Fluorescence and Failure

By Jack Sewell

Project aims

This post is about the malaria lab project that we've been working on full-time this semester. To avoid giving away too much information about what the lab is investigating, I won't mention the specific proteins under study. Essentially, the project has involved each of us studying the expression and localisation of a separate protein during the life cycle of Plasmodium, the malaria parasite. In addition, we aimed to study the morphology of the parasite when the protein was absent using a PTD construct. This removed expression of the protein from every stage in the life cycle, except for the blood stage where it is known to be essential. The point of studying the proteins was to identify a potential drug target for malaria treatment. The first thing we did was to transfect Plasmodium with a fusion gene, which coded for our protein of interest followed by the green fluorescence protein (GFP) tag. This would later allow us to see where our protein was in the cell. However, first we had to battle through the process of producing a Western blot, in order to show that the protein and GFP had been successfully fused. As Carrie has written about previously, this resulted in lots of failed attempts, with each one inevitably falling foul of a mistake that would leave us scratching our heads.

It's in there somewhere.

Life cycle imaging

Eventually though, we all had a band that we could confidently call our protein, and could then focus on the fluorescence imaging of the various Plasmodium life cycle stages. Because each attempt to transfect our PTDs into Plasmodium failed, this meant we could only study the protein localisation and not the phenotypes of the knockout. What I would consider to be the major difficulty in this part of the project was the time taken to culture the different life cycle stages. The process of growing Plasmodium in mouse blood takes around 3-4 days, after which time the mouse must be euthanised to prevent unnecessary suffering. The blood can then be used to image the parasite blood-stages (rings and trophozoites), or cultured for a further 24 hours to produce either schizonts or a mix of gametocytes, zygotes and ookinetes.

Plasmodium stages that can be imaged from mouse blood

Furthermore, studying the mosquito stages of the life cycle takes a total of 21 days following the collection of mouse blood. The mice must first be treated to eliminate all parasites except the gametocyte stages, then the mosquitoes are permitted to feed on them. At this point, the gametocytes in the mosquito gut become activated and leave the red blood cells. You then need to wait 10 days before some of the mosquitoes can be checked for oocysts lining the midgut, and mature oocysts can be imaged on day 14. After another week, on day 21, it should be possible to image sporozoites in the mosquito salivary glands. This completes the stages of the life cycle that can be studied, because the host's liver-stage parasite infections cannot be studied without complex dissection that would also render the mouse useless for study of the other stages. Unfortunately, the day 10 check of the mosquito infection for my protein showed no oocysts, meaning that it may not be possible to complete the oocyst and sporozoite imaging. This has therefore been a potential waste of 10 days, and would need the use of more mice and mosquitoes if a second attempt is to be made.

The Fluorescence Microscope

The actual process of taking parasite fluorescent images starts by mixing infected blood with DAPI, a DNA-specific stain that highlights the nucleus blue. In addition, the activated female, zygote and ookinete stages can also be tagged in red. This works by using a red fluorophore conjugated to an antibody the specifically binds to a protein expressed on the surface of these cell types. Once the fluorophores have been added, the blood can be searched for the desired stages under the fluorescence microscope. Any cells of interest must then be measured for their blue and red fluorescence, as well as the green fluorescence emitted by the GFP-tagged protein.

Activated female gametocyte with its membrane, nucleus and protein highlighted.

Getting a good image was always more difficult than you'd think, given that we had to make do with a single microscope shared between 4+ people, who often all had parasites to image at the exact same time. As well as this, we had to get the cell concentration exactly right, or they would be too sparse to find anything of interest or too dense to get the cell on its own. After many mice though, we were each able to create a complete collage to show the localisation of our protein at each of the ring-to-ookinete stages. All that remains now is to finish the study of the mosquito stages, and the project will be completed.

All in all there's been a lot of frustration and disappointment, but with some perseverance we got good results in the end. We also gained a lot from being able to adapt and learn from the things that went wrong, which in the end is a very important skill for a lab scientist to have.