PAAR proteins decide which T6SS spike arrow is being fired outside of a bacterium

Bacteria firing powerful spikes with hats

This is part 3 of the bacteria-killing-with-arrows series. The first two parts were bacteria killing each other and bacteria firing powerful spikes. You might want to read these two before delving into this current article.

This current article is based on a study that has just been published and which caused me and my former lab buddy long evenings discussing it and coming up with a great (and fun!) concept.

So I guess you remember the arrow bacteria use to fire into other bacteria. Different lethal toxins are attached to this arrow. When bacteria fire this arrow together with the toxins nearby prey bacteria, this usually leads to death of the prey bacterium.

In this study we look at the very tip of the arrow, because on top of the spike of the arrow sits another protein. We call this protein PAAR and it is a super sharp protein. This sharp needle-like protein helps to punch a hole into the prey bacterium. So this super sharp PAAR protein seems to be quite important for the firing and punching process.

Our beloved bacterium Pseudomonas aeruginosa contains seven different PAAR and ten different spike proteins. However, we still don’t know which PAAR sits on top of which spike and which of the PAARs plays a double role and occupies two spikes.

Here we focused on an arrow that is composed of three G2b spike proteins (purple in the below figure). We already knew that G2b is a special spike protein because it contains an extension which at the same time is a toxin (purple pacman). Then we could also show that the toxin Tle3 (red pacman) binds to the G2b spike and is delivered together with this spike.

This already makes this arrow pretty special, because it carries more toxins than the other arrows that I talked about before. In the previous article I discussed the G4b spike that carries the A toxin, and the G5 spike that carries the B toxin. So in many cases it is one spike – one toxin, while the G2b is one spike – two toxins.

Then we wanted to know which of the many PAARs might binds on top of the G2b spike. We indeed found that when PAAR3 was not present in the bacterium (we also tested a bunch of others!), no G2b and no Tle3 were fired out of the cell. Also, neither Tle3 nor the toxin domain of G2b was delivered into other bacteria. This likely means that PAAR3 (orange) sits on top of the G2b spike.

A typical T6SS spike consists of a VgrG timer decorated with cognate effectors and a sharp PAAR protein

By accident, we then found that when the PAAR3 is not present in the cell, the A toxin (from the G4b spike) was delivered more efficiently. This was surprising, as this means two things: the A-G4b spike has another PAAR on top than PAAR3. Second, PAAR3 seems to inhibit the delivery of the A-G4b spike.

But that is something new. The G2b-Tle3 spike seems to be preferably delivered than the G4b-A spike. But was that also the case with other spikes? Could PAAR3 together with the G2b spike and its many toxins even be the favoured spike of all arrows?

We tested that by looking at another toxin. We found that the G2a spike with the Tle4 toxin was also delivered a lot more efficient when PAAR3 was missing in the cell.

So this means that there is some kind of hierarchy when it comes to the spikes and arrows to be fired. And it looks as if PAAR3 together with the G2b spike and all those toxins is the master arrow to be fired.

Let’s take a step back and look at the whole picture here.

We know that the PAAR protein is actually the protein that is first binding to the crossbow before the rest of the arrow is loaded. We also know that to each PAAR protein a specific spike (G2b, G4b or G5…) is binding. Thus, it seems as if the PAAR protein is something like the “sorting hat” when it comes to making the decision which of the many spikes and thus which of the partner toxins are delivered. This is actually pretty cool and is shown in the picture below.

PAAR proteins decide which T6SS spike arrow is being fired outside of a bacterium

So, as I said at the beginning, those pointy PAAR proteins are really important in the whole arrow firing process. Looks as if they are even more important than we thought.

This was the third articles of the bacteria-firing- spikes series and if you want to read more, have a look at those publications or send me an email with questions or drop me a comment. I’m always happy about any type of feedback.

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