The Malaria Genome Projects

The following sections are included:

• Dedication

• Preface

• Contents

Today there are approximately 3.3 billion people — ~50% of all the people on this planet — who are at risk of developing malaria each year, with at least 500 million cases, and nearly a million deaths annually [299]. This amounts, on average, to one person dying from malaria every 30 seconds…

DNA is the stuff of which genes are made. DNA was discovered more than a century ago by an obscure Swiss physician, Friedrich Miescher (1844–1895) [162]. Shortly before completing his doctoral dissertation (1868), Miescher elected not to follow in his father's footsteps as a hospital physician and teacher of pathologic anatomy and instead pursued a career in physiological research. In part, it is believed, this was due to his partial deafness, the result of an earlier attack of typhus that certainly would have limited his abilities to use the stethoscope and perform percussion and auscultation [392]. He joined the laboratory of Felix Hoppe-Seyler (1825–1895) at the University of Tübingen, Germany, with the intention of studying what enables the cell to live and how cells are fashioned into tissues. Hoppe-Seyler, whose interests were in the chemistry of blood, was one of the first to crystallize hemoglobin and describe the interaction of oxygen with hemoglobin in the red blood cell. He was now turning his attention to a white blood cell, the lymphocyte, present in pus as well as blood. Hoppe-Seyler thought an understanding of the chemistry of the lymphocyte might lead to a better view of why pus was formed during infections. The collaboration was ideal: Miescher wanted to analyze the chemical composition of the cell and Hoppe-Seyler had the "perfect" cell for Miescher to analyze, the lymphocyte. Today a study of pus cells would be impractical inasmuch as infections are rare, but in 1869 when Miescher began his studies there were no antibiotics and antiseptic methods during surgery were non-existent, so human pus was readily available from the surgical wards of many hospitals [392]. In addition, he could take advantage of the newly invented clean, sterile, absorbent cotton used for dressing pusfilled wounds [162]. Hoppe-Seyler encouraged young Miescher; however, he also cautioned that there was no method to study pus chemistry! Miescher learned by trial and error and after many failures finally succeeded when he extracted pus-laden bandages with a weakly alkaline solution; a highly viscous, snot-like material that was impossible to handle as it would not dissolve in water, acetic acid or sodium chloride was obtained. (In hindsight we now know that this occurred because the solution he used extracted high molecular weight DNA.) He asked himself, from where in the cell did this material come? Was it the nucleus, the membrane, the cytoplasm? Examining the pus cells under the microscope he observed that the alkaline solution caused the nucleus in the pus cell to swell and break open. This suggested that the nucleus was the source of this material and hence he named the substance "nuclein." Using element analysis, one of the few chemical methods available at the time, he found the new substance to contain 14% nitrogen, 3% phosphorous, and 2% sulfur. This ratio of nitrogen to phosphorous was unique. Further, finding it to be resistant to digestion by pepsin (found in the stomach), he concluded the material could not be a protein. By August of 1869 Miescher reported that the same material was found not only in pus cells but also in other cells with nuclei such as yeast, kidney, liver and chicken and duck red blood cells. Miescher did not understand the importance of nuclein as the carrier of inheritance [392]. Indeed, he considered nuclein to be a storehouse of phosphorous for the cell and to the end of his life he rejected the idea that nuclein might have something to do with heredity…

Cells, the stuff of which life is made, were first seen 400 years ago by Antony van Leeuwenhoek (1632–1723), a Dutch linen merchant who had a hobby of grinding magnifying lenses. Using a crude microscope (with a magnification of 300 times), Leeuwenhoek was the first person to see sperm, frog and fish red blood cells, bacteria, microscopic roundworms and rotifers, the green alga Spirogyra and a variety of "animalcules" (today these are called protozoa). It was Robert Hooke (1635–1703), however, not Leeuwenhoek, who coined the term "cell" after observing a very thin slice of cork under a microscope and finding "little boxes" resembling a monk's cell. All the processes of life occur within cells and, although the "cells" Hooke observed, described and illustrated were not alive, the name stuck [597]…

In 1998, a time before the genomic sequence of P. falciparum had been completed and published, Su and Wellems stated: "Progress has been made in the genetics and genomics of malaria parasites. Numerous genes have been cloned and millions of base pairs of the parasite deoxyribonucleic acid (DNA) have been deposited in public databases; genetic maps of the P. falciparum 14 chromosomes and hundreds of linkage markers have been reported; yet despite these advances, we still know very little about the function of most genes!" [655]…

A malaria-infected red blood cell consists of several compartments much like those seen with nesting wooden Russian dolls where each doll, smaller than the previous, fits inside another. In the malaria-infected red cell, the larger compartments are separated from each other by a series of membranes. The outermost is the red cell membrane, beneath this is the parasite's parasitophorous vacuolar membrane (PVM), inner to this is the parasite plasma membrane, and finally there are the smaller membrane-lined compartments of the various organelles including the large food (digestive) vacuole, the single mitochondrion bounded by a double membrane, the vestigial plastid, the apicoplast, bounded by four membranes, and a complex of sheets of membranes studded with ribosomes, the endoplasmic reticulum. Nutrients as well as vitamins and inorganic ions necessary for growth and reproduction of the malaria parasite must be obtained from the host and, for this to occur, these critical substances have to pass across some of these membranes; in addition, for effective treatment, antimalarial drugs must also pass into the parasite across these membrane-lined compartments to hit their intended target (see Chapter 14). Conversely, the waste products of parasite metabolism must move from their place of origin outward across membranes and into the bloodstream, lest these accumulate to toxic levels…

Unlike all the other cells in our body, a red blood cell lacks a nucleus and any other organelles. It has been described as a "simple sack" containing a syrup-like solution of hemoglobin, specialized for carrying oxygen to and carbon dioxide from the tissues. Thanks to the durability of its membrane skeleton, it is able to make a 1,000-mile journey a million or more times through our bloodstream without repair. The red cell's flexible skeleton, composed of a two-dimensional meshwork of the protein spectrin, is connected to the overlying plasma membrane by a series of vertical "struts" that includes the proteins ankyrin, band 3 protein, rhesus factor antigen and glycophorin but, as P. falciparum grows and enlarges, the red cell's ability to deform itself, thereby enabling it to squeeze through the smallest confines of the capillaries without tearing itself apart, is so altered that it becomes both rigid and adhesive. Adhesiveness of the P. falciparum-infected red cells results in sequestration of these red cells in the smaller blood vessels leading to life-threatening complications; if the sequestered infected cells block the vessels in the brain it can lead to coma and death (cerebral malaria) and when blockage occurs in the placenta low birth weight and death of the offspring may occur (placental malaria). The increased "stickiness" of the P. falciparum-infected red cell coincides with its loss of biconcave shape and the appearance of pimple-like distortions, called knobs, on its outer surface. With an electron microscope, a knob can be seen to consist of an elevated plasma membrane beneath which there is an electron-dense plaque. Knobs contain the sticky "glue" responsible for sequestration, which in turn prevents clearance of the P. falciparum-infected cells by the spleen…

Malaria parasites need to live within a cell. This being so, getting inside is a must. As such, the molecular mechanisms of invasion may be the Achilles' heel to be exploited for the development of new therapies. Despite half a century of "invasion research," however, a practical and effective means for interrupting the entry process into red blood cells has not been achieved. Nevertheless, the hope for novel interventions to prevent the parasites from getting inside remains and the Plasmodium genome sequences may aid in that regard…

One spring day in 1956, William Trager removed his jacket, rolled up the sleeves of his white shirt, removed his tie, sat down in front of the microscope, and turned on the microscope lamp. From a duckling previously infected with P. lophurae, he took a sample of blood by pricking the leg vein, and carefully placed the drop of blood on a microscope slide that was held in place on a warm stage of the microscope. He rotated the objective lenses over the red liquid, removed his wire-rimmed glasses, and rotated the fine adjustment wheel to bring the specimen into focus. What he saw was thrilling. It reminded him of what it must have been like for Alphonse Laveran 75 years earlier when he discovered that malaria parasites were animalcules and not bacteria. Trager described the infected red cell becoming rounded; the pigment clustered into the residual body and within a few minutes or less the merozoites were ejected with a kind of seething motion, which seemed to scatter them. The red cell did not explode; instead merozoite release was rapid and they exited not as clusters, but spread out [682]…

Of all the species of human malaria, the deadly P. falciparum is often regarded as the most important: it is the focus of most laboratory and field research, receives the greatest amount of funding, and its control is the centerpiece of public health measures. Yet, the most geographically widespread of the human malarias is not P. falciparum, but P. vivax. Outside Africa, vivax is the dominant species with 80–250 million cases out of the annual ~515 million malaria cases. Plasmodium vivax is usually found outside tropical areas and was present throughout temperate North America and Europe until the introduction of dichlorodiphenyltrichloroethane (DDT). Despite this burden of disease, vivax malaria is oftentimes left in the shadows of research [242]. The reason for this neglect is the widely held misconception that P. vivax is relatively infrequent, benign, and easily treatable. Further, studies of the biology of P. vivax have been hampered by a lack of a continuous cultivation system for any of its life stages and the absence of a readily available and economic primate model. However, with the advances in genome sequencing, it is hoped that some of these impediments will be overcome…

In an article on 10 June 2010, Time magazine described a village in Uganda: "Lake Kwania is more of a giant swamp: shallow, full of crocodiles and choked with lily, papyrus and hyacinth. The malaria parasite loves it here. Kwania's creeks…are perfect for a deadly…mosquito, Anopheles funestus, which feeds almost exclusively on humans, with an appetite to shame a vampire. The nearby town of Apac is packed with a living blood bank of people. The average funestus bites humans 190 times a night. And, the average resident is bitten tens of thousands of times a year, including 1,586 bites — four a day — that carry malaria" [515] (http://www.time.com/time/specials/packages/article/0,28804,1995199_1995197,00.html?artId=1995199?contType=article?chn=specials)…

For the detection of genetic changes that allow malaria parasites to become drug resistant and to understand the mechanisms of immune evasion, it is essential to know their evolutionary history. Because malaria parasites lack bones or other semi-permanent structures they have left no traces in the fossil record. Without evidence of fossilized malaria parasites is it possible to create a "time machine" that would be able to reveal past encounters of the various kinds of Plasmodium with our ancestors? The short answer is: Yes. It is possible to trace the evolutionary history of human malarias using a "time machine" known as the molecular clock. It has no hands for hours, minutes or seconds, and it does not tell the time of day; nevertheless it is a timepiece able to put a series of evolutionary changes by Plasmodium into a chronological order. The remarkable feature of the molecular clock is its ability to run backwards and, in so doing, it is able to trace the history of Plasmodium and allow a family tree to be drawn…

During World War II as American and Allied forces engaged in battles in North Africa, Asia and the Pacific, the troop losses due to malaria were sometimes as great as those due to bullets and bombs. Further, with the fall of Java (today Indonesia) to the Japanese, the sources of quinine, the only effective antimalarial, became unavailable and so the Allies began to use Atabrine (also named quinacrine or mepacrine). Atabrine was a marginally effective antimalarial that turned the skin a bright yellow, caused gastroenteritis and, most disturbingly, occasionally caused temporary insanity. Atabrine was hardly the ideal antimalarial but there were no other drugs available and it did prevent death among the troops…

To understand a disease, the illness must be described objectively in a reproducible way. Since the time of Laveran it has been recognized that an objective description of the disease malaria means consistently finding the causative agent, Plasmodium. It is estimated that presently worldwide there are millions of cases of malaria. With massive increases in international travel, malaria imported from these regions can pose a significant and ever-increasing problem for those living in developed countries. Indeed, it is reported that ~30,000 travelers from industrialized countries contract malaria annually and, despite treatment, between 1% and 4% of those who acquire P. falciparum will die. Accurate and practical diagnostic tests are critical for control and treatment, and it is estimated that a sensitive and specific tool for diagnosis (requiring minimal infrastructure) could potentially avert over 100,000 malaria-related deaths and ~400 million unnecessary treatments [297]. There is a pressing need to circumvent the limitations of light microscopy — still the gold standard for diagnosis…

In the late 1800s, the great microbe hunter Robert Koch was dispatched by the German Foreign Office to German New Guinea (now Papua New Guinea) to study the pattern of disease of those living in the regions where malaria was endemic. Koch observed that malaria was more apparent in the blood of young children and almost completely absent in adults, that individuals constantly exposed to falciparum malaria develop resistance to the disease, and there may be an outbreak of disease when an area receives an influx of malaria-naïve individuals. Koch concluded that immunity to malaria requires constant exposure over a number of years and sterile protection never (or almost never) occurs. Clinical malaria, i.e. the absence of disease in the presence of blood parasites, is characterized by a reduction in parasite densities with a concomitant reduced risk of illness. "The slow acquisition of anti-parasite immunity is often thought to reflect the need for long-term exposure to the parasite" [645] as well as the age of the individual. Koch suggested that immunity against one Plasmodium species offers little protection against other species. Despite these century-old descriptions of acquired immunity, not only are the mechanisms of anti-parasite immunity unclear but so too are the targets (antigens). Indeed, despite intense research over many years, there is still no licensed malaria vaccine. Without identification of the protective antigens, the malaria vaccine is likely to remain elusive. Simply put, it is taking so long to develop a vaccine against malaria because the relevant antigens from the many life cycle stages (see p. 22) are yet to be identified, isolated, purified, and tested for efficacy in humans…

Because there is no licensed malaria vaccine, today's most desperate need is to find new medicines for the control of malaria. Two advances linked to the Malaria Genome Project have enhanced drug discovery for malaria. "First, the sequencing of the whole genomes of P. falciparum and P. vivax has been important in identifying the full range of potential drug targets [and] provided the basis for comparisons of gene expression patterns at different life cycle stages and between different species. This data set provides the ability to search for target classes that have not been pursued before in drug discovery" [727]. "The second key development is high content screening: the ability to study the viability of the parasite in 384 and 1536-well formats. This means that large compound collections can be rapidly screened, with throughputs of 100,000 per month possible even in academic centers" [727]…

A celebration of the completion of the human genome sequence was held at the White House on 26 June 2000. The President at the time, Bill Clinton, said, "it will have a real impact on all our lives — and even more, on the lives of our children. It will revolutionize the diagnosis, prevention and treatment, of most, if not all, human diseases." Those who, like Clinton, expected dramatic results overnight, would be disappointed in the years since the announcement. Among these would be Craig Venter who stood next to President Clinton and was involved in the race to sequence the human genome. In a Spiegel Online interview on 29 July 2010 he reflected on the Project: "We have learned nothing from the genome and the medical benefits so far have been close to zero." In that same year Francis Collins, who shared with Venter in the completion of the human genome sequence, and was also alongside Clinton at the celebration, put it more gently: "It is fair to say that the Human Genome Project has not directly affected the healthcare of most individuals" [147]…

The following sections are included:

• References

• Index