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Generating malaria parasite gene deletion mutants
In September 2015, the 193 countries of the United Nations adopted 17 Sustainable
Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all.
As outlined in the 2030 Agenda for Sustainable Development, specific targets are set for
each goal over the next 15 years. The fight against malaria is one of the targets of goal 3
(ensuring healthy lives and promote well-being for all at all ages). The research carried out
by Prof. Dr. Frischknecht and Mirko Singer from the Centre for Infectious Diseases at
Heidelberg University Hospital is one of several steps towards eradicating malaria.
Anopheles stephensi, one of the Plasmodium species that cause malaria, sucking blood. © University Hospital
Heidelberg/Singer
Malaria is the most common tropical disease and is caused by parasites that are transmitted
by female mosquitoes of the genus Anopheles. There are around 300 million new cases of
malaria and nearly half a million deaths from malaria every year. Researchers around the
world have been working on finding an effective malaria vaccine for many decades. The latest
of these, RTS,S, gives reason for some hope despite its limited efficacy. New research from the
US suggests that releasing genetically modified, Plasmodia-immune mosquitoes could be a
way to block the transmission of the malaria parasite. Resistance against Plasmodia was
achieved by introducing resistance genes using the powerful but controversial CRISPR geneediting technology. The idea is that the mosquitoes carrying the genes that block the
transmission of the malaria parasite will propagate and eventually eradicate the parasites. The
problem is that over 60 malaria-transmitting parasite species would need to be modified, and
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releasing genetically modified parasites could have unpredictable effects on the ecosystem.
Prof. Dr. Frischknecht from the Centre for Infectious Diseases at Heidelberg University Hospital
pursues a different route involving the genetic modification of Plasmodia. He has recently
published his results in the journal Genome Biology1. Plasmodia are mobile, unicellular,
eukaryotic organisms that have evolved a deceptive multiplication strategy: they enter the
bloodstream of human individuals through the bite of an infected female Anopheles mosquito
and invade the liver. They grow and multiply in the liver cells and subsequently in the red
blood cells. The parasites are safely enclosed in the erythrocytes where the human immune
system is unable to detect and destroy them. The parasites inside the red blood cells multiply,
the infected cells burst and the parasites continue to infect other erythrocytes, generating
cyclic symptoms. The infected mosquito also carries the disease from one human being to
another. Infected individuals become very weak: typical malaria symptoms include recurrent
fever and anaemia. Some species cause severe malaria and death; it is known that both
Alexander the Great and Tutankhamun died from malaria.
Cleaving the own genome
Liver cell containing late liver stages of the Plasmodium parasite, called merozoites, which infect red blood cells.
DNA is labelled red, actin green and the merozoites blue. © Universitätsklinikum Heidelberg/Singer
“The evolution of eukaryotic parasites is a perfect example of a development that is driven by
sexual reproduction and recombination,” says Mirko Singer, lead author of the Genome
Biology paper. His institute is equipped with several different imaging and biophysical
technologies as well as standard equipment used for molecular and genetic research. The
researchers use all these instruments to study model organisms to find out how parasites
invade the human body and how they can be prevented from causing malaria.
As part of their research, Frischknecht and Singer use genetically attenuated parasites that
lack the genes essential for the liver stage of the parasite and therefore only develop to a
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limited extent. They can be recognised and eliminated by the human immune system. An effect
similar to that achieved by polio vaccines, which actively ‘train’ the immune system to fight off
potential polio virus infections, can be achieved with genetically attenuated parasites.
The researchers have so far specifically focused on deleting between one and three parasite
genes to stop the parasite developing in the liver and make it easy prey for the immune
system. However, such parasites, along with parasites that have been attenuated with γ
radiation, can still cause breakthrough infections in rodents during immunisation and result in
pathological blood-stage infections. This is one of the major reasons why transferring the
method to humans is difficult and risky. The Genome Biology paper describes an alternative
method for producing genetically attenuated parasites (GAP). The goal is to generate parasites
that could potentially be used as malaria vaccines. “We wanted to develop a method that can
stop parasite development at a specific stage by precisely timing the point of developmental
arrest,” says Singer. In order to achieve parasites that would not lead to breakthrough
infections, Frischknecht introduced zinc-finger nuclease (ZFN) genes into one of the parasite’s
14 chromosomes. ZFNs are restriction enzymes that create double-strand breaks in DNA at
user-specified locations. When activated, ZFNs specifically recognise a chosen target site within
a genome and cleave the DNA at user-specified locations.
The arms race of the parasites
Mirko Singer presents a method that enables several hundred genes in Plasmodia to be deleted. This modification
leads to attenuated parasites. © BIOPRO/Hinkelmann
Nevertheless, some parasites survived and led to an infection in the rodents. Singer was also
able to identify the parasites’ DNA repair mechanism, which is called rudimentary
microhomology-mediated end joining, that enabled the parasites to survive, thus causing
breakthrough infections. “Little is yet known about this repair mechanism,” Singer said. It is
also possible that vaccination with genetically attenuated parasites might cause the zing-finger
nucleases to mutate. “So we need to use several, mutually supportive safety mechanisms,”
says Singer alluding to what could happen with immunisations involving insufficiently
attenuated parasites.
Plasmodia are highly adaptive single-celled organisms that have evolved resistance to almost
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all standard malaria drugs. Numerous attempts to eradicate the disease have therefore failed.
“I do not believe that malaria will be eradicated within the coming decades,” says Singer.
Although the WHO Malaria Report found that increased prevention and control measures have
led to reduced malaria mortality, Frischknecht is not so sure that it can be eradicated as
quickly as people are hoping. “In order to further reduce the number of malaria mortalities,
and work towards the eradication of the disease, the WHO is planning to provide three times
more annual funding than it actually has available for achieving this goal.”
Frischknecht and Singer have shown that in principle it is possible to attenuate the parasites
despite the fact that some of them mutate and become pathogenic again. A potential vaccine
would present the entire repertoire of surface antigens to the human immune cells. Whether
the researchers’ attenuated parasites could become an effective anti-malaria vaccine is not yet
known. “There might be a completely different solution to eradicating malaria, or none at all,”
concludes Singer.
1 Original
publication: Singer et al.: Zinc finger nuclease-based double-strand breaks attenuate malaria parasites and reveal
rare microhomology-mediated end joining, Genome Biology (2015) 16:249, DOI 10.1186/s13059-015-0811-1
Article
22-Feb-2016
Jens Hinkelmann
© BIOPRO Baden-Württemberg GmbH
Further information
Prof. Dr. Freddy Frischknecht
Tel.: +49 (0)6221 5665-37, -46
E-mail: freddy.frischknecht(at)med.uni-heidelberg.de
Research group Frischknecht, University Hospital
Heidelberg
The article is part of the following dossiers
Vaccine development
prevention
vaccine
genetic engineering
immune system
malaria
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