Scientists Report Global Survey Maps Function of Thousands of Malaria Genes

By Jason Socrates Bardi

A team of researchers led by scientists at The Scripps Research Institute (TSRI) describes in an online version of the journal Science a comprehensive global profile of genes in the malaria parasite.

This profile is a valuable tool that associates the function of the few known malaria genes with the thousands that have no known function. This should improve the prospects for designing new ways to fight the deadly disease.

"We now have potential functional roles for more than half of the previously uncharacterized genes in the genome," says TSRI Assistant Professor Elizabeth Winzeler, who is in the Department of Cell Biology at TSRI and the lead author of the study. "This type of data has the potential to dramatically accelerate the process of drug and vaccine development."

TSRI Research Associate Karine Le Roch was the first author on the paper, and in addition to Winzeler and Le Roch, the team included researchers at the Genomics Institute of the Novartis Research Foundation in La Jolla, California; the Naval Medical Research Center and the Walter Reed Army Institute of Research, both in Silver Spring, Maryland; and the National Institute for Medical Research in London.

Guilt by Association

When the genome sequence of Plasmodium falciparum was published last year, scientists saw that less than 10 percent of its thousands of genes had been characterized well enough to assign functions to them. The functions of an additional 25 percent or so of the proteins encoded by the genes in the genome could be putatively identified because they were homologous—similar in sequence and probably structure and function—to known proteins from other organisms.

But the functions of an incredible 65 percent of the genes in the Plasmodium falciparum genome were completely unknown. The research by Winzeler and her colleagues is important because it connects the majority of these mystery genes with the minority that have been characterized.

"The research published is another major milestone in advancing research against the world's most important parasitic disease. In less than a year since the genome was published we have made significant breakthroughs in proteomics and now gene expression," says Navy Captain Daniel Carucci, who is the director of the Malaria Program at the Naval Medical Research Center and one of the investigators on this paper. "Importantly, these data will greatly accelerate our understanding of the malaria parasite, it's interaction with humans and will provide new avenues for more effective drugs and vaccines."

In the research, the scientists catalogued the genes expressed in the various stages of the malaria parasite's life cycle by using technology called oligonucleotide arrays or "gene chips." Gene chips, a relatively new technology, are glass or silicon wafers onto which are deposited short fragments of DNA oligonucleotides, sometimes to concentrations of hundreds of thousand per square centimeter.

When applying a sample that contains RNA to the chip, gene "messages" that are present in the sample will "hybridize" or bind to complementary oligonucleotides on the chip. By looking to see where RNA has bound, scientists know which genes are being expressed in the sample.

DNA chips have become a standard tool for genomics research in the last couple of years, and scientists can quite easily put a large number of different oligonucleotide pieces on a single chip—even all the known genes in an organism, as Winzeler and her colleagues did in this study.

Winzeler worked with researchers at the Genomics Institute of the Novartis Research Foundation to create a malaria-specific gene chip with probes specific for the entire genome of the malaria pathogen. The chip contained over 260,000 nucleotide sequences that were taken from the Plasmodium falciparum genome.

Using these custom gene chips, Winzeler and her colleagues were able to look at the expression of genes at each stage of the parasite's life cycle.

"We have about 2,300 genes that appear to be changing throughout the Plasmodium lifecycle, and we grouped them into 15 clusters based on similarity," says Winzeler.

This should accelerate the pace of research on the parasite because it categorizes the uncharacterized genes in the Plasmodium falciparum genome in functional ways.

When they looked at the identity of genes within the clusters, they found genes that had been previously studied clustered with completely uncharacterized genes. The known genes that were clustered together were often related to one another by virtue of the fact that they were often all part of the same biological process—like invading red blood cells.

Significantly, the research suggests that by extension, the uncharacterized genes that are also clustered with these known genes may be part of the same biological processes.

Vaccine and drug development relies on identifying molecules that may be vulnerable to attack by the immune system or by man-made drugs, and this research helps to establish which of Plasmodium falciparum's unknown genes may be potential targets.

Many of the current blood stage malaria vaccine targets are in a single cluster, for instance. Perhaps the uncharacterized genes in that same cluster will prove to be potential vaccine targets as well.

The Global Scourge of Malaria

One of the greatest challenges to global public health today is the control of malaria.

Malaria is a nasty and often fatal disease that can lead to kidney failure, seizures, permanent neurological damage, coma, and death. Once endemic in the southern United States and Mediterranean Europe, malaria has largely been brought under control in these areas, but there are still occasional outbreaks, usually caused when travelers import the disease from another country. In total, about 1,200 cases of malaria are diagnosed in the United States each year.

But these numbers are tiny compared to the global incidence. In many parts of the world, malaria is a major cause of death and disability. The World Health Organization (WHO) estimates that 300 million acute cases of malaria occur annually and more than 1 million people die of malaria each year. Most of these victims are children under the age of five.

According to the WHO, in areas of intense transmission, young children may have as many as six episodes of malaria each year; the disease consumes nearly half of all annual public health care expenditures; and some 45 million years of productive human life is lost annually to the disease.

Malaria is a contributing factor in global poverty. The disease severely impacts the gross domestic product of many of the poorest countries—the broadest measure of a nation's economy. The Wellcome Trust estimates that malaria costs the global economy the equivalent of more than $31 billion each year through lost productivity and health costs.

Despite a century of effort to globally control malaria, the disease remains endemic in many parts of the world. To make matters worse, drug-resistant strains of the parasite that causes malaria have evolved over the last few decades, making malaria more deadly and more expensive to treat. There is a profound need for more drugs to treat the disease and effective vaccines to prevent it.

A Difficult Parasite to Study

There are four types of "Plasmodium" parasites that cause malaria (Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, and Plasmodium falciparum), of which Plasmodium falciparum is the most deadly. For this reason, solving the Plasmodium falciparum genome was the goal of a major six-year, $17.9-million effort by an international consortium involving researchers from the United States and the United Kingdom. This goal was achieved last year.

But the genome is only the beginning. The genome tells us where the genes are, but it may not tell us what the genes do. What scientists need to know now are which genes in the Plasmodium falciparum genome are expressed at which stages in the parasite's lifecycle and what the corresponding proteins actually do. This information should help point the way towards discoveries that will help thwart the disease.

Plasmodium falciparum has distinct stages in its lifecycle in mosquitoes and humans, and it is encountered, variously, as the extracellular sporozoite (the infectious form injected by a mosquito) and merozoite (the invasive stage); as the intracellular trophozoite and schizont forms; and as the gametocyte and gamete forms that are important for reproduction.

When a mosquito bites a person with malaria, it ingests red blood cells infected with Plasmodium "gametocytes," the pathogen's sexual stage. Inside the gut of the mosquito, the male and female gametocytes mature and mate to form "zygotes." The products of the zygote meiosis, "ookinetes" migrate through the peritrophic matrix lining the mosquito stomach and form into "oocysts." The oocysts enlarge as the nucleus divides, and eventually rupture to release thousands of motile "sporozoites," which migrate to the salivary glands of the mosquito.

If the mosquito then bites another person, the sporozoites are incidentally injected from the mosquito's mouth into the person's blood. Within 30 minutes, the sporozoites travel to the person's liver, enter the liver's hepatocyte cells, and grow, multiply, and transform into "schizonts" that will release "merozoites" to infect red blood cells.

During the time when the parasites are in the liver, the newly infected person does not yet feel sick. After some time—anywhere from eight days to several months—the merozoite form of the parasites leave the liver and enter red blood cells where, as "trophozoites," they grow and multiply, eventually forming schizonts that will release new merozoites.

The infected red blood cells eventually burst, freeing merozoites to attack other red blood cells and releasing Plasmodium toxins into the blood, causing the person to feel sick. Some merozoites form gametocytes, and if at this point another mosquito bites an infected individual, it will ingest gametocytes, the tiny sexually mature form of the parasites, and after a week or more, the same mosquito can infect another person.

Scientists who study malaria would like to isolate and purify certain genes and proteins from the various stages of the Plasmodium lifecycle, but Plasmodium is an intracellular parasite that grows only in blood cells, and it is hard to obtain sufficient numbers of parasites from other stages to carry out these studies. It is also difficult to obtain pure cultures and sufficient quantities of parasites to do biochemical studies, and doing genetic experiments with this organism is almost impossible.

Plasmodium is, in short, exceedingly hard to work with—Winzeler's colleagues had to laboriously dissect parasites from the salivary glands of mosquitoes, for instance. As a result, relatively few scientists are in a position to study many key stages of its life cycle, despite its importance for world health.

The article, "Discovery of gene function by expression profiling of the malaria parasite lifecycle" was authored by Karine G. Le Roch, Yingyao Zhou, Peter L. Blair, Muni Grainger, J. Kathleen Moch, J. David Haynes, Patricia De la Vega, Anthony A. Holder, Serge Batalov, Daniel J. Carucci, and Elizabeth A. Winzeler and appears in the Science Express online version of the journal Science on July 31, 2003 and will appear in print later this year in the journal Science. See:

This work was supported by a grant from the Ellison Medical Foundation and by the Institute for Childhood and Neglected Diseases at TSRI.




TSRI investigator Elizabeth Winzeler led the research that provides a global profile of genes in the malaria parasite. Photo by BioMedical Graphics.











These red blood cells are infected with Plasmodium faciparum, stained purple with a dye that interacts with DNA. Plasmodium falciparum is the most deadly of the parasites that cause malaria.












This malaria-specific gene chip has probes specific for the entire genome of the malaria pathogen. Image courtesy of Karine Le Roch.