ISIS Uncovers E. coli's Mechanism for Selfish Survival

Ben -  The final stop on this week's tour of ISIS shows us how it also has a lot to contribute to the study of biological systems.  In fact, ISIS has been used to shed light on how a naturally produced antibacterial protein gets into cells of E. coli. 

To find out more, I met Dr. Luke Clifton from ISIS, along with Stephen Holt and firstly, Anton Le Brun from the Bragg Institute at the Australian Nuclear Science and Technology Organisation...

Anton -   We have a toxin that's called colicin N. It's produced by E. coli to kill competing E. coli cells.  So, when the E. coli cells in nature are under stress, such as a lack of nutrients, to control the population, the E. coli will produce this toxin to kill off the competing cells so that there's enough nutrients to go around. 

The problem that we were looking at was, E. coli are what we call Gram-negative bacteria which mean they have a double membrane.  How does this toxin cross the outer membrane to get to the inner one?  It uses proteins in the outer membrane to do this.  So, we were looking at how does this toxin bind to the receptor protein and then cross that outer membrane.

Ben -   So, this is a natural antibiotic and you're trying to work out how it works.  Ultimately, what would be the point?  Why do we need to know how it actually gets into the cell?

Anton -   A number of reasons.  A membrane is what we call a hydrophobic barrier.  It kind of prevents things that we don't want to get into our cells like toxins and poisons getting in and so, one aspect of the research is, how do things cross this hydrophobic barrier in general?  Also, it's looking into how large antimicrobial agents kill cells as well, how are they transported across membranes so that in the future, we can manipulate this process to make better anti-microbial agents.

Ben -   Why do you need to use a neutron source like ISIS for this?  Why can't we just look at these cells under a microscope and try and observe what's happening?

Stephen -   Hi, I'm Stephen Holt.  Well we can certainly look at them under a microscope and we can observe physically what's happening to them when you can see their shape change, you can see that their toxin has been effective, but you don't know how they're doing it. 

So, what we want to do is we want to get down to the molecular level and get that detail.  With light, you're restricted in resolution and in other aspects as well such as determining different proteins, just can't do that directly.  With some fluorescent tags, you could, but then once again, you don't have the resolution that's really required. 

We decided to use neutrons to do this to get down to that molecular level and just try and see what each individual component was doing.  I'm sure you're all aware that water is made up of H₂O, hydrogen and oxygen.  Neutrons will interact with hydrogen in one particular way and the real strength is that if you use deuterium as opposed to hydrogen - so that's an isotope of hydrogen - you can have D₂O, so-called heavy water.  Neutrons will interact with that very, very differently.  Then you can do the same thing with your proteins, you can replace some of the hydrogens or all of the hydrogens if possible with deuterium.  So it's like tagging one of the proteins for neutrons.  Then we're able to see the two different proteins as very different entities.  When we do this to them, we can pick them out individually and that enables us to locate them within the complex or within the membrane that we're looking at and to determine how one is moving say, from the solution into the film.  Does it move all the way in?  Does it move part of the way in?  Where is it located?  And we can see very strong differences between when we've got the membrane protein there.  And when it's not there, the toxin ended up in a very different position relative to the membrane.

Ben -   Luke, what are you actually doing with ISIS in order to see these interactions?

Luke -   Well, for this particular piece of work we used two of the techniques we have available at ISIS.  One was small angle neutron scattering which allows us to examine the low resolution structural particles in solution and the other was neutron reflectometry which allows us to analyse the structure surfaces.  Small angle neutron scattering is particularly useful in looking at the structure and structural changes of proteins in solution whereas a neutron reflectometry is good at looking at the structure of a model biological membranes and interactions with these.

Ben -   I assume their names give you a clue as to what's actually happening?  So the small angle scattering involves seeing what happens when you aim the neutrons at a very acute angle and see how they scatter whereas the reflectometry involves looking at how they bounce off?

Luke -   That's correct, yeah.

Ben -   What have you actually found out about this protein?  How is it getting into those cells and doing its antibiotic work?

Stephen -   The amount of the toxin protein that went into the film, there's just no way that can be sitting inside this ion channel which is usually used just to enable individual ions - you have chlorine, etc. - to come in and out of cells.  There's just no way that we could've stuffed that amount of protein inside these channels.  It just wasn't physically possible.  So then we figured the only that can be happening is that it's sitting on the outside.  And so then, when the small angle neutron scattering work was done, the structure, the low resolution structure that came out for the complex very clearly showed that the colicin N toxin is actually going up between the membrane protein and the lipids in which are sitting in it, and it's not completely unfolding and going through this ion channel, but it's actually going around the outside and not having to unfold and perturb its structure as strongly as it would do if it went the other way.

Ben -   So, it seems to be taking advantage of a weakness between a membrane protein and the membrane itself and that's how it sneaks through.

Stephen -   Whether you called a weakness, I'm not sure.  It's clear that if you just add any protein into the presence of E. coli, it doesn't go through that barrier.  So, there's still something very special about the way that colicin N toxin can go through this.  So this is not a general transport route.  It's specific to this particular protein and then if we can understand some of that specificity, then that perhaps is a way to begin to attack bacterial cells.

Ben -   Stephen Holt from the Bragg Institute and before him, Luke Clifton from ISIS, and fellow Bragg Institute member, Anton Le Brun.