After more than a decade-and-a-half-long search, researchers have identified the gene that makes the most deadly malaria parasite resistant to chloroquine, the former mainstay, low-cost antimalarial drug. A single gene on chromosome 7 of Plasmodium falciparum holds the key, according to a just-released study headed by scientists at the National Institute of Allergy and Infectious Diseases (NIAID). Tiny mutations in this gene, known as pfcrt, associate completely with chloroquine resistance in parasite lines from affected areas in Asia, Africa and South America.
"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 accumulating.
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 research.
"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.
Reference: DA Fidock et al. Mutations in the Plasmodium falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Molecular Cell vol. 6(4), October 20, 2000.
Press releases, fact sheets and other NIAID-related materials are available on the NIAID Web site at http://www.niaid.nih.gov.
Journal
Molecular Cell