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 notthe 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

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
Figure 1. The life cycle merozoites assume a ring form calledtrophozoites. These
of the malaria parasite. organisms consume hemoglobin in erythrocytes and enlarge
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until they fill the cell completely. During their growth, the
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trophozoites metamorph into schizonts and produce new
g/016skeeter/malaria2.ht
merozoites inside the red cells. The red cells subsequently lyse
ml
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 wellknown 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
Melanesia
Elliptocytosis
Hemoglobin
Hemoglobin S
Africa, Middle East,
India
Hemoglobin C
Africa
Hemoglobin E
S.E. Asia
-thalassemia
Africa,
Mediterranean, India,
S.E. Asia, Melanesia
Africa, India, S.E.
-thalassemia
Asia
Africa,
Red cell enzymes
G-6-PD deficiency 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 wellcharacterized 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.
Figure 2 is a schematic of the natural
selection that occurs with the gene for sickle
hemoglobin in areas endemic forP.
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
Figure 2. Schematic representation of the disease at an early age, as shown in the righteffect of the sickle cell hemoglobin gene hand side of the figure.
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.

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 (O 2-) 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 (-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-6phosphate 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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