Bacteria firing powerful spikes

This week we will talk about one of my favourite bacterial devices. It is the bacterial killing machine, the type 6 secretion system, that I explained in detail in my very first post Bacteria killing each other. If you don’t remember well what is was, it is worth going back to refresh your memory, as we’re now looking closer into this fascinating nanomachine.

This post is based on a journal article which was just published and which I would like to share with you here. It was also the longest and most tedious chapter of my thesis, so I have strong feelings about this post 🙂

I hope you remember that this bacterial weapon resembles a crossbow with an arrow. At the end of the arrow sits a pointy spike which carries the toxin into the neighbouring bacterium. The spikes of these arrows are made of three of the same proteins and bacteria can have multiple of these spikes carrying different toxins. However, we do not really have a clue about how the spike carries the toxin into other bacteria.

An attacking bacterium fires its T6SS arrow into a competitor

Here, we were looking very closely at this spike and how exactly the toxin is glued to it.

We focused on two toxins which are PldA (called A from now on) and PldB (called B from now on) in our favourite bacterium Pseudomonas aeruginosa. A and B are structurally very similar which means their killing actions are very similar. However, they seem to be delivered by different spikes.

When looking at the chromosome, we saw that the gene for A is right next to a gene for the spike protein G4b and the gene for B is right next to a gene for the spike protein G5. A random connection?

For this and all the following questions, we performed two sets of experiments. With results from both experiments we could then conclude whether delivery of the toxin was successful or not.

For one, we checked secretion of the spike proteins and the toxins. This means, we grew our bacterial strain of interest for around 8 h and tested whether we would find the spike and the toxin of interest outside of the cell. If that was the case, it would mean that the crossbow indeed fired the arrow with the spike and the toxin to the outside.

We also tested whether the toxin of interest was carried all the way into neighbouring bacteria. For this we made so called prey strains. This is a Pseudomonas aeruginosa bacterium that does not have the corresponding immunity to the toxin of interest. Thus, to test delivery of toxin A, we used a prey strain that did not have the immunity for toxin A. We then grew this prey strain together with an attacking strain which was our test strain. If the attacking strain was able to deliver toxin A into the prey strain, the prey strain would die. Then after 24 h we would have a lot less prey strain in our mixture than attacker strain. And because the prey strain had a different colour that our attacking strain, we could count exactly how many prey bacteria survived. Basically, if the prey strain did not survive, the toxin was delivered by the arrow from the test bacterium.

An attacking bacterium fires its T6SS arrow into a competitor

To go back to our question, we first showed that toxin A is delivered by the spike formed by G4b and toxin B delivered by the spike formed by G5.

So it looks as if our Pseudomonas aeruginosa fires at least two different arrows with two different spikes. One spike is likely formed of three G4b proteins (green in the following pictures) together with the A toxin (green pacman) and one spike of three G5 proteins (blue in the following pictures) with the B toxin (blue pacman). Or at least that’s what we thought….

T6SS spikes consist of VgrG trimmers decorated with specific effectors

Then we saw that both the G4b and G5 proteins not only contain the spike forming parts, but also some interesting extensions (green and blue arcs). These extensions are only present in certain spike proteins in a few bacteria. Interestingly, the spike parts of G4b and G5 are 69 % identical and these extensions only 25 %. So would these extensions be the specific parts to carry their partner toxins?

We started to answer these questions by swapping the two extensions between G4b and G5. We then analysed whether the G4b spike could still deliver the A toxin and the G5 spike the B toxin.

T6SS spikes with swapped effector recognition domains

And indeed we found that A would not bind to this altered G4b spike anymore and B not to the altered G5 spike. Neither spike could now deliver their partner toxin.

Clearly, the next question was, could the G4b spike now deliver the B toxin and the G5 spike the A toxin?

Starting with the G4b spike, we saw that the B toxin was binding to the G4b spike with the G5 extension. However, it would just not get out of the cell. So somehow we changed the G4b spike too much for it to work properly anymore. Hmm, bummer…

With the second spike however, we could clearly see that the A toxin was bound to the G5 spike. We also showed that the G5 spike delivered the A toxin into other bacteria. Yes, success!

Effector domain swapping of VgrG trimers results in swapping of effector specificity

So while our swapping strategy worked for the G5 spike, it did not work for the G4b spike. Why?

We already know that the G4b spike can only be fired when the G5 spike is present inside the cell as well. However, in these experiments there was no G5 spike present just because otherwise our B toxin would always bind to G5 instead of our modified G4b spike.

Also, Pseudomonas aeruginosa still produces many other spikes that can sit on top of the arrow. With this, our beloved bug has the choice of firing many different arrows to kill other bacteria. You might ask now how this bacterium decides which arrow to fire? Well, this I will explain in my next blog article 🙂 so stay tuned.

But why do we care about those different arrows and spikes? Well, if we can understand how toxins are glued to this amazing killing machine, we might be able to use it for our own purpose. One day, we will hopefully be able to glue antibacterial components to an arrow in order for this machine to kill harmful bacteria. This, however, still requires a bit of research 🙂

Take- away message:

  • bacteria fire arrows using their killing type 6 secretion machine
  • on top of the arrows are spikes to which lethal toxins are glued
  • the spikes have specific extensions that bind their partner toxins

This is part 2 of a series about bacterial killing nanomachines. Read part 1 here.

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