A mouse is born without one certain normal gene in its body and can count itself lucky. The metabolic molecule that it lacks is pyruvate kinase. That deficiency protects the animal from the ravages of malaria.
"The significance of this seeming paradox," explained molecular geneticist/biochemist Phillippe Gros at McGill University in Montreal, "is that malaria is such a huge worldwide health issue, its proportions are just phenomenal. There are 300 million to 500 million clinical cases a year and over 1 million deaths from malarial infection. So any dent we can make in understanding how the malarial parasite replicates inside its victim's blood, or any hint as to the conditions that make this replication ineffective, will be a significant advance."
In the course of bloodsucking, malarial parasites penetrate the RBC full of hemoglobin. Sickle-cell disease genes distort that molecule, making it harder for the parasite to survive. So there's a much lower death rate from malaria for individuals who carry one copy of the sickling genes. It confers a 15 percent to 20 percent benefit in regions of very high endemic malaria, as in Africa.
Gros is senior author of an article in Nature Genetics, released online Nov. 2, 2003. Its title is the flat statement: "Pyruvate kinase deficiency in mice protects against malaria."
Gros observed, "Essentially we had been working for a number of years now on two mouse strains that we have in the lab, which showed an unusual level of resistance to malarial infection. Through a series of genetic analyses, we were able to pinpoint the region on the murine genome where this malarial trait was located. We also noted that these mice had an unusual level of erythrocyte [red blood cell] precursors called reticulocytes. An erythrocyte is essentially an empty bag that has lost its nucleus. The reticulocytes are denucleated precursors of these RBCs. In summary, they define an inactivating mutation in this enzyme that has a protective role against malaria. That's the gist of our finding."
It's All In The Scarce Glucose Metabolism
"When we determined the location of the gene controlling this high-reticulocyte number," Gros continued, "it mapped exactly at the same place where this resistance to malaria mapped. Then we cloned the gene involved and discovered that it encoded the pyruvate kinase enzyme. That enzyme is essential for glucose metabolism in red blood cells. Either directly or indirectly, mice deficient in that enzyme are resistant to malaria."
That unprecedented discovery prompted Gros and his co-authors to take the next step.
"Obviously, we now have to go into humans and see if the same antimalaria mouse-deficiency effect is valid for people," he said.
To inflict malaria on their mice, the experimenters exposed the animals to a rodent-specific parasite, Plasmodium chabaudi. "We use P. chabaudi because this is our mouse model, and it replicates in mice because it's a rodent pathogen. In human in vitro testing, we would use the human Plasmodium falciparum virulent strain. There are people out there who have this infection from whom we can obtain red blood cells easily. We can put them in tissue-culture flasks in the laboratory together with the malarial parasite and see if those red cells are also protected against that infection. That is our first and immediate goal.
"Fortunately enough," Gros went on, "some small-molecule inhibitors are known for blocking this pyruvate kinase enzyme. So we will test these inhibitors to see if they can influence the replication of the human-targeting P. falciparum parasite in vitro inside red blood cells. Contacting people, getting their red cells and their consent to do this shouldn't take too long. I would say within a year or two to answer the question: Is this relevant or not? Volunteers will be able to do that in a fairly short format."
On a philosophical note, Gros tackled the question, "What evolutionary mechanism creates this contrarian relationship?"
"Malaria is unique," he pointed out, "because this process is called co-evolution. There are a very few examples of this in nature, where two like forms influence each other to the point where they actually influence their genomes. One exercises a positive pressure for retention in the population of otherwise deleterious alleles - chromosomal variants - because those bad' alleles exert a protective effect against the malarial parasite.
"This is not meant for pyruvate kinase," Gros added, "because that is brand new, just discovered. But for sickle-cell anemia and beta-thalassemia, it's actually a co-evolution of the malarial parasite with its human host. It's also implicated in the Anopheles mosquito that delivers the parasite.
Enzyme's Inner Secret Of Malaria Protection
What (absent) property of pyruvate kinase protects the mouse from malaria, and, one hopes, the human?
"That is our next area of investigation," Gros responded. "There are two possibilities: The first and most attractive one is that the malarial parasite, when it invades and replicates inside red blood cells, needs to use glucose as a source of energy because that's the only energizer around. Maybe it requires that the host's pyruvate kinase be present inside that cell to allow glucose metabolism. So inactivating this enzyme may have that protective effect because the parasite has no other energy source. If that is the case, then we can partly or completely inhibit pyruvate kinase, thus opening a window of opportunity for drug development, perhaps. In all fairness, we have no proof that is the case. At this point, it's just a hypothesis.
"To test the hypothesis that a similar inactivation of the human homologue also has an effect on malarial parasite replication, it doesn't matter where we would get those individuals as long as they carry an inactivating mutation in their pyruvate kinase gene. We would like to replicate this in another gene mutation by screening another patient in the same gene but not in the same place.
"There have been efforts to develop a malarial vaccine for 25 years, and these have been frustratingly unsuccessful," Gros observed. "The final point is that the Bill and Melinda Gates Foundation have sponsored a number of projects with the Wellcome Trust in the UK to tackle some of these basic biology problems in malaria," Gros concluded.