It has been more than 2,000 years since Sun Tzu advised others to “know your enemy” in his strategic treatise known as The Art of War. Older Chinese records show that people have been fighting the symptoms of malaria for twice as long, since at least 2700 BC.

Despite the extended struggle, a paper in the Dec. 13 Nature shows that scientists do not know their main malarial nemesis as well as they thought. The first such analysis of the disease-causing parasite finds three distinct gene expression patterns—only one of which was familiar from studies in the lab.

Under the microscope, the parasites all look the same. Inside the red blood cells from 43 sick children in Senegal, each one-celled invader curls into a ring shape that resembles a pair of headphones. But the gene expression analysis reveals pronounced molecular differences hidden within.

One expression pattern in the study matches the one found in lab cultures of parasite isolates from all over the world. A second cluster shows the opposite genes turned on or off. A third distinctive group correlates with more severe disease, but it is too soon to know if that version of the parasite causes worse disease or is reacting to its sicker host.

“For me, the reason to do this work is to understand disease outcomes and if there is a specific parasite biology related to that,” said first author Johanna Daily, an HMS assistant professor of medicine at Brigham and Women’s Hospital, who spends most of her time in the HSPH lab of Dyann Wirth, a co-author on the study. “These results imply a new biology, but we will have to recapitulate the genetic signatures in the lab and conduct a protein analysis to verify.”

“There are a lot of steps to prove that this is relevant to the physiology of the parasite, but this result is so striking that it does not require much subtle interpretation,” said Wirth, chair of immunology and infectious diseases at HSPH, the Richard Pearson Strong professor of infectious diseases, and co-director of the infectious disease initiative at the Broad Institute of MIT and Harvard. “For all intents and purposes, this organism [associated with more severe disease] is completely different.”

Disease Agents Fight Back

Malaria now kills one to two million children a year, mostly under 5 and in Africa, and sickens another 200 to 500 million people annually. Four species of the parasite can infect people, but one of them, Plasmodium falciparum, is the potentially fatal agent in children and the subject of most research, including this study.

In the last century or so, doctors and researchers have identified the malaria parasites, discovered that mosquitoes transmit the parasites to humans, and, in the 1950s, eradicated the disease in this and other developed countries by killing the mosquito carriers and developing preventive and curative drugs against the parasite. More recently, the disease has reemerged as a major global health threat, mostly in poor countries.

Malaria has been one of the most powerful forces shaping the human genome over the millennia, other researchers have shown. Yet, Daily noticed, human mutations and acquired immunity did not seem to explain the wide range of disease severity among children. “I wondered if there were parasite correlates of severe disease,” she said. “We know lots about the host. We know a lot of human mutations. I wanted to take a parasite perspective on the clinical question.”

Finding Functions

A series of collaborations enabled Daily to study the disease in its natural setting using the latest genomic tools and computational methods for understanding complex systems.

In 2004, Daouda Ndiaye, assistant professor of parasitology and mycology at Cheikh Anta Diop University in Senegal, took Daily to a high-transmission area in the hottest part of the country, where he and his colleagues test patient blood samples for emerging drug resistance. The next year, Daily, Ndiaye, and their colleagues went back and collected more. Of 1,200 total samples screened, the researchers collected 95 with enough parasites for genetic testing.

Next, Daily sent 43 of the highest quality specimens to co-author Elizabeth Winzeler of the Scripps Research Institute in San Diego, who had developed a microarray chip for the P. falciparum genome.

Large datasets came back showing 5,200 parasite gene-expression levels in each sample. “I stared at them for a long time,” Daily said.

Daily eventually found enthusiastic collaborators at the Broad Institute. “Johanna came to my office one day and asked, ‘What should I do with this data?’” said Jill Mesirov, director of computational biology and bioinformatics at the Broad and a co–senior author on the paper. “We did some of the standard things we do with expression data for cancer,” said Mesirov, who had never worked on infectious diseases before. “We let the data sort itself out into clusters. We found three classes of profiles.”

The first and second groups were diametric opposites, a kind of yin and yang. The third group was more of a heterogeneous hodgepodge.

A growing team at the Broad worked to identify the active biological pathways in each of the three groups. Their efforts were hampered by the unknown functions of 60 percent of the parasite genes. Eventually, they used yeast as an in silico model, comparing 1,400 yeast gene profiles measured in every conceivable circumstance with the parasite clusters.

The matches revealed a surprising metabolic pathway in cluster 1: active genes associated with respiration, involving mitochondria and oxygen. In yeast, it is a starvation response. “That made us really excited,” said yeast expert Aviv Regev, a co–senior author, core member of the Broad Institute, and assistant professor of biology at MIT. “If the parasite is respiring, that is a major new thing.” The metabolic pathway also involves a parasite organelle called a plastid, which is not found in humans and so is an appealing potential target for drugs.

Cluster 2 reflected the anaerobic sugar-burning genes observed by researchers in lab dishes, akin to the glycolytic metabolism in yeast that produces wine, among other things. Only cluster 3 was associated with any clinical features, such as elevated inflammation markers, extended illness, and higher fever. In yeast, the matching pattern resembles an environmental stress response.

The findings open several lines of research for the team. Daily and her colleagues want to evaluate more samples from other places. They need to verify the results, correlate the patterns with disease outcomes, and test a suspicion that cluster 3 might actually contain several different groups. They also want to try to recreate the new-found parasite transcription patterns in the lab dish to understand the different biological states and to see if they can hunt down any fresh drug targets.