Malaria Multigene Families: The Price of Chronicity

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37 Godbold, G.D. and Mann, B.J. (1998) The involvement of the actin cytoskeleton and p21 Rho family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica. Braz. J. Med. Biol. Res. 31, 1049–1058 38 Reuner, K.H. et al. (1995) Autoregulation of actin synthesis in hepatocytes by transcriptional and posttranscriptional mechanisms. Eur. J. Biochem. 230, 32–37 39 Montminy, M.R. and Bilezikan, L.M. (1987) Binding of a nuclear protein in the cyclic AMP response element of the somatostain gene. Nature 328, 175–178 40 Onyia, J.E. et al. (1994) Identification of b-actin sequences necessary for induction by phorbol esters and calcium ionophores. Oncogene 9, 1713–1722

Focus

Malaria Multigene Families: The Price of Chronicity G. Snounou, W. Jarra and P. R. Preiser In this article, Georges Snounou, William Jarra and Peter Preiser discuss the survival strategy of malaria parasites in the light of a novel mechanism of clonal phenotypic variation recently described for a multigene family of Plasmodium yoelii yoelii. The 235 kDa rhoptry proteins (Py235) encoded by these genes may be involved in the selection of red blood cells for invasion by merozoites. The new mechanism may explain the ability of individual parasites to adapt to natural variations in red blood cell subsets, while ensuring that sufficient merozoites escape immune attack, thus maintaining a chronic infection for extended periods. This counterpoints the antigenic variation exemplified by PfEMP1 proteins (a large family of proteins derived from P. falciparum), which operates at the population level. The possibility of manipulating the expression of functionally similar genes in other Plasmodium species could lead to therapies aimed at reducing clinical severity without compromising the acquisition and maintenance of immunity. Malaria infections last for inordinately long periods of time in natural vertebrate hosts. Furthermore, experimental infections in lower primates, rodents and birds often result in lifelong infections. However, in humans, clinical episodes are unlikely to occur beyond 12 months following inoculation, unless the infection is due to Plasmodium vivax and, more rarely, to P. ovale. In these cases, relapses, due to dormant liver stages, can occur for six more years. Nonetheless, parasites can often still be found in the blood long after cessation of clinical activity1. Plasmodium falciparum parasites have been detected 1–3 years after primary infection2. The record Georges Snounou is at the Department of Infection and Tropical Medicine, Imperial College School of Medicine, Lister Unit, Northwick Park Hospital, Harrow, Middlesex, UK HA1 3UJ. William Jarra and Peter R. Preiser are at the Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK NW7 1AA. Tel: +44 181 959 3666, Fax: +44 181 906 4477, e-mail: [email protected] 28

for longevity, however, is held by P. malariae – some individuals were still infected more than 30 years after last being exposed to transmission3. A chronic, long-lasting infection offers a clear evolutionary advantage to the parasite: an increased probability of transmission to a new host. This is of particular importance to Plasmodium species because transmission can often be interrupted for long periods, during which time environmental conditions can restrict both development of the parasite in the mosquito and the density of the vector population. In order to maintain parasitic infection levels, three criteria must be met: (1) at each erythrocytic cycle, sufficient numbers of infected red blood cells (RBCs) evade the host’s immune system; (2) at least one merozoite from each mature schizont survives the short period spent free in the bloodstream; (3) the merozoites must successfully attach to, and invade, a fresh RBC. The efficiency of the mechanisms underlying these criteria must not be so great that parasite multiplication overwhelms the host. Clearly, these conditions are fulfilled in the majority of natural infections. How Plasmodium parasites achieve this longevity in the face of the host’s competent immune system is still not fully understood. Antigenic variation in Plasmodium species In the bloodstream, parasites are in direct contact with the immune system only during the brief period spanning the release of merozoites and their reinvasion of new RBCs. For the remainder of the erythrocytic cycle, parasite antigens are detectable only on the surface of infected RBCs. During the infection, the vigorous humoral and cellular responses of the host against these two forms of the parasite attest to the immunogenicity of Plasmodium. Yet the immunity acquired is rarely of a sterilizing nature, although it eventually proves sufficient in controlling parasite levels and reducing morbidity.

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Focus The discovery of antigenic variation in P. knowlesi4 (and subsequently in other primate5, human6,7 and rodent8 Plasmodium species) provided the first insight into the conundrum of how parasites evade the immune response. In P. falciparum, PfEMP1 proteins, expressed on the surface of the mature infected RBCs, were shown to be the major components underlying this phenomenon9–11. Members of the gene family (var) encoding the PfEMP1 proteins were recently cloned12–14. The parasite genome contains 40–50 highly polymorphic var genes, and their repertoire differs between isolates15. Using in vitro cultured P. falciparum, only one of the var genes is transcribed in the mature trophozoite16–17, and an overall switch rate of 2% was observed18. Clonal antigenic variation thus provides a means for infected RBCs to evade removal by the immune system. It is not known whether exposure to all the PfEMP1 variants of a given parasite line results in sterile immunity. Individuals in endemic areas still develop patent infections, despite more than 50 years of exposure to infection. This could be due to a var gene repertoire constantly changing as a result of sexual recombination, or to a short-lived immunity to the different PfEMP1 variants. Red blood cell invasion In addition to avoiding neutralization by the host defences, the merozoite has to find and penetrate a new RBC. It has long been known that parasites from various Plasmodium species have distinct predilections to invade different types of RBCs. For example, P. malariae is found mainly in mature RBC, P. vivax and P. ovale in reticulocytes, and P. falciparum will invade both. However, the number and proportion of the different RBC types alters drastically during the course of malaria infections. In addition, this blood picture is also substantially affected by infections with other pathogens, and by a number of medical conditions such as haemoglobinopathies. Thus, upon release from the liver, Plasmodium merozoites encounter diverse blood environments, yet infections rarely fail to become established following successful sporozoite inoculation. Moreover, these infections run a protracted course, despite the presence of antibodies that can be shown to react with the merozoite and, in some cases, to inhibit invasion in vitro. We have recently described a new type of clonal phenotypic variation19 that would allow merozoites to adapt to blood picture variations, escape the host defences and perpetuate the infection. Observations in P. yoelii yoelii revealed that proteins of approximately 235 kDa, found in the rhoptries of merozoites, are implicated in the selection of normocytes or reticulocytes for invasion20,21. These proteins are encoded by a multigene family of 35–50 members22,23, with at least 25 distinguishable variants (S. Khan, unpublished). Analysis of the transcription pattern of the Py235 genes in single infected RBCs revealed some notable features. Transcription of a single member of the family is seen in uni-nucleate trophozoites. However, following nuclear division, transcripts from other Py235 genes are found, such that multiple transcripts are invariably seen in mature schizonts. This distinguishes the phenomenon from the antigenic variation of the var gene family, in that an apparent switch rate of 100% per cycle is seen in the parasites. Remarkably, individual Parasitology Today, vol. 16, no. 1, 2000

merozoites isolated from the same schizont were shown to contain only one of these transcripts. Therefore, each merozoite from the progeny of a single schizont can express a different member of the Py235 family. Specificity of individual Py235 proteins to defined receptors on different types of RBC has yet to be determined. However, immunization with, or passive transfer of antibody against, Py235 proteins results in a switch of the RBC preference of P. y. yoelii20,21. At least one member of this family was shown to bind mature erythrocytes24 and to contain a 500 amino acid region with homology to part of the reticulocyte-binding protein (RBP-2) of P. vivax25. The nature of the Py235 gene diversity will be defined as sequencing of all members of this family progresses. This information will then be used to investigate the functional significance of this diversity. The novel mechanism of clonal phenotypic variation described for the Py235 multigene family confers two advantages. First, it provides the parasite with a distinct biological adaptability in the face of a changing erythropoietic environment. By providing a distinct specificity to each of the merozoites, a single schizont ensures that one or more of its progeny encounters the appropriate type of RBC and successfully invades it. Second, this phenomenon is a most efficient mechanism of immune evasion. Immune responses against one or more particular Py235 proteins will not result in the removal of all the merozoites produced by a schizont. This would be less likely if multiple members of the Py235 family were expressed by each merozoite. By adopting this tactic, the parasite maximizes its chances of survival, and the chronicity of the infection is enhanced. The preferential invasion of reticulocytes has a major impact on the virulence of the parasite. Parasites from a cloned line of P. y. yoelli (YM), which invade all RBC types and cause fulminant lethal infections, become restricted to reticulocytes when anti-PY235 antibodies are present and the mortality is consequently abolished. Reticulocytes are normally present as a small subset (approximately 1%) of the total circulating RBCs. Consequently, parasitaemias resulting from infection by reticulocyte-restricted Plasmodium species tend to remain low, and severe pathology is relatively rare. A lower rate of multiplication will also provide the host with more time to marshal immune defences against the parasite. Immunity might be further enhanced as some evidence suggests that parasites are more immunogenic when they develop in reticulocytes26–28. This suggests that a control strategy, aimed at restricting parasites to reticulocytes, would be of significant benefit to the host. The severe pathology and mortality often associated with high parasitaemias will therefore be reduced. However, since the infection will not be terminated, the resulting state of premunition will not prevent the acquisition of immunity. Multigene families in Plasmodium species Homologues of Py235 are likely to be found in the genome of other rodent malaria parasites. Crosshybridizing DNA fragments have been detected in the genome of P. berghei29, and antibodies against Py235 proteins crossreact with P. chabaudi and P. vinckei30. Whether a multigene family similar to the Py235 family is present in P. falciparum should be established with 29

Focus the completion of the P. falciparum genome project. However, the only proteins currently known to be involved in RBC invasion by P. falciparum and P. vivax are encoded by single-copy genes31. Three multigene families, in addition to the var family, have so far been found in the genome of P. falciparum: Pf60.1 (Refs 32,33), stevor and rif 34. The 140 members of the Pf60.1 family encode rhoptry proteins and appear to play a role in RBC invasion. These genes also share a motif described in rhoptry protein genes found in several Babesia species. The predicted protein structure of the highly polymorphic stevor (approximately 35 members) and rif (.200 members) genes, and their location close to the var genes, suggest that they encode variant surface antigens. It will be interesting to establish whether the expression of these multigene families is controlled by one of the two mechanisms uncovered so far, or whether the parasite has further surprises in store. Conclusion The clonal antigenic variation exemplified by the var multigene family exerts its influence at the level of the whole parasite population present in the host. When an effective antibody response is mounted against one particular PfEMP1 variant, parasites expressing this variant are eliminated. A few infected RBCs with different PfEMP1 proteins on their surface can then expand to dominate the parasitaemia. The clonal phenotypic variation discovered for the Py235 multigene family acts on the individual parasite. At each cycle of asexual multiplication, it provides the progeny of a single parasite with an enhanced probability of survival. It is likely that these two phenomena coexist in Plasmodium parasites, and the combined effect presents the host with a formidable opponent. We suggest that multigene families are a cornerstone in the survival of Plasmodium parasites, whose success relies on maintaining an infection for lengthy periods despite the immune response of the host, while at the same time limiting their multiplication rate so as to enhance the survival of that host. We are of the opinion that a means to unsettle the hold of the parasite will be found in the mechanisms that maintain this delicate biological equilibrium. References 1 Garnham, P.C.C. (1966) Malaria Parasites and Other Haemosporidia, Blackwell Science 2 Eyles, D.E. and Young, M.D. (1951) The duration of untreated or inadequately treated Plasmodium falciparum infections in the human host. J. Natl. Malaria Soc. 10, 327–336 3 Bruce-Chwatt, L.J. (1974) Transfusion malaria. Bull. WHO 50, 337–346 4 Brown, K.N. and Brown, I.N. (1965) Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature 208, 1286–1288 5 Handunnetti, S.M. et al. (1987) Antigenic variation of cloned Plasmodium fragile in its natural host Macaca sinica. Sequential appearance of successive variant antigenic types. J. Exp. Med. 165, 1269–1283 6 Hommel, M. et al. (1983) Surface alterations of erythrocytes in Plasmodium falciparum malaria. Antigenic variation, antigenic diversity, and the role of the spleen. J. Exp. Med. 157, 1137–1148 7 Biggs, B-A. et al. (1991) Antigenic variation in Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 88, 9171–9174 8 McLean, S.A. et al. (1982) Plasmodium chabaudi: antigenic variation during recrudescent parasitaemias in mice. Exp. Parasitol. 54, 296–302

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9 Aley, S.B. et al. (1984) Knob-positive and knob-negative Plasmodium falciparum differ in expression of a strain-specific malarial antigen on the surface of infected erythrocytes. J. Exp. Med. 160, 1585–1590 10 Leech, J.H. et al. (1984) Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum-infected erythrocytes. J. Exp. Med. 159, 1567–1575 11 Howard, R.J. et al. (1988) Two approximately 300 kilodalton Plasmodium falciparum proteins at the surface membrane of infected erythrocytes. Mol. Biochem. Parasitol. 27, 207–223 12 Baruch, D.I. et al. (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82, 77–87 13 Su, X-Z. et al. (1995) The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82, 89–100 14 Smith, J.D. et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82, 101–110 15 Kyes, S. et al. (1997) Genomic representation of var gene sequences in Plasmodium falciparum field isolates from different geographic regions. Mol. Biochem. Parasitol. 87, 235–238 16 Chen, Q. et al. (1998) Developmental selection of var gene expression in Plasmodium falciparum. Nature 394, 392–395 17 Scherf, A. et al. (1998) Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum. EMBO J. 17, 5418–5426 18 Roberts, D.J. et al. (1992) Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357, 689–692 19 Preiser, P.R. et al. (1999) A rhoptry-protein-associated mechanism of clonal phenotypic variation in rodent malaria. Nature 398, 618–622 20 Freeman, R.R. et al. (1980) Protective monoclonal antibodies recognising stage-specific merozoite antigens of a rodent malaria parasite. Nature 284, 366–368 21 Holder, A.A. and Freeman, R.R. (1981) Immunization against blood-stage rodent malaria using purified parasite antigens. Nature 294, 361–364 22 Keen, J.K. et al. (1990) Identification of the gene for a Plasmodium yoelii rhoptry protein. Multiple copies in the parasite genome. Mol. Biochem. Parasitol. 42, 241–246 23 Borre, M.B. et al. (1995) Multiple genes code for high-molecular mass rhoptry proteins of Plasmodium yoelii. Mol. Biochem. Parasitol. 70, 149–155 24 Ogun, S.A. and Holder, A.A. (1996) A high molecular mass Plasmodium yoelii rhoptry protein binds to erythrocytes. Mol. Biochem. Parasitol. 76, 321–324 25 Keen, J. et al. (1994) A gene coding for a high-molecular mass rhoptry protein of Plasmodium yoelii. Mol. Biochem. Parasitol. 65, 171–177 26 Poels, L.G. et al. (1977) Plasmodium berghei: selective release of ‘protective’ antigens. Exp. Parasitol. 42, 182–193 27 Jayawardena, A.N. et al. (1983) Enhanced expression of H-2K and H-2D antigens on reticulocytes infected with Plasmodium yoelii. Nature 302, 623–626 28 Schetters, T.P. et al. (1986) Plasmodium berghei: relative immunogenicity of infected reticulocytes and infected oxyphilic red blood cells. Exp. Parasitol. 62, 322–328 29 Owen, C.A. et al. (1999) Chromosomal organisation of a gene family encoding rhoptry proteins in Plasmodium yoelii. Mol. Biochem. Parasitol. 99, 183–192 30 Holder, A.A. and Freeman, R.R. (1984) Protective antigens of rodent and human bloodstage malaria. Philos. Trans. R. Soc. London Ser. B 307, 171–177 31 Barnwell, J.W. and Galinski, M.R. (1998) in Malaria: Parasite Biology, Pathogenesis And Protection (Sherman, I.W., ed.), pp 93–120, ASM Press 32 Carcy, B. et al. (1994) A large multigene family expressed during the erythrocytic schizogony of Plasmodium falciparum. Mol. Biochem. Parasitol. 68, 221–233 33 Bonnefoy, S. et al. (1997) Evidence for distinct prototype sequences within the Plasmodium falciparum Pf60 multigene family. Mol. Biochem. Parasitol. 87, 1–11 34 Cheng, Q. et al. (1998) stevor and rif are Plasmodium falciparum multicopy gene families which potentially encode variant antigens. Mol. Biochem. Parasitol. 97, 161–176

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