Chemical harpoon proteins

Over the course of this summer I looked at bacterial adhesion proteins of Gram-positive bacteria. These adhesins, proteins found on the surface of bacteria, mediate binding to host tissues which is the first step in an infection process (Figure 1). Given the rise in antibiotic resistance, adhesins are an interesting new target for drugs that combat bacterial infections because unlike antibiotics, which kill bacteria and thereby create a strong selective pressure to develop resistance, targeting adhesins does not target bacterial viability.


Figure 1. Adhesion of Lactococcus lactis to the protein fibrin which is involved in blood clotting (Photo taken by Professor Manfred Rhode, Helmholtz Center for Infection Research Braunschweig).

My research focussed on thioester domain proteins (TEDs) of vancomycin-resistant Enterococci and Clostridium difficile, two pathogens of particular interest because of the hospital acquired infections they cause and their resistance to a vast number of antibiotics. TEDs were termed ‘chemical harpoon proteins’ because of the strong covalent bonds they form to a target (this is outlined by Dr. Schwarz-Linek in a video where he explains the the chemical harpoon mechanism  http://www.st-andrews.ac.uk/stories/2017/breaking-bonds).

The first step in my project was getting from a DNA sequence, encoding the TED, to a purified protein for use in structural studies, which as it turned out required a lot of work and optimization. Purifying the VRE-TED took up the first four weeks of the project because the VRE protein turned out to not be stable in solution. This meant that crystallization trials could not be performed and as a result one of my aims, solving the structure of the VRE-TED using X-ray crystallography, suddenly became unreachable. By the time I had designed a new construct and was ready to start over from the beginning I had run out of time. Luckily, some information could be gained from the first construct, revealing which exact amino acid forms the thioester bond about the VRE-TED.

Contrarily to the VRE-TED, cloning, transformation, expression and purification of the second TED went incredibly well. Normally a lot of optimization, for example finding the optimal pH, temperature or buffer for protein expression, is required until a pure, stable protein is obtained and can be used in structural studies. The most exciting moment of the whole project was obtaining a photo of a crystal of the C. difficile TED after weeks of work leading up to this result (Figure 2).


Figure 2. Crystals of purified CdTIE-B-TED were used in X-ray crystallography to obtain diffraction images.

Another aspect of my research was looking at binding of TEDs to six different cell lines, for example to colon cells and fibroblasts. I used two C. difficile TEDs that had been engineered to contain a tag which allows detection by fluorescence. No binding was detected for any of the cell lines which made the cell binding experiments rather frustrating. Luckily my supervisor has a very pessimistic, some might say realistic, view on outcomes of experiments and reminded me that whatever experiment I was doing, it would very likely not work. This lowered my expectations and made me appreciate any results and made accepting that once again after a long day of carrying out a cell binding experiment none of the TEDs had bound to any cells more bearable.

Looking back at the eight weeks spent in the Schwarz-Linek Group has made me realise and appreciate how much I have learned. One of the most beneficial aspects was the environment I was in. Being surrounded by Master and PhD students allowed me to discuss the challenges of scientific research I experienced while carrying out my own project. It has also given me a more realistic view of the life of a PhD student and has thereby changed my expectations towards a PhD.

This experience has opened my eyes to the world of actual scientific research and made me value the few, sometimes unexpected, results which in my opinion make research worth it. I am very thankful that the Laidlaw Undergraduate Internship in Research and Leadership Programme 2017 funded this project and gave me this incredible opportunity and for the amazing supervision and guidance of Dr. Schwarz-Linek and Ona Miller.

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