The Scripps Research Institute

Scientists Identify Thousands of Proteins Associated with the Deadliest Form of Malaria

Two scientists at The Scripps Research Institute (TSRI) led a collaborative effort involving 18 researchers at half a dozen laboratories in the United States and Great Britain to determine the "proteome" of the most deadly form of the malaria pathogen— Plasmodium falciparum.

This study, in the current issue of the journal Nature, accompanies an article detailing the completion of a major six-year $17.9-million genome-sequencing effort involving 185 researchers from the United Kingdom, the United States, and Australia that sequenced the entire Plasmodium falciparum genome.

"This is the first instance that I know of where these proteomics studies have gone along side-by-side with the genome sequencing project," says TSRI Cell Biology Professor John Yates, who was the lead scientist involved in the proteomics effort, which identified the proteins in the single-celled Plasmodium that cause malaria.

These efforts will pay huge dividends in global healthcare if even a few of the newly identified proteins lead to the development of new malaria vaccines—and Yates and his colleagues found a total of more than 2,400 proteins.

"We don't exactly know the function of well over half of the proteins identified—we just know that they are there," says Laurence Florens, who is a research associate at TSRI and the lead author of the study.

Malaria is a nasty and often fatal disease, which may lead to kidney failure, seizures, permanent neurological damage, coma, and death. There are four types of Plasmodium parasites that cause the disease, of which falciparum is the most deadly.

Knowing which proteins are expressed by Plasmodium falciparum should help scientists understand how the pathogen causes malaria and, with luck, how to thwart it. That was the goal of the proteomics approach taken by Florens and Yates.

Where "genomics" maps the DNA sequence and genes in an organism like Plasmodium falciparum, "proteomics" adds the topographical information to that map by identifying which genes are actually expressed as proteins in the Plasmodium falciparum cells.

More importantly, Florens and Yates also sought to identify which proteins are expressed at which stages of the organism's lifecycle. This was no small task. Plasmodium falciparum has at least ten distinct stages in its lifecycle, and there is no way of telling which are expressed at each distinct stage of the pathogen's lifecycle simply by looking at the genes.

But Florens and Yates were able to figure out which proteins were expressed during four different stages (sporozoites, merozoites, trophozoites, and gametocytes) and, thus, which might make good vaccine targets.

Mass Spectrometry and Malaria

The process was basically to take samples of a single isolate of Plasmodium falciparum and grow three of the four different stages in blood in a way that allowed samples to be purified. The fourth stage, the sporozoites, had to be hand-dissected from mosquito salivary glands.

In purifying the samples, Yates and Florens first separated the soluble proteins from the membrane-bound proteins, then digested them (chopped into smaller "peptide" pieces with enzymes), and resolved them using liquid chromatography combined with tandem mass spectrometry.

The instrument detects the pieces and uses sophisticated software that Yates and his colleagues developed previously to search a database of predicted genes to reconstruct most of the proteins in the sample. This technique was particularly useful in this context because it allowed a very large background "noise" of mosquito and human proteins to be subtracted out. The peptides that come from the Plasmodium can be distinguished from those that come from the mosquito or the human.

Furthermore, using the technique, Florens and Yates were able to show not only which genes were expressed in each stage of the Plasmodium falciparum life cycle, but which proteins were membrane-associated, and which were inside the cell—important pieces of information for vaccine design.

One unexpected finding was that a lot of the proteins that were expressed in particular stages "co-localized" in chromosomal gene clusters possibly under the control of common regulatory elements.

Promoters are regions of DNA in front of a gene that "turn on" that gene like a switch and cause it to be expressed as protein. Normally, any given gene will have its own promoter. But Florens and Yates found many different clusters of genes that become expressed together and might be under the control of a single promoter. Florens and Yates believe that this is one of the ways that the pathogen is able to thrive in two different organisms (mosquitoes and humans).

"The switching between stages is something that happens very fast," says Florens, "and [the pathogen] needs a mechanism to express many genes quickly."

The article, "A proteomic view of the Plasmodium falciparum life cycle" was authored by Laurence Florens, Michael P. Washburn, J. Dale Raine, Robert M. Anthony, Munira Grainger, J. David Haynes, J. Kathleen Moch, Nemone Muster, John B. Sacci, David L. Tabb, Adam A. Witney, Dirk Wolters, Yimin Wu, Malcolm J. Gardner, Anthony A. Holder, Robert E. Sinden, John R. Yates, and Daniel J. Carucci and appears in the October 3, 2002 issue of the journal Nature.

This work was supported by the Office of Naval Research, the U.S. Army Medical Research and Material Command, and the National Institutes of Health.

The authors of the paper are affiliated with the following institutions: The Scripps Research Institute; Syngenta Research & Technology; the Imperial College of Science, Technology & Medicine; the Naval Medical Research Center; the National Institute for Medical Research; the American Type Culture Collection; and The Institute for Genomic Research.

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