The little creatures inside cells and the host’s response

Mostin Hu
Thursday 11 July 2019

Hello, my name is Mostin Hu and I am going to enter into my second year of Medicine at St Andrews. In this blog post, I’ll be sharing with you what I’ve been up to for the last three weeks.

One of the many fume hoods used for cell tissue culture. When culturing cells, everything must be sterile to prevent cross-contamination and infection of cells with bacteria and fungi. We keep a spray bottle of 70% ethanol to clean our gloves and any bottles before placing them inside the hood!

Background

My research is looking at the role of autophagy in intracellular infection. I know it sounds complicated, but just bear with me for a few minutes and I’ll break it down..!

 

The organisms I am looking at are called Trichomonas hominis and they are part of a larger family of protozoa called microsporidia. They are obligate intracellular parasites, which mean that they need a host cell to survive and reproduce. I know it sounds scary, but T. hominis are non-pathogenic and they usually do not cause any harm [1]. That said though, infection in individuals with a compromised immune system (perhaps due to chemotherapy or acquired immunodeficiency disease) can cause disease, making it important for us to learn more about these parasites so we can develop pharmaceuticals for treating infections!

What is autophagy?

Autophagy is a degradation process which occurs inside of cells. It is responsible for the elimination of unwanted and potentially dangerous materials such as old proteins, damaged organelles (like aging mitochondria), and foreign invading bodies [2] . There are many subtypes of autophagy, but the main goal of this process is to remove anything that is not needed within the cell, kind of like a waste disposal system.

The mechanism of autophagy within mammalian cells [3].
Research Aims

When I spoke to my professor initially, I was really surprised to find out that very little is known about this parasite. Some preliminary research done by a former PhD student of my supervisor had showed the possibility that an autophagy response is triggered when cells are infected with T. hominis but aside from that, there appears to be very little published research on this topic. In my research this summer, I am trying to confirm that autophagy is involved in T. hominis infection, and if it is, whether this response is beneficial or harmful to the parasite.

Research Methods

For the past three weeks, I have been working with rabbit kidney cells intentionally infected with T. hominis. With the addition of an autophagy inhibitor – that is, a chemical which prevents host cells from initiating the process of degrading useless proteins and foreign materials – I have been looking at whether there are any changes to the amount of infection and distribution of autophagy markers. On the molecular level, autophagy is actually quite a complex process involving many molecules recruited at specific parts of the process. One molecule that I have been focusing on is ATG5 (autophagy protein 5), which is a protein that is recruited early in the autophagy process and is involved in the subsequent formation of vesicles (the bag-like structures around materials to be degraded) [3].

The microscopy room where the immunofluorescent microscope lives! The room is dark so prevent light from damaging the light-sensitive staining on the cells.

With the help of immunofluorescence microscopy, I have been able to visualize the distribution of ATG5 within cells. Immunofluorescence microscopy is a powerful method used by many researchers to see specific structures and processes inside of cells. It takes advantage of antibodies which bind selectively to their areas of interest (called primary antibodies), in my case, the ATG5 protein. Next, a secondary antibody is added which binds to the primary antibody. This secondary antibody is fluorescent so it will glow underneath a fluorescent microscope. Finally, a fluorescent DNA stain called DAPI is added so the nucleus of each cell and parasite can also be seen [4].

Another staining technique I have been using is called Giemsa staining. Giemsa is a versatile, bluish-indigo solution used to stain a wide variety of specimens, including blood smears in the diagnosis of malaria [5]. While Giemsa does not stain selectively for any autophagy markers, it does a really good job of distinguishing nuclei from the rest of the cell cytoplasm and extracellular space, which allows me to quantify the amount of infection accurately (yes, that means I count every single cell in the images I take!).

Giemsa stained infected cells under 40X magnification. The bigger magenta-purple ovals are host cell nuclei and parasites can be seen as dots in host cytoplasm.

Preliminary Findings

When I first began my research, I had the lofty goal that I was going to solve the mystery of cellular response during T. hominis infection. Three weeks in and I’m now realizing how unrealistic that really was! Research is a slow process; the cure for cancer won’t come overnight (or in five weeks…) and I have gained a new appreciation for the meticulous nature of biomedical research. Each experiment, new method, and observation must be noted down carefully, each cell counted with specific inclusion/exclusion criteria, each chemical weighed out to the nearest thousandth of a gram. Even more, experiments revealing new findings must repeated at least three times to verify that the novel observation didn’t occur just by chance.

Next steps

More microscopy, more cell staining, and more cell counting. I need to repeat my autophagy inhibition experiment at least one more time and analyze all the pictures from there before I can make any sort of conclusion! I would also like to use antibodies which label for other autophagy markers to see if they are also localizing around the parasites, but antibodies for research are ridiculously expensive ( £300+ for 250 microlitres!!!) so I have to plan my experiments wisely and think carefully about which antibodies will give me the most information for what I’m looking for.

Acknowledgements

I have to extend my sincere gratitude to some very special people: my parents for their everlasting support and for encouraging me to apply, calming my nerves before my interview, and celebrating with me when I received my acceptance; to my professor Dr John Lucocq for taking me under his wing and allowing me (a clueless first-year medic) into his lab, for the long brainstorming sessions we have in his office and for giving me the opportunity to research; to Adam Althobaiti, a PhD candidate who has showed me the ropes around the lab and taught me so much about research techniques and methodology; to the Laidlaw team at St Andrews for giving me this opportunity to grow both as a leader and researcher; and finally, to Lord Laidlaw for generously funding the Laidlaw program which has made this experience all possible.

References

  1. Trichomonas hominis [Internet]. [cited 2019 Jul 11]. Available from: https://www.parasitetesting.com/Trichomonas-hominis
  2. Sharma V, Verma S, Seranova E, Sarkar S, Kumar D. Selective Autophagy and Xenophagy in Infection and Disease. Front Cell Dev Biol. 2018;6(November):1–17.
  3. Dikic I, Elazar Z. Mechanism and medical implications of mammalian autophagy. 2018 [cited 2019 Jul 11]; Available from: www.nature.com/nrm
  4. Hoff F. How to Prepare Your Specimen for Immunofluorescence Microscopy [Internet]. 2015 [cited 2019 Jul 11]. Available from: https://www.leica-microsystems.com/science-lab/how-to-prepare-your-specimen-for-immunofluorescence-microscopy/
  5. Jackson S, Grabis D, Manav C. Giemsa: The Universal Diagnostic Stain [Internet]. [cited 2019 Jul 11]. Available from: http://www.cellmarque.com/cmsial-literature/304-Giemsa-Academia.pdf

 

 

 

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