Growth at the tip – A successful project at the Biozentrum

During his project thesis, master student Julius Winter studied the assembly of a nano-harpoon.

Students enrolled on a master’s program in nanosciences at the University of Basel complete two projects in two different subjects before going on to write a final master’s thesis. Julius Winter, who is currently completing his master’s degree in nanosciences, completed one of his projects in the group led by Professor Marek Basler at the Biozentrum. And it was a huge success. Working alongside his supervisor, Andrea Vettiger, he studied the assembly of a nano-harpoon used by bacteria to inject toxins into other cells. The results of the project were published in Nature Communications in July.

Armed with nano-harpoons
Bacteria use a variety of “weapons” to establish themselves in their natural environment. One such weapon is a tiny harpoon that they can use to quite literally shoot other bacteria and cells and to inject toxins directly into them. Various species of bacteria use this nano-harpoon to gain an advantage over competitors or to protect themselves against host’s immune cells. The group led by infection biologist Professor Marek Basler from the Biozentrum studies the assembly and mode of action of this type of secretion system, known as the Type VI Secretion System (T6SS), in various bacterial species. Through their work, the scientists hope to identify new approaches to the treatment of bacterial infections, as the harpoons often play a crucial role in the infection process.

The scientists in Marek Basler’s group have already been able to establish that the harpoon consists of an inner dart with a sharp tip, as well as a spring-like outer sheath. The outer sheath can contract within milliseconds, firing the central dart out of the bacterium in order to puncture the cell membrane of adjacent cells. This process allows toxic substances to be injected into target cells directly using physical force. After the harpoon is fired, the entire injection apparatus is broken down into its individual parts and rebuilt. Until now, the researchers had not obtained clear proof of whether during T6SS assembly the sheath extends from its base or whether the growth takes place at the sheath tip.

Julius Winter has worked together with Andrea Vettiger (right) in the group of Professor Marek Basler (left) at the Biozentrum.

Experimenting with giant cells
This is where Julius Winter stepped in with his project. For his experiments, he worked with cells of the cholera pathogen, Vibrio cholerae. By adding the antibiotic ampicillin, which prevents synthesis of the cell wall, he first produced cells that grow without a cell wall, meaning they are no longer able to divide and instead keep on growing continuously. When treated with antibiotics, the bacteria lose their typical rod-like shape and swell up like a balloon. These cells, also known as spheroplasts, reach a size of several micrometers, making microscopic analysis easier.

To track the assembly of the harpoons, Julius Winter and his supervisor, Andrea Vettiger, selectively marked the sheaths with green fluorescent protein (GFP). This makes it possible to observe the sheath assembly in real time in living cells using fluorescence microscopy. The scientists were first able to show that the spheroplasts without a cell wall could still form working harpoons. Then, to find out how the harpoon is formed and from which end it extends, they fired a fine, high-energy laser beam at the cells for a fraction of a second, with the beam illuminating a specific part of the assembeling sheath. “The laser doesn’t damage the sheath but reduces the emission of the fluorescent protein, leaving a bleached spot that appears dark under the fluorescence microscope,” Julius Winter explains. The researchers then examined whether, during the sheath’s growth, the dark area of the sheath is displaced or the area at the top of the sheath becomes longer. This analysis provided the first experimental evidence that the harpoon sheath does not assemble by adding subunits at the fixed base, but rather at the distal end.

The results, published in Nature Communications in July, also clearly showed that the longer sheath structures assembled in the spheroplasts without a cell wall are ideal for investigating processes of dynamics and the mode of action of the bacterial harpoon. For Julius Winter, it was a pleasant surprise to become coauthor of a paper in such a renowned journal as a result of his project work.

Valuable experience at UC Berkeley
In the meantime, Julius Winter has completed another project at the University of California, Berkeley (UC Berkeley). “I took the completely unbureaucratic step of emailing Dr. Jeff Urban’s group to ask about the possibility of a research stay, and I received an offer immediately,” he says. Equipped with a travel grant from the SNI, he spent four months working on the desalination of seawater using nanoparticles. He particularly enjoyed the unique atmosphere at UC Berkeley and its proximity to San Francisco.

Now unable to let go of working in a biological laboratory and especially with fluorescence microscopy, he returned to Marek Basler’s lab for his master’s thesis. There, he continued to study the bacterial harpoon. This time, he developed a microfluidic system that allows rapid replacement of the culture medium, so that it is possible to study the influence of various components of the nutrient medium on the assembly of the bacterial harpoon. “During his short stay in our lab, Julius made fantastic contributions to our understanding of T6SS assembly. It was obvious to me that Julius has a great talent for perfoming even complicated experiments. In addition, he is very curious and keen to find answers to biological questions. I was therefore very happy that Julius decided to come back to our lab and help us to develop a new technology to study bacterial behaviours,” comments Marek Basler.

First physics and chemistry, then biology
Julius has now almost completed his studies in nanosciences. When he chose this field in 2012, it was above all the combination of physics and chemistry that interested him. Over the course of the various lectures and practical work, he has also developed a fascination with biological questions. The 23-year-old German wants to continue working in research, although he doesn’t necessarily want to start a doctoral dissertation straight away, and above all he’d like to gain more experience outside of Basel. We wish him the best of luck and enthusiasm for the future as he takes the next step in his career.