Preventing malaria from proliferating

"Plasmodium falciparum", the parasite that causes malaria, proliferates by producing dozens of daughter cells during a single cell cycle, unlike yeasts or human cells, for example. During this process, each parasite replicates its genetic material several times thanks to the specific genes being studied by the scientists. To achieve this, the team is using molecular cloning to produce DNA that contains non-functional copies of these genes.

Once the mutated DNA has been reproduced, the scientists use bacteria to amplify and obtain it in large quantity. To do so, they introduce the DNA into bacteria applied to plates coated with agar, a gelling agent that allows them to develop. The researchers can then determine which bacteria have succeeded in producing the mutated DNA of the parasite.

The scientists cultivate "Plasmodium falciparum" in vitro: it develops within human red blood cells in a rich culture medium that is similar to the blood environment. Each day, this culture medium (seen here) needs to be replaced to provide new nutrients. All manipulations are carried out under sterile conditions to prevent contamination by bacteria or other micro-organisms.

Once the red blood cells have been placed on the density gradient, and following a centrifugation step, the mature (less dense) cells will gather within a precise zone of the gradient.

To monitor the density and developmental stage of the parasites, the scientists prepare a blood smear on a glass slide. This adds contrast and improves visualisation of the pathogens within the red blood cells. Staining is a two-stage process: the smear is first dipped in a solution of eosin (a pink stain) that enables the researchers to better see certain parts of the parasite, and then in a solution of methylene blue, which stains its DNA violet. This also makes it possible to count the pathogens and determine their age thanks to the number of nuclei containing DNA.

The scientists use a fluorescence microscope to follow the development of "Plasmodium falciparum". The stained proteins enable them to determine the different stages of growth by observing where these colours appear in the cellular structure of the parasite.

A high-resolution microscope is used to understand how the parasites develop. These are placed in a well on the slide and subjected to a laser that excites a fluorescent protein they contain. The latter absorbs the laser light and in return transmits a light source that renders it visible. The results are then analysed in order to elucidate the DNA replication rate during the different developmental stages of the parasite. The aim is to eventually be able to identify targets for future treatments and contribute to the decline of malaria across the world.