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Genomic tools and drug resistance in Plasmodium falciparum
Background
Drug resistance is a big problem in the fight against Malaria. During the
1960s the hope of eradicating malaria was quelled by the emergence of Chloroquine
resistant parasites. Combination therapies offered some promise but they too are failing
(Eckland et al, 2007). P. falciparum is resistant to the most commonly used drugs, which
are chloroquine and sulfadoxine-pyrimethamine, making it predominant in Sub-Saharan
Africa and among people who visit tropical regions.
The main mechanism of drug resistance that is currently known is
polymorphisms in the genome of the malaria parasites. Pathways of drug resistance in
the parasite have not been elucidated yet. Further research is required (Mehlotra et al,
2009). Drug resistance is highly likely to result when patients are given subtherapeutic
drug levels due to unregulated use, inadequate drugs for the parasite are used and
drugs with long half lives are given to patients. These situations allow for the selection
of parasites with polymorphisms (point mutations) that reduce sensitivity to the drug
(Mehlotra et al, 2009).
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Introduction
Many genomic tools exist to help researchers identify resistant strains of
Plasmodium falciparum. According to James Mckerrow, studying the parasite at the
genomic level may help solve the problem of drug resistance faster than solely focusing
on the host genome will. I will focus on discussing four of these tools. Each tool will be
briefly described and then the role the tool plays in identifying drug resistant strains
will be explored.
Genomic tools
1. Sequencing
Sequencing is the determination of the genotype of an organism. Two
different strategies have been widely applied to sequencing genomes. One approach,
shotgun sequencing, involves fragmenting a large genome into random fragments
which are sequenced individually. Once all the fragments have been sequenced, a
powerful computer is used to order the fragments using overlaps in these fragments.
This method was successfully used to sequence the genome of, Haemophilus influenza,
the first organism to have its complete genome sequenced, in 1995. However, the
reliance of this method on finding overlaps makes it inefficient when sequencing
genomes with highly repetitive sequences (Alberts et al, 2004). Another approach to
sequencing that avoids this problem is clone by clone sequencing. Scientist use
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restriction endonucleases to cut up the genome into overlapping fragments of 100 – 200
kilobases. The fragments are inserted into bacterial artificial chromosomes which are
then put into E.coli. As each E.coli divides, it produces clones of the fragments. Based
on the positions of the restriction enzyme sites, researchers map the fragments onto a
previously generated restriction map of the organism’s genome allowing them to know
where each fragment fits in the genome. Once they know this they sequence each
fragment individually to come up with the complete genomic sequence of the organism
(Alberts et al, 2004).
The 23 Mb genome of P. falciparum was sequenced completely in 2002
using the above mentioned methods. This achievement made it possible to study
resistance at the molecular level because the genotypes of resistant and sensitive
parasites can be compared to learn which mutations lead to resistance. It would be very
costly and labor intensive to try to sequence the genome of every resistant or sensitive
P. falciparum parasite encountered in order to compare its genotype to those of other
parasites. Researchers use the presence of conserved polymorphisms in the genome as
surrogate markers for the determinacy of resistance. Microsatellites (consisting of
repeats of a short nucleotide sequence), single nucleotide polymorphisms (SNPs), or
small insertions or deletions are the most common polymorphisms found in P.
falciparum .
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After the genome of P. falciparum was sequenced researchers were able to
estimate that the number of SNPs in the parasite genome range from 25000 to 50000.
This means that there is one SNP for every 400-800 base pairs (Eckland et al, 2007). This
information is important because SNPs are responsible for a lot of drug resistance,
therefore knowing how often they occur in the genome can help scientists discover
mutations faster.
2. Microarrays
In microarray technology mRNA is isolated from the organism of interest
and then the mRNA is converted to cDNA using a reverse transcriptase. The cDNA is
annealed and then passed through a microchip with the genome or the partial genome
of the organism of interest. If hybridization occurs the degree of fluorescence can reveal
the degree of activation of a gene at any point in the life processes of a parasite
(Campbell, 2005).
Microarray technology has been utilized to explore polymorphisms in the
P. falciparum parasite. Some researchers used a P. falciparum microchip with probes
covering half of all the coding regions of the parasite. Loss of hybridization signal that is
associated with failure to match between genomic DNA was used to find the
polymorphisms in different clones of the parasite. Above normal fluorescence intensity
signaled places where gene amplification had occurred in the clones. The
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polymorphisms found have already been discussed under the sequencing section
because sequencing allowed for microarray technologies to be used in this parasite.
Microarrays are a useful tool because they allow for rapid identification of resistance
loci (Eckland et al, 2007).
Using microarrays, researchers have found that chloroquine resistance is
linked to point mutations in the chloroquine resistance transporter gene known as pfcrt,
which is found on chromosome seven of the parasite. Parasites with the non-mutated
wild-type version of the gene from all over the world are still killed by chloroquine,
confirming that, it is a mutation in this gene that confers resistance. Polymorphisms on
the pfmdr1 gene which is on chromosome 5 result in parasites which are resistant to a number
of drugs. It is possible that polymorphisms on other genes play a role in resistance; the
comparison of phenotypes made possible by sequencing and microarrays will continue to help
researchers find such genes (Eckland et al, 2007). .
3. Linkage disequilibrium
Linkage disequilibrium is the situation where alleles at two or more loci
are associated non-randomly. The alleles need not be on the same chromosome. Linkage
disequilibrium can result as a consequence of the physical structure of the chromosome,
inherent mutations that occur randomly during DNA replication and the recombination
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rate between any two given loci. A high rate of recombination can destroy linkage
disequilibrium.
As mentioned before, researchers estimate that there is one SNP for every
400-800 base pairs in the P. falciparum genome. Similar to the case in humans, these
SNPs form haplotype blocks that do not randomly segregate. These SNPs satisfy the
condition for linkage disequilibrium.
Given that P. falciparum is associated with high rates of recombination, one
would wonder how useful a linkage disequilibrium analysis can be since high rates of
recombination disrupt linkage disequilibrium. However, drug resistance is such a
recent evolutionary event that the high rate of recombination has not disrupted its
linkage disequilibrium yet. Linkage disequilibrium can therefore be used to track the
spread of the resistance gene in the population. Roper et al used linkage disequilibrium
to chart the origin and spread of the gene that renders parasites resistant to an antimalarial drug known as pyrimenthamine. The gene was found to have come from
Southeast Asia and then it spread to Africa (Eckland et al, 2007).
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4. Transfection methodologies
Transfection is the uptake of foreign DNA by a cell. The above mentioned
techniques have allowed researchers to identify the loci associated with resistance.
However, these techniques still need improvement because they do not pin-point
specific genes yet. The genomic tools implicate areas which encompass many possible
genes for resistance, many of which end up being shown to have nothing to do with
resistance after further rigorous testing. Transfection methods are used in this further
testing. A gene is thought to confer resistance if replacing a sensitive gene with it leads
to a sensitive clone surviving a drug. This genomic tool is known as allelic exchange
because one allele is replaced with another to determine whether the exchange confers
resistance (Eckland et al, 2007). .
Allelic exchange was used to show that dihydropteroate synthase (dhps)
mutants give sulfadoxine resistance. Another team of researchers convincingly proved
that pfcrt affords chloroquine resistance by exchanging the pfcrt allele of a sensitive line
with the pfcrt alleles of resistant lines from South America, Asia, and Africa. Yet another
team also used allelic exchange to show that allelic variants of pfmdr1 could alter the
degree of parasite susceptibility to various drugs including mefloquine, quinine,
halofantrine, chloroquine, and artemisinin. Gene amplification events have been
implicated in some forms of drug resistance using this method(Eckland et al, 2007).
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Another transfection method is gene intergration. A perfected version of
this technique uses virus intergrase and homologous recombination to generate
resistant clones very efficiently. The method also allows researchers to add multiple
copies to replicate gene amplification and then study which kind of gene amplifications
enhance resistance or increase sensitivity. It is evident that transfection methodologies
greatly enhance the search for resistant strains (Eckland et al, 2007). .
Conclusion
These tools offer great insight into the way the parasite works and
researchers hope this knowledge will lead to better drugs. Sequencing and microarray
technologies allow for rapid identification of resistant loci. Linkage disequilibrium
analysis allows people to chart the origin and the spread of resistant genes over time.
Transfection methodologies are more specific in pointing out which genes lead to
resistance. They even allow researchers to study how gene amplifications contribute to
resistance. These genomic tools can be used to study other parasites, especially the
parasites for the so called neglected diseases.
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References
Campbell, Neil A., and Jane B. Reece. Biology AP Edition. Upper Saddle River: Prentice
Hall College Div, 2004. Print.
Ekland, Eric H., and David A. Fidock. "Advances in Understanding the Genetic Basis of
Antimalarial Drug Resistance." PubMed. 20 Aug. 2007. Web. 23 Jan. 2010.
Mckerrow, James. "Genomic Tools and Malaria Research." Telephone interview. 3 Feb.
2010
Mehlotra, Rajeev K., Cara N. Henry-Halldin, and Peter A. Zimmerman. "Application of
Pharmacogenomics to Malaria: A Holistic Approach for Successful
Chemotherapy." Pub Med Central. Mar. 2009. Web. 23 Jan. 2010.
Mita, Toshihiro, Kazuyuki Tanabe, and Kiyoshi Kita. "Spread and Evolution of
Plasmodium Falciparum Drug Resistance." PubMed. Sept. 2009. Web. 5
Feb. 2010.