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  • JOANNE STUBBE: OK, so what I want to do today

  • is hopefully finish up or get pretty close to finishing up

  • module 6, where we've been focused on bacterial uptake

  • of iron into cells.

  • In the last lecture, I briefly introduced you

  • to gram-positive and gram-negative

  • big peptidoglycan, small peptidoglycan,

  • outer-cell membrane.

  • They both have the same goals.

  • They've got to get--

  • They take up iron the same way from a siderophore, which

  • is what we talked about last time, or by a heme.

  • And we'll talk a little bit about that.

  • And that's what you focused on in your problem set.

  • But they have different apparati to do

  • that, because of the differences between the outer--

  • because of the cell walls' distinctions

  • between gram-negative and gram-positive.

  • So we were talking, at the end of the class,

  • about, this was for the siderophores

  • which we talked about.

  • We need to take them up.

  • These are common to all uptake systems.

  • You have some kind of ATPase system and ABC ATPase.

  • We're not going to talk about that in detail,

  • but it uses ATP to bring these molecules

  • and also heme molecules across the plasma membrane.

  • And then, in all cases, you have this issue

  • of how do you get the iron out of whatever the carrier is,

  • be it a siderophore where the carriers can bind very tightly

  • or heme where you also have to do something

  • to get the iron out of the heme so that it can be used.

  • And so what I want to just say, very briefly--

  • and this you all should know now.

  • So now we're looking at heme uptake.

  • I'm not going to spend a lot of time drawing the pictures out,

  • but, if you look at the PowerPoint cartoon, what

  • you will see is there is a protein like this, which

  • hopefully you now have been introduced to from your problem

  • set.

  • So this could be IsdB or IsdH.

  • And we'll come back to that, subsequently.

  • And it sits on the outside of the peptidoglycan.

  • So this is the protein.

  • The key thing that is present in all these Isd proteins

  • is-- let me draw this differently-- is a NEAT domain.

  • OK?

  • And we'll come back to that later on.

  • But this domain--

  • So you have a big protein, and there's

  • one little domain that's going to suck the heme out.

  • And so what happens is we'll see in Staph. aureus, which

  • is what we're going to be focused on,

  • you have hemoglobin.

  • And somehow-- and I'm going to indicate heme

  • as a ball of orange, with a little planar

  • thing as the protoporphyrin IX.

  • OK, are you all with me?

  • And then somehow this gets sucked out

  • into the NEAT domain, where--

  • And again, all of these gram-positive and gram-negative

  • systems are slightly different, but in the Staph. aureus system

  • we'll be talking about today and you

  • had to think about in the problem set you basically

  • have a cascade of proteins which have additional NEAT

  • domains from which, because this is such a large peptidoglycan,

  • you need to transfer the heme to the plasma-membrane

  • transporter.

  • And what's interesting about these systems

  • and is distinct is that they end up,

  • they're covalently bound to the peptidoglycan.

  • And I'm going to indicate peptidoglycan as "PG."

  • And we'll talk about that reaction today--

  • the enzyme that catalyzes those reactions.

  • And all of these guys end up covalently bound

  • to the peptidoglycan-- which is distinct from all

  • of the experiments you looked at in your problem set.

  • Nobody can figure out how to make the peptidoglycan

  • with these things covalently bound.

  • So what you're looking at is a model for the actual process.

  • OK, so, also-- so that's the gram-positive.

  • And in the gram-negative, one has two ways of doing this.

  • And again, these parallel the ways with siderophore uptake.

  • So you have an outer membrane--

  • So this is the outer membrane.

  • And you have a beta barrel, with a little plug in it.

  • And so these beta barrels, they're at, like, 20 or 30

  • of these things in the outer membranes.

  • And they can take up siderophores,

  • as we talked about last time, but they can also

  • take up hemes.

  • OK?

  • So each one of these is distinct,

  • although the structures are all pretty much the same.

  • And so what you see in this case is,

  • there are actually two ways that you can take heme up.

  • So you can take up heme directly.

  • And we'll see that what we'll be looking at

  • is hemoglobin, which has four alpha 2 beta 2.

  • So this could be hemoglobin.

  • That's one of the major sources, and it is the major source

  • for Staph. aureus.

  • And so this can bind directly to the beta barrel--

  • gets extracted.

  • The heme gets extracted.

  • The protein doesn't get through.

  • And so the heme is transferred through this beta barrel.

  • OK.

  • So that's one mechanism.

  • And then there's a second mechanism.

  • And the second mechanism involves a hemophore.

  • And the hemophore is going to pick up the heme.

  • And so every organism is distinct.

  • There are many kinds of hemophores.

  • And I have a definition of all of these--

  • the nomenclature involved.

  • And so, after class today, I'll update these notes,

  • because that's not in the original--

  • the definitions aren't in the original PowerPoint.

  • OK?

  • So what you have, over here, is the hemophore

  • that somehow extracts the heme out

  • of hemoglobin or haptoglobin.

  • We'll see that's another thing.

  • So this gets extracted and then gets

  • transferred, in that fashion.

  • And so these hemophores come in all flavors and shapes.

  • They're different-- for example, in Pseudomonas or M.

  • tuberculosis.

  • And we're not going to talk about them further,

  • but the idea is they all use these beta-barrel proteins

  • to be able to somehow transfer the heme across.

  • And what happens, just as in the case-- if you go back

  • and you look at your notes from last time,

  • there's a periplasmic binding protein

  • that takes the heme and shuttles it, again,

  • to these ABC transporters.

  • OK?

  • So, in this system, again, you have

  • a periplasmic binding protein.

  • And this goes to the ABC transporter,

  • which uses ATP and the energy of hydrolysis of ATP,

  • to transfer this into the cytosol.

  • OK, so this is the same.

  • That remains the same.

  • And the transporters are distinct.

  • And then, again, once you get inside the cell,

  • what do you have to do?

  • You've got to get the iron out of the heme.

  • So the problems that you're facing

  • are very similar to the siderophores.

  • So, in all cases--

  • So the last step is, in the cytosol,

  • you need to extract the iron.

  • And you can extract--

  • usually, this is in a plus-3 oxidation state.

  • So you extract the iron.

  • And this can be done by a heme oxygenase, which

  • degrades the heme.

  • OK.

  • In some cases, people have reported

  • that you can reduce the iron 3 to iron 2, when the heme can

  • come out, but that still probably is not an easy task

  • because you've got four--

  • you've got four nitrogens, chelating to the heme,

  • and the exchange, the ligand exchange, rates

  • are probably really slow.

  • So I would say the major way of getting

  • the iron out of the heme is by degradation of the heme.

  • And we're not going to talk about that in detail at all,

  • either.

  • OK.

  • So that's the introductory part.

  • And here's the nomenclature, which

  • I've already gone through.

  • I've got all these terms defined.

  • And if you don't remember that, or you don't remember it

  • from the reading, you have a page with all the names--

  • which are confusing.

  • And so the final thing I wanted to say,

  • before we go on and actually start looking at peptidoglycans

  • and gram-positive bacteria and heme uptake

  • in Staph. aureus, which is what I was going to focus on

  • in this little module, is to just show you,

  • bacteria desperately need iron.

  • So what do they do?

  • This is what they do.

  • OK, so, here you can see-- and some bacteria

  • make three or four kinds of siderophores.

  • Others only make one or two kinds of siderophores,

  • but what they've done is they've figured out

  • how to scavenge the genes that are required

  • for these beta barrels.

  • So they can take up a siderophore

  • that some other bacteria makes.

  • OK?

  • And that's also true of yeast.

  • Yeast don't make siderophores, but most yeast have,

  • in their outer membranes, ways of picking up

  • siderophores and bringing it into the cell, since--

  • and remember we talked about the fact

  • there were 500 different kinds of siderophores.

  • But you can see that the strategy is exactly the same.

  • You have a beta barrel.

  • You have-- these are all periplasmic binding proteins.

  • This picture is screwed up, in that they forgot the TonB.

  • Remember, there's a three-component machine,

  • TonB, ExbB and D, which is connected to a proton motive

  • force across a plasma membrane, which

  • is key for getting either the heme or the iron

  • into the periplasm.

  • And you use a periplasmic binding protein,

  • which then goes through these ATPase transp--

  • ABC-ATPase transporters.

  • So what I showed you was heme uptake, iron uptake, but in all

  • of these cases, like Staph. aureus we'll be talking about,

  • we can also get iron out of transferrin.

  • We've talked about that.

  • That's the major carrier in humans.

  • The siderophores can actually extract the iron

  • from the transferrin.

  • And remember the KD was 10 to the minus 3,

  • so somehow, again, you've got to get iron transferred

  • under those conditions.

  • And that's how these guys survive.

  • So they're pretty desperate to get iron.

  • And inside, once they get inside the cell,

  • you have all variations of the theme to get the iron out.

  • But they're all sort of similar.

  • Somehow, you've got to get rid of whatever is tightly binding

  • it.

  • And if you're creative, you can reuse

  • whatever is tightly binding it, to go pick up some more metal.

  • OK.

  • So that just summarizes what I just said.

  • And so, in two seconds, I'm going to show you,

  • now-- we've spent one whole lecture,

  • a little more than a lecture, talking

  • about iron uptake in humans via DMT1,

  • the iron-2 transporter, and the transferrin transfer receptor.

  • So, in the plus-two and plus-three states,

  • we just started looking at the strategies by bacteria

  • and saw how widespread they are.

  • And then the question is, how do you win?

  • OK, bacteria need iron.

  • We need iron.

  • And the question is, how do you reach--

  • and we have a lot of bacteria growing in us, [LAUGH]

  • so we've reached some kind of homeostasis.