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Cell-Cell Communication in Bacteria via Quorum Sensing

Transcript of Part 2: Vibrio Cholerae Quorum Sensing and Developing Novel Antibiotics

00:00:03.05		Hi, my name's Bonnie Bassler and I'm from Princeton University
00:00:06.29		and I'm also a Howard Hughes Medical Institute investigator.
00:00:09.29		And for the second part of my talk today what I want to focus in on
00:00:13.21		is how quorum sensing is involved in pathogenesis
00:00:17.11		in a very important pathogen Vibrio cholerae
00:00:19.28		and our efforts to exploit quorum sensing in order to make new kinds of antibiotics.
00:00:26.18		So, hopefully what you remember from the first part of my talk is that
00:00:31.02		quorum sensing is this mechanism that bacteria use
00:00:33.22		to communicate with one another and to act in groups.
00:00:37.09		And so you'll recall that they make and release small molecules
00:00:40.20		that we call autoinducers, and when these autoinducers build up to a critical amount,
00:00:45.11		they recognize that those autoinducers are there which tells them that they have
00:00:49.06		lots of members of the community around, and then all the bacteria switch gene expression
00:00:53.27		which is, again, behavior in unison.
00:00:56.26		So they carry out tasks as enormous multicellular organisms.
00:01:01.03		And so what you'll remember from the first part of my talk
00:01:04.16		is that we had focused on, for that talk, this harmless
00:01:09.12		but beautiful bacterium Vibrio harveyi
00:01:11.24		which uses quorum sensing to control bioluminescence.
00:01:15.08		And you'll remember that Vibrio harveyi has two autoinducers
00:01:19.11		called autoinducer-1 and autoinducer-2.
00:01:22.05		And each of those autoinducers is detected by its own sensor.
00:01:26.00		LuxN detects autoinducer-1 and two proteins work together to detect autoinducer-2.
00:01:31.21		And the information from those extracellular signal molecules
00:01:34.22		comes to a protein called LuxU. It gets transferred to a protein called LuxO.
00:01:40.02		And LuxO's job is to control luciferase, the light producing enzymes.
00:01:44.16		And so we had studied that and figured this out in this harmless bacterium Vibrio harveyi.
00:01:49.26		But we wanted to try to think about quorum sensing in Vibrios but in pathogenic bacteria.
00:01:55.05		And so what we did is we turned out attention to this
00:01:57.19		very important pathogen Vibrio cholerae that is endemic in under-developed countries
00:02:02.09		and we figured out its quorum sensing circuit.
00:02:04.28		And what we found is that the circuit is incredibly similar to this circuit in Vibrio harveyi.
00:02:10.22		So on this next slide I'm showing you just a very slightly different circuit
00:02:14.25		but this is the quorum sensing circuit for Vibrio cholerae.
00:02:17.24		And most of it should look identical to what I just showed you for Vibrio harveyi.
00:02:21.20		So what we know is that cholera has its own autoinducer-1
00:02:27.01		and so we call that CAI-1 for cholera autoinducer-1.
00:02:30.29		And that's made by an enzyme that we called CqsA
00:02:34.10		for Cholera Quorum Sensing Autoinducer.
00:02:36.22		And this gets detected by its own sensor which we call
00:02:40.11		CqsS for Cholera Quorum Sensing Sensor.
00:02:43.28		So this circuit, this one on the bottom of my slide,
00:02:47.01		is the intraspecies communication circuit.
00:02:50.21		So in Vibrio harveyi, on my last slide, this was called AI-1 and LuxN.
00:02:55.00		In Vibrio cholerae it's called CAI-1 and CqsS and it is cholera's private language.
00:03:02.12		But then, just like Vibrio harveyi, cholera has this second circuit.
00:03:06.19		It has the LuxS enzyme that makes the generic autoinducer-2 molecule
00:03:11.02		and that gets detected by LuxP and LuxQ.
00:03:13.25		So that part of the circuit, the generic part is identical to Vibrio harveyi.
00:03:18.06		And then the downstream components, LuxU and LuxO, are also identical.
00:03:22.28		So the only point is that each Vibrio harveyi and Vibrio cholerae have
00:03:27.01		their own private autoinducer-1 system. They have the same autoinducer-2 system
00:03:32.17		and the way the information gets relayed into the cell
00:03:35.12		is identical. It's through LuxU and LuxO.
00:03:38.05		And then, of course, the job of this circuit is to turn on
00:03:41.15		and off genes when Vibrio cholerae needs them.
00:03:45.02		And so now I'll tell you how...so that's generically the system
00:03:50.06		and now I want to tell you how cholera uses quorum sensing for pathogenesis
00:03:54.04		because it's actually, sort of a unique way.
00:03:57.22		So what we've learned about cholera is that at low cell density,
00:04:01.11		so when these autoinducers are at low levels,
00:04:04.19		what happens is that information that there are very few cells around comes into the cells
00:04:11.02		and it tells Vibrio cholerae, surprisingly, to turn on all of its virulence genes,
00:04:16.25		all of the biofilm genes and other genes that are required for infection.
00:04:21.09		But then at high cell density...so that's...I should say this, be more serious...
00:04:26.15		So, remember, at low cell density cholera is turning on virulence.
00:04:31.06		But then at high cell density, when the autoinducers accumulate
00:04:35.17		and there's lots of Vibrio cholerae cells around, what happens is that the circuit
00:04:40.02		sends this information backwards and what cholera does is to turn off virulence,
00:04:45.27		off biofilms and off all kinds of other genes that are required for infection,
00:04:50.16		and it turns on genes that are involved in escape from the host
00:04:55.07		like an important protease.
00:04:56.16		And so we have to stop for a minute and talk about this.
00:04:59.09		So, cholera, unlike most bacteria that you hear about, causes an acute infection.
00:05:05.04		Most bacteria that we get sick from cause what are called persistent infections.
00:05:08.22		So their goal, if I can say things like that,
00:05:11.09		is to get in you to cause an infection and to stay there.
00:05:14.29		So all of the bacteria that we've learned about other than cholera
00:05:17.29		that cause persistent infections use quorum sensing to turn on virulence
00:05:23.09		at high cell number when they know they're going to be able to make the host succumb.
00:05:27.09		Cholera has this very insidious strategy.
00:05:30.12		What it does is that you get it from eating contaminated water or contaminated food
00:05:35.10		and it gets into your intestine and it immediately turns on this entire battery of virulence genes.
00:05:42.05		So it turns on a gene called Tcp for Toxin Co-regulated pilus
00:05:46.20		that allows it to adhere to your intestinal epithelial cells.
00:05:50.02		It turns on the toxin, which is the thing that makes you sick.
00:05:53.01		And then what happens is it starts growing like crazy.  It makes the person very sick.
00:05:57.21		And people get a terrible diarrhea from cholera.
00:06:00.16		But then, when cholera is at high number, it turns all of those genes off,
00:06:05.20		the ones that really make you sick.
00:06:07.09		And it turns on this protease, which is a "detachase" that cuts the bacteria
00:06:13.02		off your intestinal epithelial cells and out they come to infect the next host.
00:06:18.10		So it's using quorum sensing for virulence but it simply has the mechanism reversed.
00:06:23.24		It turns on the virulence genes at low cell density.
00:06:26.20		It makes you really sick. It grows to the gazillions.
00:06:29.16		It turns off virulence genes. It turns on an escape pathway and out it comes
00:06:34.08		back into the environment to make many more hosts sick.
00:06:37.08		And so it's a fabulous strategy from cholera's perspective,
00:06:39.28		but this is why cholera causes an acute disease.
00:06:43.14		If you can actually survive that phase of the disease, you're cured
00:06:47.12		because it's self limiting. It gets in and it gets out.
00:06:50.14		And so the important point for today that you need to understand is
00:06:54.05		that the autoinducers shut off virulence.
00:06:57.27		And so as we talked in the first part of my talk, there's lots of groups
00:07:01.29		now trying to work on strategies to interfere with quorum sensing
00:07:06.22		to work on bacteria that cause persistent infections.
00:07:10.15		If we could make anti-quorum sensing molecules or inhibitors of these enzymes
00:07:16.27		that make theses autoinducers maybe those could be new therapies.
00:07:19.26		And there's a tremendous amount of work going on about that in the field.
00:07:23.18		But of course, it's kind of difficult. We have to figure out what molecules
00:07:26.22		interfere with the circuit. We have to try to make inhibitors of these enzymes.
00:07:29.22		And so that's fun and that's interesting but it's taking a little bit of time.
00:07:33.19		And so what we thought about this funny cholera circuit
00:07:37.12		is that because the autoinducers turn off virulence, we could use this as a test case
00:07:44.03		to see if we could manipulate quorum sensing and actually shut down virulence.
00:07:48.13		Because cholera uses this weird circuit to turn off virulence at high cell density
00:07:54.01		what that means is that the autoinducer itself is the drug.
00:07:58.11		If we add the autoinducer, we should be able to shut down virulence.
00:08:01.22		And so that's backwards of what we're trying to do in all these other systems
00:08:05.15		where we want to get antagonists of quorum sensing.
00:08:08.08		So we thought, as a proof of principle, to find out is there any
00:08:11.28		merit in the idea of trying to interfere with these quorum sensing systems?
00:08:16.04		Cholera gave us a unique chance because we could just add the autoinducer
00:08:21.04		and see whether or not we could shut down virulence.
00:08:23.15		And so of course to measure these kinds of genes, virulence and biofilm, that's kind of tricky.
00:08:28.29		And so what we thought we would do to get at that is to use bioluminescence.
00:08:33.00		So you'll remember from the first part of my talk,
00:08:34.28		that quorum sensing controls bioluminescence in Vibrios.
00:08:38.15		And so what we did is we engineered into Vibrio cholerae a quorum sensing activated
00:08:43.24		luciferase reporter. So now when we add autoinducers bioluminescence turns on.
00:08:50.13		And we thought we could use this as a read out to try to purify this molecule
00:08:54.19		and see if we could control quorum sensing with it.
00:08:57.12		OK, so now we have a Vibrio cholerae that makes light in response to cholera autoinducer-1
00:09:03.05		and to autoinducer-2. But we just wanted to focus on cholera autoinducer-1
00:09:07.21		because that would be a test that was simply particular to cholera.
00:09:12.06		And so remember I told you in the first part of my talk
00:09:15.02		that these molecules are on the outside of cells.
00:09:17.12		And so we wanted to find out what this cholera autoinducer was.
00:09:20.12		So the strategy we did was to just grow up a lot of cholera.
00:09:24.01		Spin the cells out of solution. Filter them out. Collect the cell-free supernatants.
00:09:29.00		And we could see that it had a lot out autoinducer activity in it
00:09:32.29		because it turned on luciferase when we added it back to the cells.
00:09:36.24		And so then to purify it what we did was we did a number of extractions
00:09:41.22		of the media and some sort of fancy column chromatography and then finally in the end
00:09:46.17		we put our cleaned up, cell-free supernatants on to an HPLC column
00:09:51.05		and just measured bioluminescence as a measure of activity.
00:09:54.18		And so here you're looking at bioluminescence and this is just fraction number
00:09:58.24		dripping off that column. And what you can see is that nothing comes off
00:10:03.00		and then all of a sudden a big peak of activity comes off.
00:10:06.15		And so the bacteria make a lot of light in response to the stuff that's in these tubes.
00:10:11.23		So, sure enough, we could show that all of the
00:10:14.16		cholera autoinducer-1 activity was in this one peak.
00:10:17.22		So we pooled those test tubes full of stuff and there was a lot of activity in it.
00:10:23.20		And then we could just take that and do techniques like mass spectrometry,
00:10:26.25		NMR, ORD, CD, just different sort of techniques that would
00:10:30.24		tell us what the molecule is.
00:10:32.24		And sure enough, this peak had one molecule in it and it was very clean.
00:10:37.04		And so, just from this experiment we could purify the cholera autoinducer-1
00:10:40.27		activity and identify the molecule.
00:10:43.20		And this is the molecule. It has a funny name, 3-(S)-hydroxy-4-tridecanone
00:10:49.00		which is why we call it cholera autoinducer-1 because that's simpler.
00:10:52.04		And what you can see, I hope, is that it is a molecule that has 13 carbons in it
00:10:56.12		and only two functional groups. And so the way that this molecule is made by
00:11:01.05		the CqsA enzyme is that it takes a C-10 moiety from fatty acid biosynthesis
00:11:07.24		and connects it to a C-3 moiety to make this C-13 molecule.
00:11:14.10		The only stereochemistry in the molecule is right here at this carbon.
00:11:19.02		And what we did after we purified the molecule was we synthesized this molecule
00:11:24.10		both in the S form, which is shown here and also in the R form.
00:11:27.24		And then using chiral chromatography we showed
00:11:30.07		we could separate those two molecules.
00:11:32.00		Then we took the real thing and what we saw was that, indeed, cholera only makes this S moiety.
00:11:38.13		So this is the only molecule that cholera makes that is cholera autoinducer.
00:11:43.02		OK, so now we had it and we wanted to see if we could start
00:11:45.26		messing around with quorum sensing by having this
00:11:48.12		molecule and being able to synthetically prepare it.
00:11:51.05		So what we did was to test the specificity of the response.
00:11:56.15		And so again, you're looking at bioluminescence and now we're using synthetic molecules.
00:12:01.07		So this top one, the C13S, this is the real molecule
00:12:05.13		except that now we've made it.
00:12:06.19		So we purified it from cholera, identified it, and then we made it using chemistry.
00:12:10.19		We also, I told you, made the R isomer.
00:12:13.09		And then, going down, we're simply chopping off one carbon or another.
00:12:17.09		So we have a C12 molecule, a C11 molecule.
00:12:20.01		And we made many more, this is just a sample of the kinds of molecules that we tested.
00:12:23.26		And so what you can see if you look at the activity of the molecule
00:12:27.13		is that the C13S molecule is the most active.
00:12:30.26		The R molecule is slightly less active.
00:12:33.09		And then if you start chopping off carbons the molecules get less and less active.
00:12:37.17		And so, indeed, nature has selected for the most active of the molecules.
00:12:42.14		The C13S molecule, which is the molecule that cholera makes
00:12:45.24		for its autoinducer, is the most active in our activity assays.
00:12:49.19		OK, so that shows you that, indeed, we can find out what cholera autoinducer-1 is.
00:12:55.20		And what I should say is that even though that molecule looks very simple,
00:12:58.17		it's a brand new molecule to biology.
00:13:01.00		So that molecule has never been seen before and apparently it's special.
00:13:05.04		It's just this cholera autoinducer, but cholera, I guess, is the one with
00:13:09.08		CqsA that invented making this particular molecule as a signaling molecule.
00:13:14.16		So now we have it. We know we can make it.
00:13:17.12		We know that it can turn on this engineered luciferase reporter
00:13:22.02		that's responding to quorum sensing.
00:13:23.17		But the real test and the real goal of this set of experiments
00:13:26.19		was to ask: Can we add this molecule and turn off virulence as a new therapeutic?
00:13:32.11		And so, of course, we wanted to go on to do that
00:13:34.26		and so what we decided to do in our first experiment is to measure
00:13:39.02		production of this pilus that I told you about, which is called TcpA.
00:13:43.22		And so that's the pilus that lets cholera attach to your intestine
00:13:47.17		and then it delivers the toxin once it's infected you.
00:13:50.29		And so there's a Western blot assay for that.
00:13:53.28		Right, so we can do a Western blot for TcpA.
00:13:56.22		And that's shown on this slide. And so if we just look at wild type cholera,
00:14:01.08		and these black lines, of course, are the TcpA production.
00:14:06.19		And so what we have... and these sort of have fancy names but it doesn't really matter
00:14:10.22		is that we have mutants that are locked at low cell density.
00:14:14.03		And so you'll remember, at low cell density, cholera turns on virulence.
00:14:18.07		And so, indeed, in a mutant that's locked at low cell density
00:14:21.16		you see a lot of TcpA, this virulence factor.
00:14:24.12		We also have a mutant that's locked at high cell density.
00:14:27.14		And so what you can see is if the mutants are locked at high cell density
00:14:31.04		cholera never turns on virulence factors.
00:14:33.27		So it doesn't turn on TcpA.
00:14:36.05		But now if we just take the wild type cell and we add
00:14:39.09		increasing amount of our synthetic autoinducer CAI-1 what you can see is as
00:14:44.12		we add more of the synthetic molecule, this virulence factor production turns off.
00:14:49.23		So that was hopeful. And then what we also notice
00:14:53.16		is that if we did this in a mutant that was a CqsA mutant, and
00:14:57.08		so you'll recall, that's the enzyme that makes CAI-1, the autoinducer.
00:15:01.25		So if it's not making its own autoinducer, we get a much more dramatic affect
00:15:06.19		because of course, we're not fighting against
00:15:09.02		the endogenously produced cholera autoinducer-1.
00:15:13.11		So, indeed, our synthetic molecule can turn down TcpA production.
00:15:18.25		And to show that the molecule is actually working the way we think it should,
00:15:22.17		we tested the exact same experiment but we did it on a mutant
00:15:26.07		that was mutant in CqsS, which you'll recall is the detector for CAI-1.
00:15:32.06		So what we did was we deleted that detector and now what you see is
00:15:37.03		if we add the cholera autoinducer, nothing happens.
00:15:39.23		And that makes sense. If the bacteria don't have the detector
00:15:42.26		to transduce that information in, they don't respond.
00:15:46.15		So, sure enough, CAI-1 can turn down virulence, in vitro
00:15:50.14		and CqsS, the receptor, is required for that.
00:15:54.00		So it's working exactly the way we think it ought to.
00:15:56.24		And so that's an in vitro test for whether or not the cholera autoinducer
00:16:01.06		can be used as a therapeutic. But of course the real test is
00:16:04.11		not whether we can turn off TcpA, the pilus, in a test tube of bacteria
00:16:10.11		but can we make bacteria not be infectious?
00:16:13.13		So the next experiment we did was to use a mouse model.
00:16:17.03		So there's a very well established mouse model for cholera infection.
00:16:20.21		And so what we know is that if we infect wild type Vibrio cholerae
00:16:24.21		into this mouse, the mouse dies.
00:16:27.06		And that's been used for many years in the cholera field to think about infection.
00:16:32.11		And so the question is: If we infect cholera...excuse me, infect the mouse
00:16:37.18		with wild type cholera but we add cholera autoinducer-1,
00:16:41.16		which we now know is this molecule, can we, in fact, get the mouse to live?
00:16:45.26		And the answer is yes.
00:16:47.15		So, indeed, if you give both these things together, it keeps the mouse alive.
00:16:53.09		You'll also recall from my slides that there's another autoinducer involved, autoinducer-2.
00:16:58.07		And it turns out, if we add both CAI-1 and autoinducer-2 together
00:17:03.04		that works even better. And so together, those two autoinducers
00:17:06.26		fully turn off the cholera virulence cascade and they look promising
00:17:10.18		for making a new therapeutic for treating cholera
00:17:13.19		in countries in which it's endemic.
00:17:15.24		And I should say that, you know, these molecules
00:17:19.14		even though they work, they're not perfect in terms of what one would like
00:17:22.24		when one thinks about the properties that molecules that are used as drugs have.
00:17:27.15		And so what we've begun to do is to make a series of molecules
00:17:30.25		that are related to the real molecule.
00:17:32.23		And so this is just showing you a few of the molecules where we've attached
00:17:36.09		different groups on them to see if we can get a molecule
00:17:39.22		that acts even better than the real CAI-1.
00:17:42.18		And so all of these now are being tested in vitro and in vivo
00:17:46.05		to see if we can get a good combination of an autoinducer-2-like molecule
00:17:50.07		and a CAI-1-like molecule to control pathogenesis
00:17:54.14		in Vibrio cholerae which infects a million people a year.
00:17:57.27		And so that's the state of affairs right now.
00:18:00.06		I hope from my two seminars what you've learned
00:18:02.09		is that bacteria talk with a very complicated chemical lexicon.
00:18:06.09		They all, we think now, have at least two molecules.
00:18:09.06		One that says me, one that says other.
00:18:11.22		So, an autoinducer-1 and autoinducer-2.
00:18:14.12		And they use that information to control group activities and act like big multicellular organisms.
00:18:20.21		And in the case of many pathogens, including cholera, what they do is
00:18:24.24		they use those molecules to infect human and animal and plant hosts.
00:18:28.26		And so the goal of the field is to move toward being able to disrupt
00:18:32.17		quorum sensing by making agonists or antagonists of these molecules
00:18:37.07		And the first idea that this could actually work, I've show you in this last, short seminar
00:18:41.29		about how we've used the cholera autoinducer to shut down
00:18:45.14		virulence in an animal host.
00:18:47.19		And so that's the state of affairs and of course
00:18:49.23		we're working on these and other topics right now.
00:18:52.03		And I thought what I would finish my two seminars by doing
00:18:55.06		is to show you my lab because I'm very proud of these people.
00:18:58.01		All of these people are undergraduates, graduate students and post-docs from Princeton.
00:19:03.12		And so I need to make the confession that...
00:19:05.26		So here they are, my gang.
00:19:07.07		and that of course they did all of the work that I showed you today.
00:19:10.10		I didn't do very much of that at all.
00:19:11.29		I get to give the talks but they did all the pipetting
00:19:14.17		and crystallography and molecular analysis and mutant analysis that you've seen.
00:19:19.11		And it's really wonderful gang of people all between their twenties and thirties years old
00:19:24.12		and they're just the engine that drives this kind of science.
00:19:27.01		And I'm lucky, here I am over here, I'm really lucky to get to work with them
00:19:31.08		because they're incredibly creative and have, essentially,
00:19:34.02		figured out all of this idea that bacteria can talk to each other.
00:19:37.24		So again, thanks for listening to me.
00:19:39.12		And I'm Bonnie Bassler from Princeton University and the Howard Hughes Medical Institute.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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