Humbio 153: Parasites and Pestilence
Prof. Scott Smith
In Search for an Ideal Target
Malaria research and genomic tools
Malaria is a disease that has been around for 4,000 years. Meaning “bad air” in Italian (mal aria), malarial symptoms were first described in ancient Chinese medical writings in 2700 BC. The disease continues to plague to globe, the focus of research and public health campaigns worldwide. Caused by the Plasmodium species, 3.3 billion people live in areas at risk from malaria transmission, and it caused an estimated 863,000 deaths in 2008 (Centers for Disease Control and Prevention). It disproportionally affects Africa, especially Sub-Saharan Africa, with 89% of the mortality occurring in the continent (CDC). As the fifth leading cause of death from infectious diseases worldwide, it causes catastrophic barriers to economic development and is an enormous, modern public health challenge. Despite the grim outlook of malaria, genomic tools are a major driving force in discovering more about this parasite and investigating new paths for drug discovery.
While malaria is preventable and treatable, there is no effective, cheap vaccine that can be easily disseminated to the billions of people who need it. As a result, millions of dollars are being funneled into malaria research. Malaria research is a focal point of the Millennium Development Goals, the Gates Foundation, and the Institute for One World Health, just to name a few. With the sequencing of the Plasmodium falciparum genome in 2002, the possibilities for genomic research expanded exponentially with significant implications. Genetic analysis is essential for understanding how the parasite attacks its human host and what mechanisms can prevent the onset of disease.
There are five species of the protozoan parasite, Plasmodium, that affect humans; P. vivax is the most common with 80% of all infections, and P. falciparum is the most deadly with 15% of the caseload. The life cycle of Plasmodium falciparum is of the most interest to researchers, since it causes the most disease and mortality out of the five species. The P. falciparum life cycle exhibits a unique double-cycle within the host that provides opportunities for the parasite to evade host immunity and lay dormant for periods of time. The two cycles within the human host are known as the exo-erythrocytic (liver stages) and the erythrocytic cycles (blood stages). Infected female Anopheles mosquitoes inject infective sporozoites into the blood stream, which next invade liver cells. Schizogony, asexual reproduction of the parasite, occurs in human liver cells, until the schizonts burst and release merozoites. The parasite subsequently enters the blood stream, invading red blood cells and carrying out more asexual reproduction. The invaded red blood cells eventually burst with more merozoites that repeat the cycle, or develop into gametocytes that will be taken up by mosquitoes. A third, sexual cycle of the parasite occurs within the female mosquito. All of the Plasmodium developmental stages are programmed in its genome, and thus research targeting these stages has been especially fruitful. (John and Petri 2006)
Only specific parts of the P. falciparum life cycle cause disease within a human host. While malaria may result in a variety of symptoms that resemble influenza, disease is caused when red blood cells burst and release merozoites and other toxic materials into the blood stream. Current drugs to combat and cure malaria are organized into three categories, depending on which part of the cycle they target, namely blood schizonticides, tissue schizonticides, and gametocyticides (Weekley and Smith). However, there are major challenges in malaria treatment due to the mutation of the Plasmodium parasite. Mutations cause existing drugs to be ineffective against malaria parasites, and subsequent drug resistance is a major problem in endemic areas of malaria. Growing evidence is showing that resistance is developing even to artemisinin, a WHO recommended drug, therefore research at the genetic level is incredibly important to find new potential drug targets.
Why is it important to do genomic research? According to James McKerrow, professor and vice-chair for research affairs in pathology at University of California, San Francisco, the ideal drug targets something in the parasite that does not exist in a human host. In order to indentify this target, analyzing the differences between human and parasite genomes is absolutely critical. While in reality, gene functions are much more complicated than either having or not having a gene; in many cases a homologous gene shared by humans and parasites can still make an effective drug target. In relation to malaria, this means that understanding and analyzing the P. falciparum genome and biology is a crucial area of research in the ongoing search for a new cure or vaccine.
Sequencing the Plasmodium falciparum genome was the first step to conducting genome-wide analyses on the malaria parasite and understanding its clinical manifestation in humans. The recent accessibility of the genome has created a new paradigm where drug targets can be found in the genome, potentially accelerating the process of drug development (Jomaa, et al. 1999). The international push for the entire sequencing of the Plasmodium falciparum genome was initiated in 1996, and was finally completed in 2002. In the particular example of the P. falciparum clone 3D7, completed in 2002, a chromosome shotgun sequencing strategy was used to analyze the genome (Gardner et al. 2002). At the time, a whole genome shotgun strategy was not feasible due to technological and monetary constraints. Chromosomes were separates on pulse field gels, and then 1-3 kilobase fragments were used to construct shotgun DNA libraries. Subsequently, several other strains of P. falciparum have been sequenced.
The genome itself reveals an incredible wealth of valuable information about the malaria parasite. The genome consists of 22.8 million bases, 14 chromosomes, and approximately 5,300 protein-encoding genes (Gardner, et al. 2002). Interestingly, the genome is comprised of approximately 80% of Adenine and Thymine pairs, compared with about 67.7% (A+T regions) in P. vivax. This could indicate that genes in both species especially high in Adenine and Thymine may be more recombinogenic, providing more of an opportunity for host immune system evasion (Winzeler 2008). Around two thirds of the proteins the genes produce have not been linked to genes in other organisms, thus they are unique to P. falciparum.
The protein-coding DNA sequences also bring to light information about the metabolism, evolution, DNA repair mechanisms, membrane transport, and immune evasion of the organism. The genome provides a key look into the metabolism throughout the entire life cycle, which is usually unobservable except during the intra-erythrocytic stage. The enzymes necessary for glycolysis were all found, but those needed for gluconeogenesis were not, as well as other enzymes needed for glycogen and carbohydrate stores. In comparison studies to other genomes of eukaryotic and prokaryotic organisms, the malaria parasite was found to have no proteins in common with any prokaryotic proteome. Further analysis revealed what other organisms the genes were derived from. The P. falciparum genome encoded for a limited number of membrane transporters, and instead researchers concluded that several novel pathways must be used in transport. Additionally, three unique gene families were found that produce proteins essential for host immunity (Gardener, et al. 2002). Despite all of these findings, many of the genes identified in the P. falciparum genome do not have homologues to other commonly studied organisms. As a result, the functions of such genes are still in question, and additional research in this area could indirectly assist with drug research (Winzeler 2008).
With the entire P. falciparum genome now available in public online databases, a variety of genome-wide techniques for analyzing the parasite’s gene expression have been utilized. DNA microarrays have been one of the most widely used and useful genomic tools in this endeavor. Most commonly, microarrays have been used for transcript profiling, figuring out what tissues are expressing what genes at a certain time. They are incredibly beneficial, because they allow researchers to analyze expression for thousands of malarial genes from just one experiment (Rathod, et al. 2002).
How do microarrays work? On the most basic level, a microarray is prepared by filling different wells on a chip with thousands of copies of different single-stranded DNA sequences. These probes are DNA fragments replicated through PCR and then grouped together by gel electrophoresis. These fragments are consolidated into the wells of the array. mRNA expressed in two different tissues or during two different parts of development is extracted, isolated, and then complementary DNA, or cDNA, is made by reverse transcription. The cDNA is put onto the microarray chip, where it can hybridize with any complementary DNA sequences in the wells. Using fluorescent dyes labels on the cDNA, one can tell where the cDNA binds from where it lights up on the well. This indicates what DNA sequences are being transcribed to make proteins in that specific tissue. (Sadava, et al. 2008). Three different types of microarrays have been used in malaria research thus far. Random, or shotgun, P. falciparum microarrays are constructed from unknown genomic libraries (gDNA) that have been digested and cloned for unique coding regions. Gene-specific microarrays use known P. falciparum gene sequences within the wells. Lastly, double-stranded ordered arrays are used to discover what proteins or transcription factors bind to the different DNA sequences in double-stranded form. Rathod et al. describes these three methods, in addition to direct printing of microarrays through the use of long oligonucleotides.
The studies of the malaria parasite using DNA microarrays have elucidated crucial insights about how the organism controls gene expression and the unique timing of certain transcripts. In particular, microarrays have helped discover specific genomic activity at different stages in the P. falciparum life cycle. Bozdech et al. used microarrays to investigate the intraerythrocytic development cycle (IDC), where the parasite undergoes asexual reproduction in host red blood cells. The researchers targeted the IDC stage, because this stage of development is responsible for the clinical symptoms of malaria. They created a P. falciparum specific microarray with predicted open reading frames from the entire genome as probes. Through looking at gene expression across one-hour time intervals during the 48- hour cycle, this study discovered that P. falciparum expressed the majority of its genes during this stage. Most importantly the study also showed that more than 75% of the genes expressed during this cycle are only turned on once during the IDC. Only a few genes were expressed continually. Through analyzing the transcriptome, or total RNA expression, P. falciparum gene expression can be described as a simple cascade where during the cycle a gene is expressed just once at the exact right time (Bozdech, et al. 2003).
Additional studies have used the same methods to look at other parts of the P. falciparum life cycle in terms of gene expression and active protein synthesis. Silvestrini, et al. used microarrays for a genome-wide expression analysis to identify genes specifically utilized during the early stages of gametocytogenesis. Through this study the researchers discovered which genes were designated for gametocytes, and also identified two novel membrane associated sexual stage-specific proteins. An important general observation they made was that there are very few differences in gene expression between the malaria parasite and a gametocyteless derivative, implying that early sexual reproduction could rely on control of post-transcriptional control of gene expression. In sum, these findings provide a very specific facet in the parasite biology that could be utilized for future drug research.
Northern blotting is a genomic technique commonly used in studies also utilizing microarrays. In genetic profiling, microarrays use RNA to see what proteins will be expressed at a certain point during development. The northern blotting technique is commonly used to confirm microarray results. Northern blotting involves extracting RNA samples from a specific tissue sample. The RNA is then separated by size through gel electrophoresis, and transferred onto a nylon membrane (this step is where the entire technique takes its name from). UV light or heat fixes the RNA to the membrane, and specific labeled probes hybridize with the RNA on the membrane. The probes that hybridize are visualized onto an X-ray film, and since the probes are known sequences, you can determine what RNA were expressed in a tissue at a certain time (Trayhurn 1996). Bozdech, et al. used northern blot hybridizations to confirm the microarray results of six genes showing differential expression from the trophozoite and schizont stages of P. falciparum. Their microarray results identified several genes activated during these two stages through expression profiling, and the northern blot was an additional method to reaffirm their findings. Silvestrini, et al. also used northern blotting as a genomic technique to look more in depth at several genes identified by microarray involved in gametocytogenesis.
Genomic tools have not only been able to identify general patterns of gene expression throughout P. falciparum development, but also function of specific genes and proteins. Transgenic parasites have been used in this pursuit. Transgenic parasites are created by directly inserting foreign DNA into the parasite’s genome, which can result in promoting the transcription of a protein or modifying the normal genetic function, such as knocking out a gene (Tallquist). The transgene can be a modified or in-activated gene or a reporter gene under control of the target gene's promoter, and is passed on within the original genome. In one study, transgenic parasites were created for the analysis of nek-2 function, a protein kinase expressed in gametocytes. In order to determine its function, the gene for nek-2 was disabled, and the findings showed that nek-2 was necessary for ookinete development during meiosis (Reininger, et al. 2009).
Transient transfection is another genomic tool similar to inserting transgenes. However, transient transfections allow insertion of a specific gene, but are not passed on within the genome to subsequent generations of parasites, hence their transient nature (Biowww.net). This technique of altering parasites has been used to determine transcriptional regulation of P. falciparum cysteine proteases, known as falcipains. These proteases are specifically involved in parasite infection, including hemoglobin degradation and red blood cell invasion. Through the use of transient transfection, researchers were able to identify DNA sequences sufficient for transcription of the 4 genes responsible for producing falcipains (Sunil, Chauhan, and Malhotra 2008). This ability to target sequences not only involved in transcription, but regulators of transcription, is another piece in the puzzle of P. falciparum function. The transcriptional regulation of cysteine proteases is especially important, due to their role in erythrocyte invasion.
More recently, multiple strains of P. falciparum have been sequenced. This has allowed for comparisons of different strains by using the same genomic tools. Such studies can take geographically designated strains and identify genomic similarities and differences. One such study incorporates many of the elements previously discussed. Llinas, et al. did transcriptional analysis of three different malaria strains through the use of microarrays and northern blotting. Even though the strains originated from different geographical locations, they exhibited a fairly conserved pattern of transcription during the IDC phase, with differences primarily in surface-antigens involved in parasite-host interactions (Llinas, et al. 2006). With the full analysis of multiple strains of P. falciparum transcriptomes, further research should be devoted to investigating the 60% of the genome with unknown purpose (Llinas and DeRisi 2004). The unique malaria parasite genes are prime material for finding novel drug targets.
The world of malaria research is vast and far from finished. The genomic tools described thus far are only a few of the techniques used to analyze the P. falciparum genome, but arguably some of the most important. Sequencing and DNA microarrays, among others, have helped researchers understand how the malaria parasite infects humans so well, and what exactly makes it so unique. The complete sequence of the P. falciparum genome alone opened an entire avenue of research for understanding the biology of the parasite.
In depth studies into the genomic expression and function of how malaria works inside the body is the first step to creating an effective drug or vaccine. Drugs need very specific targets that will affect only the parasite and not the human hosts. To find these targets, researchers need to know the intricacies of Plasmodium function, right down to the level of what genes are activated at what point during the life cycle. Genomic studies are providing the foundation for a fully comprehensive picture of this extremely complex parasite. While individual studies might seem to discover inconsequential, minute details, such as a single protein that is expressed during only one hour of one stage of development, this could be what drug researchers need to devastate parasite development and halt subsequent malaria transmission. Malaria’s extremely high fatality rate is providing an incentive to researchers around the globe to persevere in this endeavor. Research is an ongoing process, and labs from San Francisco to Australia are devoting their time, money, and energy to this disease. With the use of genomic tools and the entirety of the P. falciparum genome, the possibility of finding a treatment for malaria is a definite possibility and necessity.
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