revised April 2, 2002

Malaria and the Red Cell

For thousands of years malaria swept through the ranks of humankind like a scythe in the hands of an angry god. Falciparum malaria (P. falciparum) , transmited by the female anopheles mosquito is the most deadly of the four types of of the disease (the other three being P. vivax, P. ovale, and P. malariae). Each year, malaria attacks about 400 million people, two to three million of whom succumb to the illness. Most malaria victims are children. An understanding of the origin of sickle cell disease and several other red cell disorders requires knowledge of a few of the basics about malaria and something about the process called natural selection.

Natural Selection

Human defense systems and pathogens (organisms such as bacteria that produce disease) engage in constant stuggle, with the outcome being health or illness. Each year, for instance, millions of people battle the rhinoviruses that produce the common cold. The prize for the winners is a day at school or work. The penalty for the losers often is a miserable day at home. Fortunately, the losers survive and have a chance to redeem themselves when battle resumes the following year.

Sometimes the stakes are higher, however. People and pathogens also engage in struggles where the prize is life itself. Each winter, along with the rhinovirus wars, humankind engages in battles with the influenza viruses. The influenza viruses are far more potent than are the rhinoviruses, however. Death from influenza is a well-know risk, particularly for the elderly and people whose defense systems are weakened by chronic illness.  Fortunately, the elderly and infirm now have medical science as a powerful ally that bolsters their defenses with immunizations to prevent influenza attack or drugs that suppress the influenza virus once it has struck.

These pathogen wars take place in the context of constant changes in our physical characteristics. Our genetic essence resides in a molecule called DNA which is the master blueprint to our makeup. We receive half our DNA from each parent. However, a few spontaneous alterations occur in everyone's DNA so that we are not a perfect melding of our two parents. These changes in the DNA molecule are called "mutations". The overwhelming majority of mutations are minor and inconsequential. The individual with the mutation neither suffers an illness nor dies as a result. By the same token, the mutation imparts no survival advantage over other people. These are "neutral" mutations.

Rarely, mutations are detrimental to health. If the mutation is so severe that the person dies before procreation, the muation dies with them and is not passed into the next human generation. These are "negative" mutations. A mutation that severely impairs the body's defense system against bacterial infection, for instace would fall into this category.

Even less common are mutations that give the recepient an advantage over other people. Sometimes the advantage improves the ability to survive a potentially deadly illness.  The affected individual can then pass his/her genes to the next generation more efficiently than other people because they are more likely to reach reproductive age. This increases the chance that the modified gene will survive into the first generation (that of the children) and from there move into the following generation (that of the grandchildren). This is a "positive" mutation.

Natural selection has to be considered in the context of "pre-modern" societies. Modern medicine has altered the balance of nature and often allows us to rescue people who otherwise would die of their condition. A case in point is juvenile diabetes. Untreated, the disorder often is fatal in childhood. Modern medicine allows most people with juvenile diabetes to live essentially normal lifes. Natural selection consequently is no longer an issue for most people with juvenile diabetes. The same is true for many other hereditary disorders. When we examine traits in the human population that rose to high frequency through natural selection, we are in effect peering through the looking glass at our distant past.

A common misconception is that natural selection somehow produces a desirable change: "giraffes grew long necks in order to reach leaves high in trees." This is not the way in which natural selection works, however. Natural selection does not promote or produce a change in an organism. Rather, a change occurs because of spontaneous alterations or mutations in the DNA genetic code. Changes in the genetic code can alter the physical characteristics of the organism. If the new trait gives the organism a survival or reproductive advantage over its fellows, the new trait will be represented in the second generation more frequently than it was in the first generation. This is the natural process by which  advantageous characteristics are selected.

Malaria

Schematic of Malaria Life
Cycle
Figure 1. The life cycle of the malaria parasite. From "Mosquito Bites"; http://whyfiles.org/016skeeter/malaria2.html

The plasmodium parasite that causes malaria is transmitted from mosquitos to men. The parasites spend part of their life cycle in the mosquito and part of it in the human host (Figure 1). The infective plasmodial sporozoites enter the bloodstream from the saliva of the feeding female anopheles mosquito. The Kupfer cells of the liver clear the sporozoites from the blood stream and kill many of the organisms. A fraction of the sporozoites escape destruction however, and penetrate the hepatocytes where they take up residence.

The parasites within the hepatocytes transform into a new entity called schizonts. The nuclear genetic material in the schizonts replicates to the point that the hepatocytes are totally filled with new forms called merozoites. A single schizont can produce thousands of merozoites. Erumpent hepatocytes release the merozoites into the bloodstream where they invade circulating red cells. After penetrating the red cells the merozoites assume a ring form called trophozoites. These organisms consume hemoglobin in erythrocytes and enlarge until they fill the cell completely. During their growth, the trophozoites metamorph into schizonts and produce new merozoites inside the red cells. The red cells subsequently lyse and release merozoites that can penetrate new red cells and restart the pernicious process.

Some of the trophozoites in the red cells take a different developmental pathway and form gametocytes. Gametocytes are the sexual form of the parasite and do no lyse the red cells. A mosquito taking a blood meal from a person whose red cells contain gametocytes acquires the malarial parasite. The sexual reproduction cycyle then  begins in the mosquito. The mosquito subsequently transmits the parasite when it attacks another human host.

Malaria Defenses

The complex nature of the malaria parasite life cycle in the human host presents several points at which the organism could be targeted for destruction. The sporozoites injected into the blood stream with the initial mosquito bite are attacked there by components of the immune system. These include antibodies, lymphocytes called "natural killer cells" as well as lymphocytes that attack the malarial parasites because of prior exposure to the organisms (conditioned lymphocytes).

Host immunity is crucial to survival of people infected with the malaria parasite. This is particularly true with respect to the nocuous falciparum parasite. The immune system works best when it has been primed against the invader. Children who suffer their first or second bout of malaria have not developed the immune response needed to provide adequate defense against the parasite. This explains in part the high mortality seen in children infected with P. falciparum. Vaccines are a common way of achieving host immunity prior to pathogen exposure. Polio immunization is a well-known example. Unfortunately, the malarial parasite constantly changes its immune makeup, thereby frustrating efforts to produce an effective vaccine.

The intrahepatic phase of malarial parasite growth presents another potential point at which to attack the organism. No mutation in the structure or function of hepatic cells that kills the malarial parasite or retards its growth is known.

The last point at which life cycle of the malarial parasite can be frustrated in humans is at the phase of red cell invasion and multiplication. Red cells are constantly created and destroyed as part of their life cycle. A mutation that somehow destroys both the infected red cells and the parasite could therefore eliminate the malaria parasite.  The destroyed infected cells would be replaced by new, healthy cells.

Table 1: Red Cell Defenses Against Malaria
Cell Component
Alteration
Global Distribution
Membrane
Duffy antigen null
Africa
 
Melanesian Elliptocytosis
Melanesia
Hemoglobin
Hemoglobin S
Africa, Middle East, India
 
Hemoglobin C
Africa
 
Hemoglobin E
S.E. Asia
 
ß-thalassemia
Africa, Mediterranean, India, S.E. Asia, Melanesia
 
a-thalassemia
Africa, India, S.E. Asia
Red cell enzymes
G-6-PD deficiency
Africa, Mediterranean, India, S.E. Asia

Table 1 lists some of the red cell defenses against malaria that have arisen by natural selection. The mechanisms by which some of these alterations thwart the malaria parasite are well-characterized while others are not.

At the red cell membrane, the Duffy antigen is the molecule used by the parasite P. vivax to enter the red cell. The high association of Duffy antigen null rell cells in some groups of people with sickle cell trait suggested that the Duffy antigen might provide some protection against malaria (Gelpi and King, 1976). Later investigations showed the Duffy antigen to be the receptor by which the merozoites of P. vivax enter red cells. People who lack the Duffy antigen (FY*O allele) are resistant to P. vivax (Hamblin and Di Rienzo, 2000). The Duffy null phenotype is most common in people whose ancestors derive from regions in Africa where vivax malaria is endemic.

Sickle hemoglobin provides the best example of a change in the hemoglobin molecule that impairs malaria growth and development. The initial hints of a relationship between the two came with the realization that the geographical distribution of the gene for hemoglobin S and the distribution of malaria in Africa virtually overlap. A further hint came with the observation that peoples indigenous to the highland regions of the continent did not display the high expression of the sickle hemoglobin gene like their lowland neighbors in the malaria belts. Malaria does not occur in the cooler, drier climates of the highlands in the tropical and subtropical regions of the world. Neither does the gene for sickle hemoglobin.

Sickle trait provides a survival advantage over people with normal hemoglobin in regions where malaria is endemic. Sickle cell trait provides neither absolute protection nor invulnerability to the disease. Rather, people (and particularly children) infected with P. falciparum are more likely to survive the acute illness if they have sickle cell trait. When these people with sickle cell trait procreate, both the gene for normal hemoglobin and that for sickle hemoglobin are transmitted into the next generation.
 
Schematic of Effect of the
Sickle Gene on Survival of Malaria
 Figure 2. Schematic representation of the effect of the sickle cell hemoglobin gene on survival in endemic malarial areas. People with normal hemoglobin (left of the diagram) are susceptible to death from malaria. People with sickle cell disease (right of the diagram) are susceptible to death from the complications of sickle cell disease. People with sickle cell trait, who have one gene for hemoglobin A and one gene for hemoglobin S, have a greater chance of surviving malaria and do not suffer adverse consequences from the hemoglobin S gene.

Figure 2 is a schematic of the natural selection that occurs with the gene for sickle hemoglobin in areas endemic for P. falciparum malaria. The left-hand side of the panel shows the situation in people with two genes encoding normal hemoglobin A (designated by red). These people have a significant chance of dying of acute malarial infection in childhood. In contrast, people with two genes for sickle hemoglobin (shown in green) are likely to succumb to sickle cell disease at an early age, as shown in the right-hand side of the figure.

In the center are people with sickle cell trait who possess one gene for normal hemoglobin and one gene for sickle hemoglobin. These children are more likely to survive their initial acute malarial attacks than are people with two genes for normal hemoglobin. Also, they suffer none of the morbidity and mortality of sickle cell disease. Therefore, the people with sickle cell trait are more likely to reach reproductive age and pass their genes on to the next generation (Ringelhann, et al., 1976).

The genetic selective scenario in which a heterozygote for two alleles of a gene has an advantage over either of the homozyous states is called "balanced polymorphism". A key concept to keep in mind is that the selection is for sickle cell trait. A common misstatement is that malaria selects for sickle cell disease. This is not true. A person with sickle cell disease is at an extreme survival disadvantage because of the ravages of the disease process. This means that a negative selection exists for sickle cell disease. Sickle cell trait is the genetic condition selected for in regions of endemic malaria. Sickle cell disease is a necessary consequence of the existence of the trait condition because of the genetics of reproduction.

The precise mechanism by which sickle cell trait imparts resistance to malaria is unknown. A number of factors likely are involved and contribute in varying degrees to the defense against malaria.

Red cells from people with sickle trait do not sickle to any significant degree at normal venous oxygen tension. Very low oxygen tensions will cause the cells to sickle, however. For example, extreme exercise at high altitude increases the number of sickled erythrocytes in venous blood samples from people with sickle cell trait (Martin, et al., 1989). Sickle trait red cells infected with the P. falciparum parasite deform, presumably because the parasite reduces the oxygen tension within the erythrocytes to very low levels as it carries out its metabolism. Deformation of sickle trait erythrocytes would mark these cells as abnormal and target them for destruction by phagocytes( Luzzatto, et al., 1970).

Experiments carried out in vitro with sickle trait red cells showed that under low oxygen tension, cells infected with P. falciparum parasites sickle much more readily than do uninfected cells (Roth Jr., et al., 1978). Since sickle cells are removed from the circulation and destroyed in the reticuloendothelial system, selective sickling of infected sickle trait red cells would reduce the parasite burden in people with sickle trait. These people would be more likely to survive acute malarial infections.

Other investigations suggest that malaria parasites could be damaged or killed directly in sickle trait red cells. P. falciparum parasites cultured in sickle trait red cells died when the cells were incubated at low oxygen tension (Friedman, 1978). In contrast, parasite health and growth were unimpeded in cells maintained at normal atmospheric oxygen tensions. The sickling process that occurs at low oxygen tensions was presumed to harm the parasite in some fashion. Ultrastructural studies showed extensive vacuole formation in P. falciparum parasites inhabiting sickle trait red cells that were incubated at low oxygen tension, suggesting metabolic damage to the parasites (Friedman, 1979). Prolonged states of hypoxia are not physiological, raising questions about degree to which these data can be extrapolated to human beings. However, they do suggest mechanisms by which sickle hemoglobin at the concentrations seen with sickle cell trait red cells could impair parasite proliferation.

Other investigations suggest that oxygen radical formation in sickle trait erythrocytes retards growth and even kills the P. falciparum parasite (Anastasi, 1984). Sickle trait red cells produce higher levels of the superoxide anion (O2-) and hydrogen peroxide (H2O2) than do normal erythrocytes. Each compound is toxic to a number of pathogens, including malarial parasites. Homozygous hemoglobin S red cells produce membrane associated hemin secondary to repeated formation of sickle hemoglobin polymers. This membrane-associated hemin can oxidize membrane lipids and proteins (Rank, et al., 1985). Sickle trait red cells normally produce little in the way of such products. If the infected sickle trait red cells form sickle polymer due to the low oxygen tension produced by parasite metabolism, the cells might generate enough hemin to damage the parasites (Orjih, et al., 1985).

The immune system is key to weathering attacks by P. falciparum. Maternal antibodies passed to newborns prior to birth provide some protection from malaria for the first several months of life. Thereafter, the onus is on the toddler's immune system to provide the needed defense. Epidemiological studies performed in regions with endemic malaria show that antibody titers to P. falciparum are lower in children with sickle cell trait than in children with genes only for hemoglobin A (Cornille-Brogger, et al., 1979). The investigators speculated that lower levels of immune activation might reflect a lower parasite burden in children with sickle cell trait due to clearance of the infected red cells. Analysis of people with sickle cell trait and people homozygous for hemoglobin A in the regions with endemic malaria in fact show a lower mean parasite burden in people with sickle cell trait relative to hemoglobin A homozygotes (Fleming, et al., 1979). In contrast, children with sickle cell disease have a high fatality rate, with acute malarial infections being a chief cause of death (Fleming, 1989).

Hemoglobin C is also believed to protect against malaria, although data on this point were not conclusive until recently. Hemoglobin C lacks the in vitro antimalarial activity of hemoglobin S. Some epidemiological studies found no evidence for protection against malaria in people with either homozygous or heterozygous hemoglobin C (Willcox, et al., 1983). The relatively small number of patients with hemoglobin C in these studies left the conclusions open to question, however.

The issue was finally settled in an investigation that included more than 4,000 subjects (Modiano, et al., 2001). Hemoglobin C heterozygotes had significantly fewer episodes of P. falciparum malaria than did controls with only hemoglobin A. The risk of malaria was lower still in subjects who were homozygous for hemoglobin C. Homozygous hemoglobin C produces a mild hemolytic anemia and splenomegaly. The much milder phenotype of the condition relative to homozygous hemoglobin S led the investigators to speculate that without medical intervention for malaria, hemoglobin C would replace hemoglobin S the over the next few thousand years as the dominant "antimalarial" hemoglobin in West Africa.

The thalassemias also reached levels of expression in human populations by protecting against malaria. The inbalance in globin chain production characteristic of thalassemia produces membrane oxidation by hemichromes and other molecules that generate reactive oxygen species (Grinberg, et al., 1995;Sorensen, et al., 1990). Reactive oxygen species also injure and kill malaria parasites (Clark, et al., 1989).

In vitro malaria toxicity of thalassemic red cells is most easily seen in cells containing hemoglobin H (ß-globin tetramers) (Ifediba, et al., 1985; Yathavong, et al., 1988). Hemoglobin H occurs most often in people with three-gene deletion alpha-thalassemia (Zhu, et al., 1993). The compound heterozygous condition of two-gene deletion alpha thalassemia and hemoglobin Constant Spring also produces erythrocytes that contain hemoglobin H (Derry, et al., 1988). Two gene deletion alpha thalassemia also protects the host from malaria, however. The process is difficult to demonstrate with in vitro cultures of malaria parasites. Alpha thalassemia may protect against malaria in part by altering the immunue response to parasitized red cells (Luzzi, et al., 1991) In any event, epidemiological studies show clear evidence of protection provided by two-gene deletion alpha thalassemia (Flint, et al., 1986; Modiano, et al., 1991).

One of the key reasons for the high fatality rate in P. falciparum malaria is the occurence of so-called cerebral malaria. Patients become confused, disoriented and often lapse into a terminal coma. Clumps of malaria-infested red cells adhere to the endothelium and occlude the microcirculation of the brain with deadly consequences. The P. falciparum parasite alters the characteristics of the red cell membrane, making them more "sticky". Clusters of parasitized red cells exceed the size of the capillary circulation blocking blood flow and producing cerebral hypoxia. Thalassemic erythrocytes adhere to parasitized red cells much less readily than do their normal counterparts (Carlson, et al., 1994). This alteration would lessen the chance of developing cerebral malaria.

The rise to high frequency of alleles that produce red cells deficient in glucose-6-phosphate dehydrogenase activity is one of the most dramatic examples of the selective pressure of malaria on humankind (Ruwende, et al., 1995; Tishkoff, et al., 2001). Reactive oxygen species are formed continually as erythrocytes take up oxygen from the lungs and release it to the preriperal tissues. As noted above, malaria parasites are easily damaged by these reactive oxygen species (Friedman, 1979). Glucose-6-phosphate dehydrogenase prevents oxidation of the heme group. In its absence, hemichromes and other species that generate reactive oxygen species accumulate in erythrocytes (Janney, et al., 1986). P. falciparum grow poorly in erythrocytes that are deficient in G-6-PD (Roth JR, et al., 1983). Malaria continues to battle back in this struggle, however. The advent of P. falciparum parasites that produce their own G-6-PD provides ample evidence of the continuing moves and counter-moves in the battle between man and malaria (Usanga, et al, 1985).


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