In a step towards a possible taming of the malaria parasite, researchers at the Hyderabad-based CSIR-CCMB have developed a novel and economical way of gene delivery within Plasmodium falciparum (P falciparum) cells.
The ease of making genetic alterations in the parasite will aid in better understanding of the biology of the malarial pathogen, and thereby help in its control, says Rakesh Mishra, Director, Centre for Cellular and Molecular Biology (CCMB).
The Lyse-Reseal method
Called the Lyse‐Reseal method, the CCMB technique helps in easier delivery of DNA (deoxyribonucleic acid) into the cells of the parasite’s nucleus. It is as effective as the electroporation method that is presently in use, according to Puran Singh Sijwali, Principal Scientist at CCMB, and his team.
The researchers have demonstrated the technique with two different strains of P falciparum. It works with 10 times less DNA than what is required in electroporation, they claim.
Gene delivery into the target cells is a popular choice to manipulate and study gene functions. However, there are several technical challenges in studying Plasmodium genes.
Plasmodium causes malaria when it grows in the oxygen‐carrying red blood cells (RBCs). While growing inside the RBCs protects the parasite, it poses a huge challenge to biologists, as it requires crossing through four membrane layers to reach the Plasmodium’s genes.
A widely used method to manipulate genes is electroporation, which uses electric shocks to introduce DNA into the cells. It is a standard gene transfer technique used in genetic modification of crops, mice etc.
The main advantage of the CCMB method is that it does not require an expensive electroporation device and other proprietary accessories. Hence, it can be used for Plasmodium genetic studies even in low‐resource labs, which are more common in malaria-endemic regions.
How the method works
Explaining the method, Puran Singh says: “RBCs open up or lyse when they are in hypotonic solutions (with salt concentration lower than inside the cell). This allows them to fill in the lysed RBCs with circular DNA of their choice.”
Increasing the salt concentration in the solution again lets them close the RBCs, getting ‘resealed RBCs’. They infect the resealed RBCs containing the particular DNA with Plasmodium falciparum. The parasite goes inside the RBC and passively takes up the DNA from the RBC there. The DNA eventually ends up in the parasite’s nucleus with its own genes.
The CCMB team of Puran Singh, Deepak Kumar, Zeba Rizvi and Gokulapriya Govindarajulu have also demonstrated that RBCs of blood group O+ provide the most efficient delivery of DNA into P falciparum in-vitro. This study was recently published in Scientific Reports.
A tough parasite to crack
No parasite has frustrated human efforts to tame it more than Plasmodium falciparum. For over a few thousand years, it has not just survived, but seems to be thriving.
The WHO estimates 3.4 billion people across 92 countries to be at risk of malarial infection. It’s a huge health burden on developing countries such as India.
A statistic as alarming as this makes it imperative to study the parasite biology with a focus on understanding the functions of its genes and identifying targets for the development of drugs and vaccines.
The parasite has not only become resistant to drugs, it also foxes products that try to keep it off homes, and has successfully avoided an effective vaccine. In recent years, though, the fight against Plasmodium has become more intense with several novel medical techniques being tried out.