Malaria Genome Projects, The: Promise, Progress, And Prospects

Promise, Progress, and Prospects

Irwin W Sherman

• Publisher: ICP
• Year: 2012
• ISBN:
  9781908977021
• Topic: Medicine
• Subtopic: Infectious Diseases

The year 2012 marks the tenth anniversary of the announcement of the genome sequence of the human malaria parasite Plasmodium falciparum and that of its mosquito vector Anopheles. The genome sequences were a result of the Plasmodium falciparum Genome Project.

This book covers in detail the biology of malaria parasites and the mosquitoes that transmit the disease, how the Genome Projects came into being, the people who created them, and the cadre of scientists who are attempting to see the promise of the Projects realized. The promise was: a more complete understanding of the genes of the parasite (and its vector) would provide a rational basis for the development of antimalarial drugs and vaccines, allow a better understanding of the regulation of the complex life cycle in the red blood and liver cells of the human, identify the genes the parasite uses to thwart the host immune response and the ways in which the parasite evades cure by drug treatments, as well as leading to more effective measures of control transmission. The hope was that cracking the genetic code of Plasmodium and Anopheles would reveal the biochemical Achilles heel of the parasite and its vector, leading to the development of novel drugs and better methods of control, and by finding the targets of protective immunity could result in the manufacture of effective vaccines.

Through a historic approach, this book will allow for those new to the field, or those with insufficient background in the sciences, to have an easier entry point. Even scientists already working in the field may better appreciate how discoveries made in the past can impact the direction of future research.

Contents:

• Introduction
• Respice: Before the Genome Project
• The Nature of Plasmodium falciparum and Its Genome
• Chipping Away at the Genome
• The Importance of Import
• Remodeling the Genome's Home
• Getting on the Inside
• The Great Escape
• The Neglected Malaria: Plasmodium vivax
• The Anopheles Genome and Transmission Control
• The Monkey's Tale
• A Not So Sweet Solution
• To Search and Find
• The Elusive Vaccines
• New Medicines, Old Problems
• Prospice: Looking to the Future

Readership: Biochemists, parasitologists, microbiologists, molecular geneticists, immunologists, hematologists, malariologists and tropical physicians and graduate students.

Preface

To decode the genome — the entire sequence of genes — of the most deadly human malarial parasite Plasmodium falciparum took six years of effort by a multinational team of scientists, driven by lofty expectations and hubris. The feat was announced on 3 October 2002 in Nature magazine and was heralded by a New York Times headline: “Genetic decoding may bring advances in worldwide fight against malaria.” The need for the Malaria Genome Project seemed obvious to many: each year the disease causes hundreds of millions of people to become sick and more than a million — mostly African children under the age of five — die. Few infectious diseases can rival malaria's ability to kill and cripple. Then, as today, “it has ducked every vaccine attempt and shaken off most of the drugs developed to knock out the disease” [512].

In 1996, the proponents of the Malaria Genome Project argued that it was more than an academic exercise; in their view it was a global public health imperative. Its grand promise was that a more complete understanding of the malaria parasite's genes would provide a rational basis for the development of antimalarial drugs and vaccines, allow a better understanding of the regulation of the complex life cycle in the red blood and liver cells of the human, identify those genes that the parasite uses to thwart the host immune response, and explain the manner by which it is possible for the parasite to evade cure after drug treatments. The hope of this new breed of “gene hunters” was that cracking the life code of P. falciparum would reveal the novel biochemical Achilles' heels of the parasite, lead to new drugs and enable pharmaceutical companies to manufacture protective vaccines through the discovery of the targets of protective immunity.

Support for the Malaria Genome Project was not wishful thinking. It was based on previous scientific successes beginning in 1995 when it was possible to “read” the genome of the free-living microbe Haemophilus influenzae, followed a year later by the pathogens Mycoplasma genitalae, Borrelia burgdorfi, the causative agent of Lyme disease, Helicobacter pylori , implicated in gastric ulcers, and Mycobacterium tuberculosis, responsible for tuberculosis. Further, there were cost-effective technologies to isolate DNA from individual chromosomes, machines to “read” the sequences of genes and computer programs for aligning the pieces to produce a linear map. The cost, the project promoters suggested, would be substantially less than that spent on the Human Genome Project. Deciphering all 23 million letters in the P. falciparum genome would be much cheaper than the much larger human genome —∼3 billion letters. Indeed, improvements in instrumentation and powerful computers would not require $28 billion, as did the Human Genome Project; nevertheless tens of millions of dollars would still be needed. As no single funding source was large enough to take on such an immense project, several agencies were tapped, including the U.S. Department of Defense, the Wellcome Trust and the National Institutes of Health (NIH). In order to get the sequencing work done, an international consortium of scientists was assembled in the United States at The Institute for Genomic Research (TIGR), Sloan Kettering Institute, Stanford University, Harvard University and University of Florida, in the UK at the National Institute for Medical Research (NIMR), the Wellcome Trust Sanger Institute, Edinburgh University and Oxford University, and in Australia at the Menzies School of Health Research and the Walter and Eliza Hall Institute.

My own involvement with Plasmodium DNA began several decades earlier and was by accident rather than by design. In the summer of 1960 I began post-doctoral studies in the laboratory of William Trager (1910–2005) at the Rockefeller University (formerly the Rockefeller Institute). This was several decades before the human malaria P. falciparum could be grown continuously in large quantities in laboratory dishes, so a convenient model for studying human malaria was the bird malaria P. lophurae. Plasmodium lophurae had been isolated in 1938 by Lowell T. Coggeshall (of the Rockefeller Foundation) from the blood of a Borneo fireback pheasant, Lophurae igniti igniti, kept captive in the Bronx Zoo. Thereafter the parasite was maintained by transferring infected blood into chickens and ducks. The growth of P. lophurae in young ducklings was phenomenal. At five days after inoculation (by hypodermic syringe) of suitably diluted infected blood almost 90% of the recipient's red blood cells were infected. It was also possible to obtain 100 ml of whole blood from a single duckling; after spinning in a centrifuge it was possible to obtain 10–20 ml of packed red cells with a wet weight of∼15 g — an amount equivalent to that from a luxuriant culture of bacteria. My research focus was biochemical, i.e. enzymes. At the time, purifying an enzyme required fairly large quantities of crude material. Plasmodium lophurae was perfectly suited for this, and I became quite adept at isolating substantial quantities of parasites removed from their hemoglobin-rich habitat — the red blood cell.

At the time of my arrival at Rockefeller, Trager had two small laboratories and an office on the third floor of Theobald Smith Hall. The equipment was minimal for biochemistry — a spectrophotometer placed in an abandoned broom closet, a few refrigerated centrifuges, incubators, glassware, cotton-plugged test tubes and pipettes, an assortment of balances for weighing out reagents, and a wood-framed glass hood and bunsen burners for sterile transfer. All of this was in support of Trager's attempts to grow P. lophurae in glass dishes both within and removed from red blood cells. Taking all this in I imagined myself in the microbiology laboratory of Robert Koch (1843–1910) or Paul Ehrlich (1854–1915), yet here I was a century later at the center of medical research, “the Rockefeller” in New York City, working with the doyen of malaria, William Trager. Once resident at Rockefeller, Trager and I had a conversation about a meeting he had attended in Florida where another participant was the Brandeis University molecular biologist Julius Marmur (1926–1996). Marmur, following hard on the discovery by the Rockefeller Institute physician Oswald Avery of pneumococcal DNA (called transforming principle) as the genetic material, had worked with Avery's colleague, Rollin Hotchkiss (1911–2004), at Rockefeller (above us on the fourth floor of Theobald Smith Hall!) and demonstrated that the DNA of pneumococcus coded for an enzyme. Later, after developing highly reproducible methods for DNA purification, Marmur was able to show that upon heating purified DNA the helical strands separated (called melting) and the melting temperature was dependent on the composition of the DNA, that is, the percent of guanine plus cytosine (G + C). Marmur was attempting to use the melting temperature of DNA from a variety of bacteria to be used “as a valuable asset in their classification.” He told Trager he needed some one-celled microbes that were not bacteria for comparative purposes, and he asked whether Trager could help supply these. Trager had no intention of spending his valuable time in preparation of gram quantities of malaria parasites as this would require abandoning his beloved cultures for several days. So he “volunteered” me to send Marmur some of my precious samples of P. lophurae. I did so and when the results were published I was thanked for “useful discussions” but unexpectedly the data for the P. lophurae DNA were not included [584]. When I asked Marmur about the omission he simply replied, “It was so low in G + C that it didn't fit.” Indeed, what Marmur had found was that the G + C content was 20%!

Eight years later, when I was on the faculty of the University of California at Riverside, Charles Walsh, a graduate student in my laboratory, confirmed Marmur's unpublished findings, but our publication received little attention. Others regarded it to have no real significance as this malaria parasite came from a bird with little in common with the human malaria, P. falciparum. It was gratifying many years later, when P. falciparum could be grown in culture so its DNA could be analyzed, to find that its G + C was 18–20% (similar to that of P. lophurae) and very different from the malarias infecting rodents and monkeys, which were 24% and 37%, respectively. Although my venture with malaria DNA might have led me to join the ranks of the “gene hunters” it did not. Rather I continued to study the products of genes — protein enzymes — and only at various intervals did I direct my research toward DNA, RNA and protein synthesis. However, never in the succeeding years did I ever lose interest in the research being carried out by others on the genes of malaria parasites and culminating in the Malaria Genome Project.

For whom is this book intended? My aim is to tell a story for those who want to know more about the biology of malaria parasites and the mosquitoes that transmit the disease, how the P. falciparum Genome Project came into being, the people who created it and the cadre of scientists — biochemists, parasitologists, microbiologists, molecular geneticists, immunologists — who are attempting to see the promise of the 10-year-old project realized. I have approached the subject in an historic fashion so that those new to the field, or those with insufficient background, will have an easier entry point. Hopefully, through this approach even those already in the field may better appreciate how discoveries made in the past can impact the direction of future malaria research.

Chapter 1

Introduction

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.

Here's how one victim describes the disease: “I wanted to sit up, but felt that I didn't have the strength to, that I was paralyzed. The first signal of an imminent attack is a feeling of anxiety, which comes on suddenly and for no clear reason. Something has happened to you, something bad. If you believe in spirits, you know what it is: someone has pronounced a curse, and an evil spirit has entered you, disabling you and rooting you to the ground. Hence the dullness, the weakness, and the heaviness that comes over you. Everything is irritating. First and foremost, the light; you hate the light. And others are irritating — their loud voices, their revolting smell, their rough touch. But you don't have a lot of time for these repugnances and loathings. For the attack arrives quickly, sometimes quite abruptly, with few preliminaries. It is a sudden, violent onset of cold. A polar, arctic cold. Someone has taken you, naked, toasted in the hellish heat of the Sahel and the Sahara and has thrown you straight into the icy highlands of Greenland or Spitsbergen, amid the snows, winds, and blizzards. What a shock! You feel the cold in a split second, a terrifying, piercing, ghastly cold. You begin to tremble, to quake, to thrash about. You immediately recognize, however, that this is not a trembling you are familiar with from earlier experiences — when you caught cold one winter in a frost; these tremors and convulsions tossing you around are of a kind that any moment now will tear you to shreds. Trying to save yourself, you begin to beg for help. What can bring relief? The only thing that really helps is if someone covers you. But not simply throws a blanket or quilt over you. The thing you are being covered with must crush you with its weight, squeeze you, flatten you. You dream of being pulverized. You desperately long for a steamroller to pass over you” [360]. Indeed, “a man right after a strong attack…is a human rag. He lies in a puddle of sweat, he is still feverish, and he can move neither hand nor foot. Everything hurts; he is dizzy and nauseous. He is exhausted, weak, and limp. Carried by someone else, he gives the impression of having no bones and muscles. And many days must pass before he can get up on his feet again” [46].

Plasmodium discovered

For over 2,500 years the prevalent belief was that “the fever” (malaria) was due to miasmas — deadly vapors arising from wetlands and hence the French name paludisme from the Latin meaning swamp. In England in the 15th and 16th centuries the fevers were seasonal. They were called ague (meaning acute fever) and were common in low lying and marshy areas called the unwholesome fens in Kent, Cambridgeshire, Essex and the Thames estuary. The word malaria, literally “bad air,” was unknown in the English language until the writer Horace Walpole in 1740, during a visit to Italy, wrote: “There is a horrid thing called malaria that comes to Rome every summer and kills one.” Other diseases such as cholera, tuberculosis and diphtheria, in addition to malaria, were believed to be due to “bad air” but in the mid-19th century, after Louis Pasteur (1822–1895) and Robert Koch (1843–1910) identified microbes as the cause of anthrax, cholera, tuberculosis, diphtheria and tetanus, it became clear that germs, not miasmas, were the cause. Once germs (microbes) were shown to be responsible for a variety of infectious diseases it provoked intrepid physicians to find the germ responsible for malaria.

There were various reports of a sighting of the microbe causing malaria and of particular interest was one in 1879 by two investigators in Italy, Edwin Klebs and Corrado Tommasi-Crudeli, who found a rod-shaped bacterium (a bacillus) in the mud and waters of the Pontine marsh in the malarious Roman Campagna. When the bacteria were injected into rabbits, fever resulted, the spleen was enlarged, and the same bacteria could be re-isolated from the sick rabbits. They named the germ Bacillus malariae. This finding, quite naturally, attracted worldwide attention. Klebs and Tommasi-Crudeli were well-respected scientists. Klebs held the professorship of pathology in Prague and had conducted research on the relationship of bacteria to disease with Koch. Tommasi-Crudeli, who had studied with the eminent pathologist Rudolph Virchow in Berlin, was Director of the Pathological Institute in Rome with a specialty in malarial fevers. Initially, support for B. malariae as the agent of malaria was forthcoming. In 1880 Ettore Marchiafava, a loyal first assistant to Tommasi-Crudeli, and Giuseppe Cuboni reported isolation of the same bacillus from the bodies of patients who had died from malaria, and when studies in Klebs' laboratory found that the antimalarial drug quinine killed B. malariae there appeared little doubt that malaria was a bacterial disease [294].

In the United States, the U.S. Board of Health commissioned Major George Sternberg to try to repeat the experiments of Klebs and Tommasi-Crudeli in the malaria-affected area around New Orleans. He found bacteria similar to B. malariae in the mud from the Mississippi delta; however, after the bacteria were injected into rabbits the fevers produced were atypical of malaria in that they were not periodic. Sternberg was also able to produce the disease in rabbits by injecting them with his own saliva. He concluded that the disease was septicemia, not malaria, and suggested the bacteria were contaminants [294]. Others raised questions when they were unable to grow B. malariae outside the body using the blood of patients with malaria, and there was a suggestion that the killing effect of quinine was not specific. This lack of reproducibility certainly acted to undermine the belief that malaria was a bacterial infection; more telling, however, was the discovery made by an obscure French military physician, Charles-Louis Alphonse Laveran (1845–1922).

In 1875, after reading the literature on malaria and becoming acquainted with physicians who had worked in the French territory of Algeria where cases of malaria were numerous, Laveran wrote a treatise on military epidemiology. He noted that malaria could occur in countries where the climate was cold and could be absent from tropical climes, but that the fevers became more severe as one moved from the poles to the equator. Although swamps and low humid plains were the most favorable environment for malaria, he concluded that swamps themselves did not cause the fever, as even in hot countries not all swamps gave rise to fever. Laveran's treatise made a prescient statement: “Swamp fevers are due to a germ” [347].

After several military assignments in France, Laveran was transferred in 1878 to Bone, Algeria. The North African coast was rife with malaria and Laveran spent most of his time looking at autopsy specimens from those who died from the disease. One of the signal characteristics of malaria in cadavers is the enlarged and blackened spleen and liver, due to accumulation of a brownish-black pigment, called hemozoin. Laveran has been described as “bespectacled with sharp features and a small trim beard” [425]. He was reputed to be extraordinarily precise, meticulous, singularly sharp-minded, incisive and self-opinionated. In short, he did not suffer fools gladly. Laveran spent most of his time looking at preserved dead material from the deceased who had succumbed to “the fever,” but he also examined fresh specimens. His microscope was not a good one but he was patient and determined. On 6 November 1880, while examining a drop of fresh liquid blood from a feverish artilleryman, he saw several transparent mobile filaments — flagella — emerging from a clear spherical body. He recognized that these bodies were alive, and that he was looking at an animal, not a bacterium or a fungus. Subsequently he examined blood samples from 192 malaria patients: in 148 of these he found the telltale crescents. Where there were no crescents, there were no symptoms of malaria. Laveran also found spherical bodies in or on the blood cells of those suffering with malaria. Remarkably, and in testimony to his expertise in microscopy, Laveran's discovery was made with a microscope having a magnification of only 400 diameters. He named the beast Oscillaria malariae and communicated his findings to the Société Médicale des Hôspitaux on 24 December 1880 [362].

Laveran was anxious to confirm his observations on malaria in other parts of the world, and so he traveled to the Santo Spirito Hospital in Rome where he met with two Italian physicians (one of whom was Marchiafava, Tommasi-Crudeli's assistant, and the other Angelo Celli, Professor of Hygiene) and showed them his slides. The Italians, whose chief interest was B. malariae, were unconvinced and told him that the spherical bodies he had seen were nothing more than degenerating red blood cells caused by B. malariae or some other cause. Consequently, few of those attending the sick and suffering with “the fever” paid attention to Laveran's observations.

In 1883, Marchiafava and Celli claimed to have seen the same bodies as described by Laveran but without any pigment granules. They also denied the visit by Laveran two years earlier. The Italians were unsuccessful in growing the bodies outside the body of the malaria patient. The lack of consensus ...

Table of contents

• Cover
• Half Title
• Title
• Copyright
• dedication
• Contents
• Chapter 1 Introduction
• Chapter 1 Introduction
• Chapter 2 Respice: Before the Genome Project
• Chapter 3 The Nature of Plasmodium falciparum and its Genome
• Chapter 4 Chipping Away at the Genome
• Chapter 5 The Importance of Import
• Chapter 6 Remodeling the Genome's Home
• Chapter 7 Getting on the Inside
• Chapter 8 The Great Escape
• Chapter 9 The Neglected Malaria, Plasmodium vivax
• Chapter 10 The Anopheles Genome and Transmission Control
• Chapter 11 The Monkey's Tale
• Chapter 12 A Not So Sweet Solution
• Chapter 13 To Search and Find
• Chapter 14 The Elusive Vaccines
• Chapter 15 New Medicines, Old Problems
• Chapter 16 Prospice: Looking to the Future
• Title
• Title