No Abstract is available for this chapter, therefore we present the first paragraph of the chapter..
The Birth of the Project
Malaria is responsible for more than a million deaths a year, and up to 500 million people suffer from
this disease. In response to this growing global crisis a group of 30 international researchers and
funders gathered in May 1996 to discuss an exciting but daunting new project- obtaining the complete
genome sequence of a malaria pathogen. At that time only small viral genomes and the bacterium
Haemophilus influenzae (1.83Mb) had been completely sequenced. Although there were on-going projects to
sequence some larger eukaryotic genomes of model organisms, for example the 12 Mb genome of the
yeast Saccharomyces cerevisiae and the 97Mb genome of the nematode worm,
Caenorhabditis elegans, their completion required a very large international effort; and in 1996 it was inconceivable that a draft
human genome sequence would be available by 2000. Sequencing the 14 chromosomes (30Mb of DNA)
of Plasmodium falciparum, the most lethal of the malaria parasites infecting humans, was then a
considerable technical challenge because of the AT-rich nature of the DNA, but the potential value of genomic approaches
for drug and vaccine development were strong motivators for the multiyear, multimillion-dollar
investment. Initially the Burroughs Wellcome Fund (BWF), The National Institute of Allergy and Infectious
Diseases (NIAID), the U.S. Department of Defense (DoD) and the Wellcome Trust (WT) agreed to work
collaboratively and complementarily on funding the project (Hoffman et al., 1997). The World Health Organization
(WHO) joined the project in 1998.
Abstract
The Plasmodium
falciparum genome project, initiated in 1996, was the first attempt to sequence a
genome of a eukaryotic pathogen. Not only has the genome told us a lot about the biology of the parasite but
since the sequence data started to be released onto public web sites, it has changed the way many
Plasmodium laboratories have approached their research. Here we outline how the sequencing project was
approached, and describe what the major findings have been from being able to look at the unusual genome of
P. falciparum. We also attempt to foresee what impact genomics and comparative genomics will have on
the field of malaria research.
Abstract
The field of comparative genomics of malaria parasites has recently come of age with the completion of
the whole genome sequences of the human malaria parasite
Plasmodium falciparum and a rodent malaria model,
Plasmodium yoelii yoelii. With several other genome sequencing projects of different model
and human malaria parasite species underway, comparing genomes from multiple species has necessitated
the development of improved informatics tools and analyses. Results from initial comparative analyses
reveal striking conservation of gene synteny between malaria species within conserved chromosome cores,
in contrast to reduced homology within subtelomeric regions, in line with previous findings on a smaller
scale. Genes that elicit a host immune response are frequently found to be species-specific, although a
large variant multigene family is common to many rodent malaria species and
Plasmodium vivax. Sequence alignment of syntenic regions from multiple species has revealed the similarity between species in
coding regions to be high relative to non-coding regions, and phylogenetic footprinting studies promise to
reveal conserved motifs in the latter. Comparison of non-synonymous substitution rates between orthologous
genes is proving a powerful technique for identifying genes under selection pressure, and may be useful for
vaccine design. This is a stimulating time for comparative genomics of model and human malaria parasites,
which promises to produce useful results for the development of antimalarial drugs and vaccines.
Abstract
The recent publication of a complete reference sequence for the
Plasmodium falciparum genome is a momentous event for malaria researchers. In addition, genomic and functional genomics data is now
available for six further Plasmodium species and eight
non-Plasmodium species of apicomplexan parasites.
These datasets can greatly expedite the identification of candidate targets for drug, vaccine and
diagnostic development, in addition to enhancing our basic understanding of malaria parasites. But how can
researchers most effectively access and exploit genomic-scale data, integrating this information with the results
from other experiments? Bioinformatics research is fundamentally no different from 'wet lab' experiments
conducted at the bench, requiring an understanding of the starting reagents (databases), the strengths and
weaknesses of experimental (computational) methods, and a critical analysis of the results obtained. This chapter
discusses the nature and organization of data resources, strategies for data mining, and the interpretation
of computational results.
Abstract
Genome manipulation, the primary tool for assigning function to sequence, will be essential for
understanding Plasmodium biology and malaria pathogenesis in molecular terms. The first success in
transfecting Plasmodium was reported almost ten years ago. Gene-targeting studies have since flourished, as
Plasmodium is haploid and integrates DNA only by homologous recombination. These studies have shed new light on
the function of many proteins, including vaccine candidates and drug resistance factors. However, many
essential proteins, including those involved in parasite invasion of erythrocytes, cannot be characterized in the
absence of conditional mutagenesis. Proteins also cannot be identified on a functional basis as random DNA
integration has not been achieved. We overview here the ways in which the Plasmodium genome can be
manipulated. We also point to the tools that should be established if our goal is to address parasite infectivity in a
systematic way and to conduct refined structure-function analysis of selected products.
Abstract
Toxoplasma gondii is closely related to the
Plasmodium species sharing with them multiple structural
and functional features but differing significantly in their mode of transmission and differentiation. The broad
host range specificity and the robust nature of the extracellular form of
T. gondii have facilitated its propagation
in cell culture and led to the rapid development of genetic tools. Genetic manipulation has also
revolutionized the research on Malaria and rather unexpectedly, some genetic properties differ significantly between
T. gondii and P. falciparum imposing limits as to which strategies are specifically applicable to each of
these parasites. Advantages, constraints are intrinsic to both systems and consequently our understanding
of both parasites is not only complementary but also synergistic. The information obtained from the
Plasmodium and several other Apicomplexa genomes have led to the identification of exciting new classes of genes
that are restricted to the phylum and thus of considerable interest as potential targets for intervention.
T. gondii is particularly amenable to ultrastructural and biochemical studies and some important findings regarding
the composition and function of subcellular structures and organelles, unique metabolic pathways
and mechanisms leading to the establishment of intracellular parasitism are of great significance for the
malaria research.
Abstract
Microsatellites are short (2-6 bp in length) tandemly repeated sequences. They have been shown to
be abundant in the Plasmodium falciparum genome and as such will play a significant role in the study
of malaria in the post-genome era. Microsatellites are useful for dissecting gene function, identifying
vaccine candidates and drug targets, and elucidating the evolutionary history of the malaria parasite. With
the availability of a high-density genetic marker map for P. falciparum, it is now possible to perform
genome-wide allelic association studies for genes contribution to such specific phenotypes as drug resistance
and virulence. Additionally, the application of a large number of microsatellite markers to field samples
promises to reveal important information about population genetic structure, demographic history, and
transmission dynamics.
Abstract
Substantial gene synteny has been observed between malaria species. However, new data on the
organization of plasmodial subtelomeres has recently become available and demonstrates that chromosome ends
show, unlike the central region, a very dynamic evolution of its DNA sequence. Sequences of the
subtelomere elements vary greatly among malaria species. Duplications among subtelomeres have created large
families of expressed genes often encoding variable surface antigens.
Fluorescence in situ hybridisation (FISH) combined with three-dimensional microscopy has demonstrated that chromosome ends in Plasmodium
are not randomly arranged in the nucleus. Telomeres form clusters of 4 to 7 heterologous chromosome
ends and are associated with the nuclear periphery. The physical alignment of subtelomeres promotes
frequent recombination between members of telomere-associated virulence factor genes in
heterologous chromosomes. This has important implications for the parasite survival and its adaptation to
environmental stress.
Abstract
The study of gene expression in malaria parasites has blossomed in recent years with the development
and use of reporter gene constructs in transfection experiments and the recent release of the annotated
sequence of the P. falciparum genome. The promoters and regulatory regions of many genes from several
Plasmodium species have been characterized, however the exact nature of the transcription factors that bind them
remain ill-defined. In addition, how expression of the large multigene families found in all
Plasmodium genomes is regulated presents a particularly interesting problem. Recent findings with regard to the
var gene family of P. falciparum may shed light on the topics of allelic exclusion and transcription switching that are
important for understanding how expression of these gene families is controlled.
Abstract
A remarkable feature of
Plasmodium parasites is that they express structurally distinct sets of rRNA in
a developmentally specific manner. The expression of three different rRNAs parallels the
developmental cycle of the parasite. Parasites missing the rRNA genes predominantly expressed in the mosquito
develop at a slower rate than wild type parasites but never the less develop fully. The normal cyclical pattern
of expression is influenced by temperature and ambient glucose concentrations and changes in these
factors can result in dramatic changes in the parasite's physiology. Variable forms of ribosomes are not unique
to Plasmodium species. They are often found in plants and bacteria but alteration in these cases result
from changes in the protein complement of the ribosome. Alteration of the rRNA itself occurs in
Plasmodium species and presents opportunities to study a novel biochemical property of the parasite and to gain
insights into RNA as a functional moiety.
Abstract
The molecular mechanisms regulating cell proliferation and development in malaria parasites are still
largely unknown. Phenomenological observations, pertaining to the organisation of the cell cycle during
schizogony or to the signal transduction pathways whose activation is responsible for the developmental stage
transitions, can now be complemented with information gathered from genomic databases. The PlasmoDB
database has been used extensively to identify putative homologues of a number of eukaryotic cell cycle
regulators such as cyclins, cyclin-dependent kinases, factors involved in the control of DNA synthesis, and
components of signal transduction pathways. However, gene identification based on sequence homology is limited
by the fact that any Plasmodium-specific functional homologue will be missed by this approach.
Furthermore, experimental data indicate that the structure of some regulatory pathways (unlike that of metabolic
pathways) cannot be deducted directly from database mining. Because of these limitations in the direct exploitation
of genomic database, elucidation of the organisation of the signal transduction and cell cycle
machineries requires experimental, proteomics-based approaches such as the characterisation of protein
complexes containing cell cycle regulators and the establishment of a map of protein-protein interactions
involving these elements.
Abstract
The apicoplast is a semi-autonomous plastid organelle that is homologous to chloroplasts of plants. It
occurs throughout the Apicomplexa and is an ancient feature of this group acquired by the process of
endosymbiosis. Like plant chloroplasts, apicoplasts are semi-autonomous with their own genome and expression
machinery. In addition, apicoplasts import numerous proteins encoded by nuclear genes. These nuclear genes
largely derive from the endosymbiont through a process of intracellular gene relocation. The exact role of a
plastid in parasites is uncertain but early clues indicate synthesis of lipids, heme and isoprenoids as
possibilities. The various metabolic processes of the apicoplast are potentially excellent targets for drug therapy.
Abstract
In order to navigate its complex lifecycle, the malaria parasites must interact with a range of host cells.
Examples of this are the invasion of hepatocytes by sporozoites and erythrocyte invasion by merozoites.
This requirement for cell recognition brings with it the need to display cognate ligands on the
parasite surface, and therefore the capacity of the host to develop defences against the infection. Even at a
stage where the intracellular nature of erythrocyte development would appear to offer an opportunity for the
parasite to be immunologically "silent", parasite-derived proteins are found on the surface of the infected erythrocyte.
This review will discuss the proteins found on or associated with the surface of the infected erythrocyte
and the resulting phenotypes.
Abstract
The merozoite of the asexual stage parasite is adapted for the invasion of the red blood cell.
Specific structures of the merozoite such as the filamentous surface coat and the secretory organelles of the
rhoptries, micronemes and dense granules participate in this process. The repertoire of proteins in these
locations participate in a specific set of receptor-ligand interactions, some still poorly understood, that trigger
the secretion of products from specialized organelles and the activation of a molecular motor. There is
redundancy in the parasite ligands involved and genomic analysis is revealing a large set of gene products that
are potentially involved in invasion.
Abstract
Malaria parasites switch from asexual proliferation to a sexual cycle of development in the blood of a
vertebrate host in order to transmit between the vertebrate and mosquito host. The central role of sexual
development in the life cycle and transmission of
Plasmodium parasites make the sexual stages, such as the
gametes and zygotes, attractive targets for interruption strategies to prevent transmission of the disease (for
example transmission blocking vaccines). Here we review the knowledge on the cellular and molecular
processes involved in sexual differentiation, i.e. the development of gametocytes and gametes, fertilization and
zygote formation. In contrast to rapidly expanding molecular knowledge of asexual multiplication of malaria
parasites, the molecular mechanisms of sexual differentiation are still largely unknown. However, with the
availability of the genome sequence of different
Plasmodium species combined with post-genomic technologies
this situation will change. Insight into molecular processes underlying sexual development and the
proteins involved will maximise numbers of candidate proteins available for consideration as transmission
blocking vaccine candisdates and will allow researchers and policy makers to make rational, informed
decisions about the proteins considered for components of a multi-stage vaccine.
Abstract
Recent advances in our understanding of the molecular and morphological development of the
malarial zygote into a mature invasive ookinete are described. The similarities and more particularly the
differences of this development to sporozoite and merozoite differentiation are emphasised. Notable differences
include the absences rhoptries, the formation of just one apical complex during meiosis, and the formation of a
pore-containing inner pellicular complex. The impact of both vertebrate and mosquito components of the
bloodmeal on ookinete development are discussed. The destructive events of invasion of the midgut wall and
oocyst differentiation are described and the potential interactions between the parasite and the mosquito
immune system outlined. The very successful efforts to develop vaccines targeted to the ookinete are
summarised. The contrasting lack of drugs that block transmission through the vector is highlighted.
Abstract
For decades, the 4-aminoquinoline chloroquine
(CQ1) was the mainstay of antimalarial chemotherapy,
with its characteristic properties of rapid efficacy, safety and affordability. However, resistance to this drug
has now become prevalent in the majority of malaria-endemic countries and is severely limiting its use as
an effective antimalarial. CQ is believed to act against CQ sensitive (CQS) malaria parasites by
accumulating inside the digestive vacuole (DV) of the intra-erythrocytic parasite, where CQ binds to heme
(ferriprotoporphyrin IX, or FPIX) that is liberated during hemoglobin degradation. The resulting impairment in heme
detoxification results in parasite death. Chloroquine resistance (CQR) in
Plasmodium falciparum is defined in vitro
by elevated CQ IC50 values, reduced CQ accumulation and the ability of verapamil (VP) to reverse
(or "chemosensitize") these two traits. Molecular genetic and epidemiological investigations have recently
revealed an association between P.
falciparum CQR and the presence of multiple amino acid mutations in the
DV transmembrane protein PfCRT (P.
falciparum chloroquine resistance transporter). Allelic
exchange experiments have now definitively demonstrated that mutant
pfcrt alleles present in different
malaria-endemic continents can confer CQR to CQS parasites. Investigations into other candidate genetic determinants
have demonstrated that mutations in the parasite protein Pgh1
(P. falciparum P-glycoprotein homologue 1,
also localized to the parasite DV membrane and encoded by the
pfmdr1 gene) can modulate the CQR phenotype of parasites, but are not sufficient to confer CQR
in vitro. Recent clinical studies demonstrate an
increased risk of CQ treatment failure in patients harboring parasites with CQR-associated mutations in
pfcrt and sometimes pfmdr1. Other factors, including host immunity, are also important in determining clinical
outcome. In vitro, the CQR phenotype may be intimately associated with changes in the physiological processes
of the DV, such as altered DV pH. These changes may critically impact the access of CQ to hematin
dimers that act as a drug receptor in the DV. Alternatively, mutant PfCRT may contribute to CQR by
physically interacting with CQ, leading to increased drug efflux. Ongoing studies, bolstered by recent advances
in Plasmodium genetics, genomics and proteomics, can be expected to significantly deepen our
understanding of the genetic basis of P.
falciparum CQR, the relationship between CQR and DV physiology, and the
effect of CQR on the parasite response to other heme-binding antimalarials. The knowledge gained from
these studies will be of greatest benefit when they can be translated into new therapies to treat
drug-resistant malaria.
1 ADQ, amodiaquine; AO, acridine orange; CQ, chloroquine; CQS, chloroquine sensitive; CQR,
chloroquine resistance (or chloroquine resistant); DV, digestive vacuole; FPIX, ferriprotoporphyrin IX; iRBC, infected
red blood cells; Pgh1, P-glycoprotein; RBC, red blood cells, SP, sulfadoxine-pyrimethamine; UTR,
untranslated region; WHO, World Health Organization.
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