These findings, published in the August issue of Nature Methods, “should substantially speed up research efforts to bring malaria under control,” says Dr. David Fidock, senior author of the paper and an associate professor of microbiology & immunology at Einstein.
Malaria is caused by a single-celled parasite, Plasmodium, which is transmitted through the bite of the Anopheles mosquito. The disease kills an estimated 1.2 million people every year.
The Einstein scientists focused on the most deadly Plasmodium strain—P. falciparum—which is proving increasingly resistant to treatment. Their research has led to the first efficient technique for inserting any gene of interest into the P. falciparum genome to gain biological information that could lead to more effective treatments.“This opens up a whole new window into the genetic manipulation of this lethal parasite,” says Dr. William Jacobs, Jr., who is a Howard Hughes investigator and professor of molecular genetics and microbiology & immunology at Einstein and a major author of the Nature Methods paper. “Malaria researchers finally have an efficient way to shuffle genes into P. falciparum, which should lead to valuable information about the parasite’s virulence, how it’s transmitted from mosquito to humans and how it develops resistance to antimalarial drugs.”
The research effort was conducted primarily by Louis Nkrumah, an MD/PhD student at Einstein. Central to this effort was a bacterial phage (virus) that Dr. Jacobs isolated from soil in his backyard in the Bronx and dubbed the “Bronx Bomber.” It infects Mycobacterium smegmatis, a bacterial species closely related to Mycobacterium tuberculosis, which causes tuberculosis. Dr. Jacobs has used the Bronx Bomber to gain important knowledge about tuberculosis bacteria.
Bacterial phages are adept at integrating their genes into the DNA of their bacterial hosts. Phages typically rely on host proteins for gene integration. But the Bronx Bomber does the job all by itself, using one of its own enzymes. Dr. Jacobs realized that this unique property of his tuberculosis virus could be used for “breaking into” other microbial species—in particular P. falciparum, which has proven notoriously resistant to attempts to develop efficient methods of genetic manipulation.
Einstein researchers wanted to see if they could use the Bronx Bomber’s enzyme to introduce any gene of interest into P. falciparum. So they fashioned a plasmid (circular loop of DNA) containing several elements: the gene for the Bronx Bomber enzyme; a section of DNA that would bind the plasmid to a complementary section of DNA inside P. falciparum; and a marker gene fused with a green fluorescent protein that would light up if the marker gene became functional.
The Bronx Bomber transfection technique proved remarkably successful. “Using standard methods of gene manipulation, we wouldn’t know for four or five months whether we had successfully achieved a stable recombinant organism—and many experiments failed,” says Dr. Fidock. “But with this technique, recombinant parasites are typically produced within two to four weeks, and their identification and characterization has become far more streamlined. This method should significantly benefit genetic strategies for exploring the biology of this parasite.”
The other Einstein researchers involved in this study were Rebecca A. Muhle and Pedro A. Moura. Their collaborators, from the University of Pittsburgh, were Pallavi Ghosh and Graham F. Hatfull.
Karen Gardner | EurekAlert!
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