I know I know, by now you probably think you have heard and read enough about the fact that bacteria can kill each other. But let me tell you a new story of how we tried to manipulate a bacterium to use new toxins to kill other bacteria.

It all started in the very first weeks of me embarking on that PhD road back in 2014. Time’s flying when you’re having fun, right?!

My supervisor wanted me to mutate our house bacterium Pseudomonas aeruginosa and give it new killing powers. We thought that if we use the killing machine of this bacterium and make it fire even more toxic arrows, Pseudomonas might become the superstars between the arrow shooting bacteria.

The type 6 secretion system and its spike

You remember the so-called type 6 secretion system that is this super cool killing machine in some bacteria? And how this machine looks more or less like a bow and arrow like in the picture below? When you look at the model of this arrow (in green), it does indeed have the shape of an arrow or a spike.

Some of these arrows are longer than others because they have so called extensions that can be toxins.

Changing the spike

Well, we wanted to see whether we could use one of these arrows from our beloved bacterium and fuse any random extension to it. Then, we wanted to check whether our bacterium can use and fire this modified arrow with extension. The idea was, if the modified arrow would behave the same as the “normal” non-modified arrow, we might have found a way to manipulate bacteria to shoot any kind of arrow we want.

So, we started easy and took two very similar strains. One strain is called PAO1 and this one has an arrow with an extension; the other strain is called PAK and none of its arrows has any extension. So, we took this extension (which we will just call E here and in purple in the pic below) and fused it to an arrow without extension in PAK. We then saw that PAK can indeed shoot this E-arrow out of the cell. First success 🙂 Happy times!

We then tried a pretty random extension, an enzyme from the bacterium Escherichia coli. Let’s call this enzyme Bla – which actually stands for beta-lactamase, but I just love this abbreviation 🙂 So, we fused this Bla enzyme to our arrow in Pseudomonas aeruginosa and again, we could see that our bacterium can fire the Bla-arrow out of the cell.

… and into another cell

Now, Bla is also a so-called reporter enzyme which means we can follow its location. This is possible, because Bla breaks down certain chemical compounds (which I will call CC here). This broken compound then turns blue. So, we set up an experiment, in which we infected tumor cells with our bacteria. We then gave CC to the cells, which the cells can easily take up.

If our bacteria fired the Bla-arrow into the cells, Bla would break down CC and we could measure the colour change towards blue. This colour change would not occur in cells that did not fire Bla, as well as all the other controls we needed to set up – and there were LOADS of them!

However, when we did the experiment, we could not see a change in colour. This meant that our bacterium could not fire our Bla-arrow into tumor cells. Okay, I tried, sad for a while but let’s move on.

(You also need to talk about your negative results, even though it hurts a bit.)

From the tube to the tip

And we came up with something crazy.

You probably remember that our bow and arrow sits on a long tube within the bacterium, and that the contraction of this tube is actually what drives the arrow out of the bacterial cell like in this video below.

by Prof. Marek Basler, Biozentrum University of Basel – Bacterial nano-sized speargun – the so-called Type VI secretion system works like a power drill.

Because bacteria are super efficient (we should all learn from them!), they even put some of these toxins inside the tube. So when bacteria fire the whole arrow out of the cell, these toxins inside the tube are also fired outside.

What we now wanted to test, is whether we can use one of these toxins (let’s call this one T) for our arrow shuttle. So, could we make our bacterium fire T together with the arrow spike instead of the tube? To try this, we fused T to the arrow instead of having it as a protein on its own.

Again, we could see that our bacterium can fire this new T-arrow, but in a less efficient way than the other modified arrows we made before. And we also saw that the T-arrow was not very stable. So, for some reason T seemed a bit unhappy at the top of the arrow. We assumed that T was used to contact his friends, the tube proteins, and was probably still doing that.

Hence, we decided to inhibit this interaction between T and the tube proteins, because another group before already showed how to do that.

But this was when the whole mess started. The T-arrow was not stable anymore. There was no firing of the T-arrow anymore. Our machine would not work properly and I had no idea what was going on.

It was a very confusing time, for both the bacteria and for me.

So, we decided to call it and leave it as it is.

Let me kill you

But then there it was – another idea evolved.

We knew that T was toxic, so it could kill!

Could our bacterium fire the T-arrow into other bacteria and actually kill them just with this arrow?

So, I tried and yes it killed, even though only in very very very small amounts. But this was understandable, because the T-arrow was only fired in small amounts anyway. Obviously, if not many T-arrows leave the cell, not many will be pushed into another bacterium and kill there.

So, this was a success – small, but relevant!

This meant, that our initial hypothesis was actually true!

We mutated our bacterium and gave it the power to fire new arrows.

These new arrows could have all sorts of powers. One can imagine, fusing other toxins to the arrow that might specifically kill certain bacteria (maybe just use a better one than T!!!).

And maybe one day, one will find an arrow that can be fired into cancer cells and then one can load this arrow with a drug to fight cancer cells.

But this still requires a lot of research. Like… a lot.