"To date all evidence supports the notion, and nothing yet contradicts it,
that this could be the only gene necessary to confer chloroquine
resistance," says Thomas E. Wellems, M.D., Ph.D., chief of the malaria
genetics section at NIAID.
Dr. Wellems led the effort along with David A. Fidock, Ph.D., former
visiting scientist in his lab who recently joined the faculty of the Albert
Einstein College of Medicine, and Paul D. Roepe, Ph.D., of Georgetown
University. Their report appears in the October 20 issue of Molecular Cell.
"This important advance," says NIAID Director Anthony S. Fauci, M.D., of the
new study, "will not only help researchers further explore the mechanisms of
P. falciparum chloroquine resistance and ways to overcome that, it also will
assist scientists surveying population groups in malarious regions for
strains of chloroquine-resistant P. falciparum."
In fact, such field research, already under way but not yet published, is
providing confirmation of the new finding.
Each year, malaria strikes some 300 to 500 million people, and more than 1
million people die. Most of those affected are young children in Africa.
In the past few decades, the increasing spread of chloroquine-resistant
malaria across several continents has often left doctors with no choice but
to prescribe other drugs that can be more toxic and more expensive.
The idea that chloroquine resistance could be determined by one rather than
multiple genes represents a dramatic change in dogma, notes Dr. Fidock. For
years, he explains, researchers have believed that chloroquine resistance
must involve multiple genes because it arose independently in the Old and
New Worlds and was an exceedingly rare event: the first cases of
chloroquine-resistant malaria turned up in Asia and South America 10 years
after the drug was first introduced in the 1940s. It took another two
decades for chloroquine resistance to first appear in East Africa, where it
soon spread rapidly.
The new study found that all chloroquine-resistant strains from Asia and
Africa have one of two related pfcrt variants that differ from the
chloroquine-sensitive gene by seven or eight tiny mutations, or single
"letter" changes, in the DNA. In South America, chloroquine resistance
associates with other pfcrt variants having multiple mutations, which
supports the independent genesis of chloroquine resistance in the New World.
Importantly, however, all chloroquine-resistant pfcrt variants from the
three regions include two specific mutations in pfcrt, known as K76T and
A220S, accompanied by up to six other single letter changes.
The NIAID group developed the first methods to genetically manipulate P.
falciparum and has been leading the research effort to find the genetic
basis of chloroquine resistance since the mid-1980s. In 1991 they reported
they had narrowed down their hunt to some 100 genes on chromosome 7 of P.
falciparum. The group then began searching this stretch of DNA for the
specific gene or genes involved. In 1997 they reported in the journal Cell that one gene, cg2, appeared to be
a candidate. But subsequent attempts to prove this hypothesis by
modifying this gene and another less promising one, cg1, in
chloroquine-resistant parasites in an effort to reverse drug resistance
did not yield the hoped-for results.
"When we disproved our initial hypothesis that cg2 is necessary for
resistance," says Dr. Wellems, "that led to a lot of sleepless nights." But
despite this disappointing finding, they remained confident that the genetic
determinant of chloroquine resistance had to reside in that same stretch of
DNA they had been examining, based on other data that Dr. Wellems' lab were
A new approach helped solve their quandary. When looking for the cg2 gene,
they used a computer tool that only picked out protein-coding regions that
were more than 100 amino acids long. Subsequently, they developed methods
to spot smaller protein-coding regions. This enabled them to identify the
presence on chromosome 7 of a highly interrupted gene that is, a gene
whose protein-coding segments were broken up in small clusters along the
chromosome. These clusters, when assembled together, gave the complete protein involved
in chloroquine resistance.
Subsequently, they for the first time converted sensitive parasites to
resistant ones by introducing a mutant pfcrt gene. Currently, the
researchers are conducting more definitive gene modification experiments
along these same lines.
Although they are confident they have identified the key player in
chloroquine resistance, Drs. Wellems, Fidock and Roepe continue to
collaborate on unanswered questions regarding pfcrt's precise role and the
function of the protein coded for by this gene. Such questions include,
What role does the gene play in resistance to other antimalarial drugs? And
what is the mechanism by which the protein confers chloroquine resistance?
It is this latter question that Dr. Roepe focuses on. It is known that the
protein coded for by pfcrt sits in the membrane of the digestive compartment
of the parasite. It is also known that chloroquine acts by binding to the
free heme of red blood cells. In the current paper, Dr. Roepe suggests that
the pfcrt mutations increase the acidity of the digestive compartment,
making more of the heme insoluble and therefore unable to form complexes
with chloroquine. But this does not explain how certain modified forms of
chloroquine can be effective against chloroquine-sensitive as well as
chloroquine-resistant parasites. So a second model proposes that mutations
in the pfcrt protein may directly or indirectly change a structurally
specific drug interaction affecting chloroquine movement across the membrane
of the digestive compartment. Both areas are the subject of intense
"We are starting to understand why modifications of chloroquine could work
against the parasite," says Dr. Wellems. "These studies also raise the
possibility that with stronger guidelines for chloroquine use the
institution of public health measures in a more focused way the drug,
perhaps in combination with so-called reversal agents, could have a new
lease on life."
NIAID is a component of the National Institutes of Health (NIH). NIAID
supports basic and applied research to prevent, diagnose, and treat
infectious and immune-mediated illnesses, including HIV/AIDS and other
sexually transmitted diseases, tuberculosis, malaria, autoimmune disorders,
asthma and allergies.
Press releases, fact sheets and other NIAID-related materials are available
on the NIAID Web site at http://www.niaid.nih.gov.