The Hopes and Realities of the Plasmodium falciparum Genome

Photo: Courtesy of the World Health Organization, P. Virot THE REALITY OF ETHIOPIA: Trying to survive malaria in Ethiopia, on Africa's east coast. In 1998, Ethiopia's infant mortality rate was 116 per 1,000 live births (WHO) compared to 7.2 per 1000 in the US (CDC). Sequencing a 23-megabase genome hardly sounds like a triumph--that's just twice the size of an average yeast genome and one-hundredth of the human genome. Yet, there was cause for celebration after a high-profile team of collaborators closed all but 93 gaps in the sequence of Plasmodium falciparum, the most deadly and endemic human malarial parasite. Besides infecting nearly 500 million people and killing an estimated 2 million annually, this single-celled terror presented a formidable sequencing task: Its genome's dense A-T content made it difficult to clone. Now, with the genome in hand,1 the sequences for its mosquito2 and human hosts available, a rodent model3 in the ready, and a slew of proteomic data4 already compiled, scientists hope to tease out enough information to defeat a disease rivaled only by AIDS and tuberculosis as the world's most important health problem. But, scientists are tempering their giddiness. "We don't want to glorify the malaria genome too much," says researcher Jane Carlton of The Institute for Genomic Research (TIGR) in Rockville, Md., recognizing the challenges in developing drugs for which there is great need, but no lucrative market. Still, ambivalence rules: Synchronized press conferences and special journal issues heralded the sequence's release. Moreover, optimism abounds that new therapies and vaccines will ensue from information that has been trickling out since sequencing began. At least one treatment, an existing antibiotic, is directly attributable to the sequence and has entered clinical trials. Now back to the facts: Of 5,268 genes predicted by annotation software, scientists can find homologs from known systems in only 40%. That leaves a lion's share of uncharted territory in an organism that has looked downright foreign from the get-go. Researchers say that better partnerships are needed and hope that the camaraderie that sequenced malaria and its primary mosquito host, Anopheles gambiae, forecasts future successes. The collaboration "was refreshing to those of us who lived through the conflict of the human genome era," says Robert Holt, a former researcher with Rockville, Md.-based Celera, who unveiled the Anopheles genome in Washington, DC, on Oct. 3. The collaborating groups at the Wellcome Trust Sanger Institute, UK, TIGR, Stanford University, and others working to sequence P. falciparum ran into problems even before they started. Says Malcolm Gardner, who joined TIGR when the project began in 1996: "There were many people who thought that because the genome was so A-T rich and because the DNA was so difficult to clone that we would never be able to sequence the genome." The reasons for the 80% A-T content and why it makes cloning a challenge are largely speculative. Dyann Wirth, who heads the Harvard University Malaria Initiative, says, "It may be that they're recombinogenic. Maybe they form secondary structure; we don't know the mechanism." One hypothesis she's investigating is that repeating structures in A-T rich regions may contribute to the antigenic diversity that helps malaria escape destruction by human and mosquito immune systems. "A consequence of this A-T richness may be the ability of the parasite to bury certain segments of proteins quite extensively through a relatively simple replication slippage and repair mechanism," Wirth says. The sequencing consortium proved its mettle in 1998 with the release of chromosome 2.5 Shotgun libraries were constructed from tiny fragments 1-2 kilobases long. To get through long stretches of A or T, researchers employed creative strategies such as transposon walking, where known sequences are inserted randomly to break up monotonous stretches and serve as distinct markers from which to sequence. Image: Courtesy of Drew Berry, The Walter and Eliza Hall Institute MALARIAL PROPAGATION: An infected red blood cell ruptures open, releasing another generation of malaria merozoites. SEQUENCES IN HAND--WORTH A CURE IN THE BUSH? The genome has also turned up new information about malaria's strange little organelle, the apicoplast. Discovered in the last decade and believed to be a product of endosymbiosis, this chloroplast-like structure has its own 35-kilobase genome, and roughly 10% of malaria's nuclear-encoded proteins appear to target this organelle. Many see it as fertile ground for drug targets. In 1999, a group from Justus-Liebig University in Germany mined existing P. falciparum data to find a pathway for isoprenoid synthesis in the apicoplast, similar to one found in blue algae and bacteria. Fosmidomycin, an antibiotic targeted to this pathway, suppressed P. falciparum in vitro, as well as a rodent strain of malaria in live mice.6 Initial results in humans show that the parasite clears quickly, says Hassan Jomaa, CEO, Jomaa Pharmaka, Germany. And, he notes, the cure persists when fosmidomycin is used with other drugs. Many say, however, that only a vaccine can eradicate the disease from endemic zones. Daniel Carucci, malaria program director, Naval Medical Research Center, Silver Spring, Md., points to the var and rif genes as being particularly interesting. Scientists knew that this family of proteins is involved in immune evasion when malaria infects red blood cells. Expression data published with the genome show that these proteins also appear in the sporozoite stage, the form injected by mosquitoes that go straight to the liver.4 Carucci says he and others are working to develop vaccines that target multiple stages of the parasite's life cycle. Somewhat less promising data came from the concurrent release of the genome for Plasmodium yoelii yoelii,3 a rodent malarial parasite used often as a model to study P. falciparum. Comparative analyses showed that subtelomeric regions, involved in antigenic variation, were very different in P. falciparum and P. yoelii. "In yoelii," says Carlton, TIGR's project leader. "There's actually a different subset [of genes] involved in antigenic variation. ... That might make the model system not so useful for the study of falciparum." She adds, however, that significant similarities exist between P. yoelii and Plasmodium vivax, a severe but less fatal form of human malaria, which TIGR is now sequencing. GENOMES, GENOMES EVERYWHERE Researchers are looking at other wild plasmodium species and strains, hoping that information gleaned from them will fill the data void regarding drug resistance and immune system evasion. TIGR and Sanger, for example, are eyeing Plasmodium berghei, Plasmodium chabaudi, Plasmodium knowlesi, and the distantly related Toxoplasma gondii, among others. The P. falciparum genome was sequenced from a parasite taken from a Dutch schoolgirl who contracted malaria in 1979. The clone, 3D7, has spent nearly 20 years in a laboratory. Significant differences are expected in field strains. Genetic variation poked its ugly proboscis into the sequencing of the Anopheles genome. Without an inbred mosquito strain to start with, groups at Celera, Genoscope--the French sequencing institute--and the European Molecular Biology Laboratory had some trouble resolving DNA from hundreds of mosquitoes into one consensus sequence. "That was a bit confounding for the assembly algorithms," says Holt in an interview, "and it resulted in us having about 7-7.5% of the genome being artificially duplicated." Populations in the wild will vary even more, but Holt says he doesn't believe it will affect management strategies. "Insecticides are targeted against proteins that are very well conserved within a species, and even across species." Regardless of what information the genome gives up, it is "not going to produce these drugs, and it's certainly not going to make sure--if these tools are developed--that they actually get applied where they're needed," says Gardner. Carlton points out a decades-old problem: "Malaria studies and projects have always sort of been the poor cousins. They haven't received much money because there was so little money in the pot." The P. falciparum genome was sequenced for about $30 million (US), Anopheles for less than half that--a piddling sum in the billion-dollar genome game. Yet, getting treatments for those who need them most will certainly cost more. At least one man believes it's feasible to immunize Africa for $1 billion a year. Stephen Hoffman, who has been studying P. falciparum since before the sequencing project's inception, recently left Celera to found Sanaria (for 'clean air'; malaria translates as 'bad air'). A few years ago, some brave volunteers allowed themselves to be bitten by thousands of irradiated, infectious mosquitoes. The initial immune response protected them--some for up to 42 weeks.7 After looking at the data, he recalls, "I sat back and said, 'Wow! What if I'd worked on this for the past 15 years instead of genomics.'" Hoffman wants to create a vaccine, based on attenuated malaria, and market it to travelers at $200 a person. "So knocking that down to $40 and you multiply 40 times 25 million births in a year--that's a billion dollars. I don't think it's unreasonable." Brendan A. Maher can be contacted at bmaher@the-scientist.com. References 1. M.J. Gardner et al., "Genome sequence of the human malaria parasite Plasmodium falciparum," Nature, 419:498-511, Oct. 3, 2002. 2. R.A. Holt et al., "The genome sequence of the malaria mosquito Anopheles gambiae," Science, 298:129-49, Oct. 4, 2002. 3. J.M. Carlton et al., "Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii," Nature, 419:512-9, Oct. 3, 2002. 4. L. Florens, et al., "A proteomic view of the Plasmodium falciparum life cycle," Nature, 419:520-6, Oct. 3, 2002. 5. M.J. Gardner et al., "Chromosome 2 sequence of the human malarial parasite Plasmodium falciparum," Science, 282:1126-1132, 1998. 6. H. Jomaa et al., "Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs," Science, 285:1573-76, 1999. 7. S.L. Hoffman, "Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites," The Journal of Infectious Diseases, 185:1155-64, 2002.

The Hopes and Realities of the Plasmodium falciparum Genome

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