact with the host cytoplasm. Thus, they may have been derived either through direct invasion, escape from a phagosome or host-mediated loss of the phagosomal membrane. Although a pathogenic origin is implicated, the association ultimately resulted in bringing respiration to the host. The origin of chloroplasts may have been analogous to the association of Chlorella with Hydra ~3, where the host is the aggressive partner. Intervention
Pclmsitolody Toddy ,.ol 0 no ! 2 19~'~
in lysosomal fusion would probably have been necessary for survival of the endosymbionts that evolved into plastids. Often lacking highly specialized metabolism capabilities such as photosynthesis or nitrogen fixation, parasites may seem to possess little that might have positive evolutionary consequences for their host. Yet parasites carry an array of metabolic capabilities and unique gene products that might exert a positive effect on their host under certain conditions. It is instructive to recognize that, in addition to photosynthesis and respiration, plastids and mitochondria both carried with them other important metabolic functions. Mitochondria provide [3-oxidation of fatty acids, urea cycling, pyrimidine biosynthesis and intermediates in amino acid biosynthesis >4. Likewise, plastids perform other essential functions that could explain their sometimes cryptic persistance, including the presence of plastid-like DNA 5s (the 35 kb circle) in coccidian parasites. With the exception of Euglena, which can be cured of their plastids with streptomycin, possessing plastids appears to be obligatory for plastid-containing hosts. Although achlorophyllous plants do not to rely on their plastids for carbon assimilation, remnants of their plastids are still maintained. Similarly, Chlamydomonas can be maintained in the dark on acetate, but treatments to eliminate plastids are lethal. The reasons for this are not proven but probably lie in the other metabolic functions of plastids, including the reduction of sulfate and nitrite and involvement in biosynthesis of amino acids, vitamin B1, starch, pyrimidines, fatty acids, tetrapyrroles and isoprenoidsS6; representative of more rudimentary functions with which many parasites might complement their hosts. Parasites not only possess essential biosynthetic pathways but may also carry the capacity to synthesize unique secondary compounds that might also be deployed for the benefit rather than the demise of the host. Killer Paramecium carry symbiotic bacteria (eg. Caedibacter), which both secrete a toxin and confer resistance to that toxin s7. When a non-symbiotic Paramecium comes in contact with a Paramecium carrying Caedibacter, the non-symbiotic one is killed. Possessing a toxin to which the Paramecium is susceptible, it may be speculated that these bacteria were once pathogenic, yet the toxin now acts as a selective advantage to the Paramecium carrying it. Likewise, one selective advantage of the x-bacteria and A. proteus association at the initial stages of parasitism may lie in the ability of the bacteria to kill non-resistant amoebae. Conclusions The modes of interaction between host and parasite are not rigid, and can evolve to become either more or less virulent. The interrelationship of mutualism and parasitism is reflected in the parallels used for accessing and persisting within host cells, demonstrating that intracellular persistence does not imply virulence. In those cases where pathogens become less virulent, association can represent a potential opportunity for the host to gain metabolic functions. This perspective suggests that questions concerning widespread persistent parasites should include possible contributions of the parasite towards host adaptation. We also speculate that, perhaps like A. proteus
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and its x-bacterial symbionts, it may even be found that some host cells become dependent upon their parasites. We would expect to observe this as a complication of curative therapy, in which the destruction of the parasite also results in the loss of the infected host cell, although w e know of no examples at this time.
Acknowledgements We wish to thank Betsey Dyer, Genevi@ve Milon and Isabelle Tardieux for helpful d~scussions, and Ted Pat-dy for supplying the photograph for Fi,~. 2. References
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I
R A P D in the Genus Trichinella We agree with Zadenga and Nun-ell' when they urge researche,-s to type isolates of Tnchinella before publishing experimental data. Host pubhshed studies of this parasite do not mention th~s essential informatlon and recent wod< has shown that antigenic differences between ~olates can result in diffecent host responses. D N A bandqng patterns, [DNA probes t~ and isoenzyme analysis' have p,-o'ven their ability to type isolates or identif/species of TtfchJne/Id. but these methods are time consuming and often t-equate large amounts of D N A and the use of radioactive probes. We and others have used a conventional polymerase chain reacbon (PCR) to identify genom< sequences of T spnaks -~: but, with the pnmers used, vve wece unable to
differentiate the other four- species'. Recently, PlacPhecson and Gajadhad reported the value of random amplified polymo~?hic D N A (P,APD) (arbitra~7 primed PCR) foc fingerprinting parasitic organisms. We used RAPD to type 18 isolates of TnchJnello from various oc@ns, most of them kindly provided by Pozio (Istituto Superiore di SanitY, Rome, Italy), ,who had identified them as T. sprahs. T nelson. 7 pseudospirr3hs. 7 ndt~vd. T bnto,.~ or FnchJnella type S D N A was extracted by a conventional method from muscle lapvae and RAPD assays were caned out accordin~ to Wilhams e; r;I Several arbitrarily chosen pnme~°s were used. With some primers, PCR amplification (45 cycles at 94°C for I rain. 36°C for I rain and 72°C for 2 rain) yielded several D N A fragments r'anglng fl-om 0.2 to 3 kb that were polymorphic between the sl~ groups of'solatestFl~ I).lnaddltion,
isolates were compared by calculating similarity coeffcients based on both the TI T5 T2 T2 T2 T2 T3 T3 T3 T3 T3 T3 T4 T7 mw
Fig. I. Fragments amplified using primer TGGTCGCGGC on DNA extracted from the following isolates: T I (Trichinella spiralis), 1"5 ('rrichinella type 5), T2 (T. nativa), T3 fT. britovi), T4 (T. pseudospiralis), T7 fT. nelsoni). PCR products were electrophoresed on a 1.4% agarose gel and stained with ethidium bromide. Mw, molecular weight marker III from Boehringer, ranging from 0.56 to 21.2 kb.