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: http://www.sciencemag.org/sciencexpress/recent.shtml.
This work was supported by a grant from the Ellison Medical
Foundation and by the Institute for Childhood and Neglected
Diseases at TSRI.
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