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Cell-Cell Communication in Bacteria via Quorum Sensing

Transcript of Part 1: Bacterial Communication via Quorum Sensing

00:00:03.14		Hi, my name's Bonnie Bassler and I'm a professor at Princeton University
00:00:07.01		and I'm also an investigator at the Howard Hughes Medical Institute
00:00:10.17		and I'm delighted to be here to get to talk to you about bacterial quorum sensing.
00:00:15.00		So the goal of my two seminars is to try to convince you that bacteria can talk to each other
00:00:21.10		and to try to show you that what this chemical communication does
00:00:24.29		is to allow bacteria to act as enormous multicellular organisms and
00:00:30.12		accomplish tasks that they could never accomplish if
00:00:33.13		they simply acted as individuals, because they're too tiny.
00:00:37.07		And so what I thought I would do today is to give you sort of a historical tour
00:00:42.10		of how we got into this idea that bacteria can talk with each other
00:00:45.13		and then try to give you some ideas of where this field is going
00:00:48.18		and how we're trying to do something that's medically relevant
00:00:51.22		by interfering with bacterial communication.
00:00:54.01		So, to get going, the idea that bacteria can talk to each other all started
00:00:59.21		about 40 years ago when a man named Woody Hastings made an amazing observation.
00:01:04.00		So what he noticed was that there were two bioluminescent bacteria,
00:01:08.15		so bacteria from the ocean that made light, sort of like firefly light,
00:01:13.06		but what he noticed was a special property of the light.
00:01:16.05		That they only made light when they were at high cell density.
00:01:18.29		And so this is actually depicted on my first slide.
00:01:21.15		What you're looking at is a flask.
00:01:23.13		This is just a person from my lab holding a flask of a liquid culture of one of these two bacteria.
00:01:29.08		They're called Vibrio fischeri and Vibrio harveyi.
00:01:32.20		And what you can see is that the flask...or the liquid in the flask is glowing in the dark.
00:01:37.22		And that light is made by the bacteria, in this case, Vibrio harveyi.
00:01:42.04		And what Woody Hastings noticed was that at low cell density,
00:01:45.27		so when the bacteria were alone, they didn't make light.
00:01:48.29		But, when they grew to a certain cell number, all of the bacteria turned on light together.
00:01:53.29		And so the question that he wanted to know, and what we're going to talk about today is:
00:01:57.21		"How can it be that a bacterium knows the difference between times when it's alone
00:02:02.20		and times when it's in high number?"
00:02:05.04		And what's fantastic about bioluminescence is that you can just see it.
00:02:09.14		So, we're geneticist in my lab and we want to understand how these bacteria
00:02:13.16		perceive when they're alone and when they're together.
00:02:15.22		And so what we can do is to just to make mutants, turn the lights off,
00:02:19.27		look at the Petri plates and look for bacteria that are glowing when they shouldn't be
00:02:24.01		or not glowing when they should be.
00:02:25.22		And just by doing that simple experiment, we and many other groups in the field
00:02:29.26		have been able to understand how these bacteria tell the difference between
00:02:33.26		times when they're alone and times when they're together
00:02:36.12		and then initiate a signal transduction cascade,
00:02:39.13		the output of which is this beautiful bioluminescence.
00:02:42.23		So, I told you that Woody Hastings noticed this both in two bacteria,
00:02:46.23		one was named Vibrio harveyi, which is this free living bacteria that you're looking on at this slide
00:02:51.14		and the other one was its very close relative Vibrio fischeri.
00:02:55.07		So like Vibrio harveyi, Vibrio fischeri is a bioluminescent marine bacterium.
00:03:00.16		But it lives a very different lifestyle than Vibrio harveyi. Vibrio harveyi's free-living
00:03:05.06		but Vibrio fischeri lives as a symbiont in a number of marine animals
00:03:09.15		one of which is this one, which is called the Hawaiian bobtailed squid.
00:03:12.26		So what you're looking at is a squid that's been turned on its back
00:03:16.13		and dissected down the middle and I hope that you can see are these two glowing lobes
00:03:21.23		that make up a specialized light organ in the squid
00:03:24.16		and this is the place that the bacteria live.
00:03:26.29		So the way this symbiosis works is: the squid is,
00:03:29.29		this light organ is under the bottom or the mantel of the squid.
00:03:32.24		This light organ is under there and it houses Vibrio fischeri
00:03:36.06		at something like ten to the eleventh or ten to the twelfth cells per mL.
00:03:40.07		We have no idea how the squid grows the bacteria to this high cell number.
00:03:45.00		But what we know is that the bacteria are trapped inside the light organ
00:03:48.26		and in there they make a molecule that you can think of like a hormone
00:03:53.04		or a pheromone and we call it an auto-inducer.
00:03:55.25		So they make and they release this molecule
00:03:58.12		and since the bacteria are trapped inside the light organ of this squid
00:04:02.16		the molecule gets trapped inside with them
00:04:05.06		and it tells the bacteria "You're inside, not outside. So, you should make light."
00:04:09.28		And so Vibrio fischeri glows because it perceives this molecule
00:04:13.15		which helps it count its neighbors.
00:04:16.20		And so the reason Vibrio fischeri  does that is because this squid feeds it.
00:04:20.17		So this light organ is loaded with amino acids and nutrients and all kinds of goodies
00:04:25.10		that makes the life of the bacterium much easier than
00:04:28.10		if the bacterium was living free-living in the ocean.
00:04:31.04		So the selection from the bacterium's point of view is that it gets fed
00:04:34.23		and gets to grow to high numbers in that squid.
00:04:37.02		Now the reason the squid is interested in this is because it wants the light from that bacteria.
00:04:41.18		So the way this work from the squid's perspective is:
00:04:44.13		this little squid, its only about this big full grown
00:04:47.05		and it lives off the coast of Hawaii, that's why its called the Hawaiian bobtailed squid
00:04:51.07		and the squid is nocturnal. So, during the day it buries itself in the sand and it sleeps
00:04:56.22		but then at night it has to come out to hunt.
00:04:58.27		And so on bright nights when there's lots of starlight or moonlight,
00:05:02.16		since this squid lives just off the coast, so just this sort of shallow, knee-deep water
00:05:06.22		since its living in this shallow water, the light from the moon and the stars
00:05:10.20		can penetrate the water that the squid lives in.
00:05:12.23		And so what the squid has developed is a shutter.
00:05:15.12		It's the squid's ink sack that it can open and close over this specialized light organ.
00:05:20.17		And so what happens is that the squid has detectors on its back
00:05:24.00		so it can sense how much moonlight and starlight is hitting its back
00:05:27.07		and then it opens and closes this shutter so the amount of light
00:05:30.15		coming out of the bottom, which is made by the bacteria,
00:05:32.24		exactly matches how much light hits the squid's back.
00:05:36.05		So this way the squid doesn't make a shadow.
00:05:38.16		So it actually uses the light from the bacteria to counter-illuminate itself
00:05:42.29		as an anti-predation device.
00:05:44.05		And so the selection for the light from the squid's point of view is that
00:05:48.00		it gets protected, for its lifetime, from predators.
00:05:50.17		So the bacteria make the light, they get fed and the squid uses the light to protect itself.
00:05:56.09		But of course this squid has this terrible problem
00:05:58.19		because it's got this dying, ten to the twelfth cells per mL culture of bacteria
00:06:02.18		in this little light organ and it can't maintain that.
00:06:04.29		And so what happens is every morning, when the squid goes back to sleep,
00:06:08.24		when it buries itself in the sand, its got a pump that's attached to its circadian rhythm.
00:06:13.02		And so when the sun comes up, what the squid does is it pumps out 95% of the bacteria.
00:06:18.26		And so now the bacteria are dilute and of course that little molecule,
00:06:22.04		that hormone, auto-inducer molecule I told you about, is gone.
00:06:25.18		And so now the bacteria can't perceive that molecule, so they don't make light.
00:06:29.12		But then as the day go by, the bacteria start doubling, they keep releasing this hormone molecule
00:06:34.18		and at night, they perceive it, and the light comes on exactly when the squid needs it.
00:06:39.13		And so the reason to tell you about these two, first stories is because
00:06:43.09		now we understand that hundreds of species of bacteria make auto-inducers
00:06:47.11		and talk to one another with these chemical communication systems.
00:06:52.22		So, the way we understand this in the simplest terms is that at low cell density,
00:06:57.25		so this is over here, when a bacterium is alone or in dilute suspension,
00:07:01.21		it's making and releasing these molecules that I have as these little blue dots.
00:07:05.16		But, of course, if the bacteria is dilute, the molecules just diffuse away.
00:07:09.13		But as the cells grow in number, when the population density increases,
00:07:13.19		since all the bacteria are releasing these molecules into the environment
00:07:17.05		the more cells there are, the more of this molecule there is.
00:07:20.12		And at a particular concentration of the molecule, which is indicative of cell number,
00:07:25.02		the bacteria perceive that the molecule is there
00:07:27.16		and then they all change their gene expression in unison.
00:07:31.03		And so in the case of Vibrio fischeri and Vibrio harveyi, they turn on light.
00:07:35.08		And so what you can see then is that these bacteria are acting like an enormous
00:07:39.23		multicellular group and turning on and off gene expression,
00:07:43.04		which is really changes in behaviors, on a population wide scale.
00:07:47.00		And so now we have a fancy name for this: we call it quorum sensing.
00:07:50.22		The bacteria vote with these little chemical votes, they count the votes,
00:07:54.20		and then everyone responds to the vote.
00:07:57.08		And what I hope you'll think by the end of this seminar is that this had to be
00:08:00.28		one of the first steps in the development of multicellular organisms.
00:08:05.29		So this is sort of how it works in generic terms and we also know a lot now.
00:08:10.20		In these past few years we've learned a lot about the mechanisms
00:08:14.13		that underlie these communication systems.
00:08:16.16		And so there's a couple of sort of general themes that are emerging.
00:08:20.29		And so what we now understand is that in gram negative bacteria,
00:08:24.23		they typically have systems like the one I have depicted on this slide.
00:08:29.00		So this...these ovals will be my cells
00:08:33.23		these are supposed to mean bacterial cells from here forward.
00:08:36.14		So what we know about gram-negative bacteria,
00:08:38.22		is that they have an enzyme which is called LuxI, for the inducer.
00:08:43.17		And this is the enzyme that makes the signal molecule, which on this slide are these red diamonds.
00:08:48.25		And all of these signal molecules are what are called acyl homoserine lactones.
00:08:52.24		And I'll tell you more about those on the next slide.
00:08:54.26		So what we now understand is that hundreds of species of gram-negative bacteria
00:08:59.09		all have highly conserved LuxI enzymes
00:09:02.07		that make an acyl homoserine lactone signal molecule
00:09:05.03		and that freely diffuses in and out of the cells
00:09:07.24		so the more cells there are the more the molecule there is.
00:09:10.16		And when the molecule hits a particular amount
00:09:12.25		it gets bound by a partner protein that's called LuxR for regulation.
00:09:17.13		And what happens is when LuxR binds the auto-inducer molecule
00:09:21.05		that unveils a DNA binding domain on the LuxR protein.
00:09:25.04		So this complex of the LuxR protein and the signal molecule bound
00:09:29.13		can sit at the promoters of genes that the bacteria want to express
00:09:33.23		when they're in a community and they turn them on.
00:09:36.17		And so what this allows bacteria to do is to count their numbers
00:09:40.07		and then turn on genes that are useful only when the bacteria are acting as a group,
00:09:45.06		but are not useful when the bacteria are acting as individuals.
00:09:48.24		So again to reiterate, we now know that there are hundreds of systems like this
00:09:52.20		in gram-negative bacteria. So that's how the systems work.
00:09:56.26		We also know a lot about these signals that are made by these LuxI-type enzymes.
00:10:01.16		So what I did on this slide was just put a few different species of bacteria,
00:10:05.24		these are gram-negative bacteria that have quorum sensing.
00:10:08.27		And then I have the molecule that they use to talk with next to them.
00:10:12.22		And so what I hope you can see is that all of the molecules are related.
00:10:16.23		So again these are called acyl homoserine lactones.
00:10:19.20		And so what you see is that the right hand part of all of the molecules is identical.
00:10:24.07		That's the acyl homoserine lactone or the homoserine lactone.
00:10:27.15		But this left part, see these carbon chains?
00:10:30.04		Each one is a little bit different in every single species of bacteria.
00:10:34.18		And so what that does is to confer exquisite species specificities to each of these languages.
00:10:40.20		So what I mean by that is that only the bacterium that makes the signal can respond to it
00:10:45.23		and no other bacteria can. So for example,
00:10:48.19		if we take the Pseudomonas signal and but it on Vibrio fischeri nothing happens.
00:10:52.26		And likewise, Pseudomonas is impervious to the Vibrio fischeri signal.
00:10:57.18		So these molecules fit like locks and keys with their partner LuxR proteins
00:11:02.24		and there is no cross-talk in these systems.
00:11:05.15		They are for intra-species communication
00:11:07.29		and so what we think is that this kind of quorum sensing
00:11:10.29		allows bacteria to count their siblings.
00:11:13.15		It allows them to count and talk within their own species.
00:11:17.11		So that's how gram-negative bacteria do it.
00:11:20.05		We also now know that gram-positive bacteria have quorum sensing.
00:11:23.23		And so the ideas are similar to what I've already told you
00:11:26.16		but the mechanism that gram-positive's use to communicate is a little bit different.
00:11:30.19		So here I have sort of a model for gram-positive bacterial quorum sensing.
00:11:35.18		So gram-positive bacteria use peptides as their words or as their signals.
00:11:40.28		And so the way that this works is that the peptides are encoded in genes
00:11:45.13		and they get made as a precursor protein. So sort of a big protein.
00:11:48.27		And then there's a processing system that cuts that big protein into a smaller peptide
00:11:53.14		and then that gets secreted outside of the cell by a dedicated secretion machinery.
00:11:58.04		But again, the idea is the same:
00:12:00.05		the more cells there are, the more of this peptide there is accumulating in the environment.
00:12:04.08		And in this case, when the peptide hits a critical amount,
00:12:07.08		it gets bound by a transmembrane bound protein
00:12:11.10		so sort of a sensor that connects the inside of the cell to the outside.
00:12:15.03		And when the peptide gets bound, that initiates a phosphorylation cascade.
00:12:19.18		The bottom line of which is that, ultimately, a transcription factor gets phosphorylated.
00:12:24.16		That activates it, it lets it sit on the DNA and turn on the genes that the bacteria want
00:12:29.25		to express when they're a community.
00:12:31.26		So even though the mechanism is different than in the gram-negative bacteria,
00:12:35.20		the idea is really the same. The number of auto-inducer molecules, peptides in this case
00:12:41.10		increases with cell number increasing,
00:12:43.22		and then information gets sent in to a DNA binding protein
00:12:47.15		that turns on all of the genes the bacteria need to carry out group behaviors.
00:12:52.15		So that's how these work in gram-positive bacteria.
00:12:55.02		And just like I told you for gram-negative bacteria, we also know about the peptide signals.
00:13:00.00		So again, these are just a couple of peptides that we know are auto-inducers
00:13:03.23		for different gram-positive bacteria. And they can be a little bit fancy.
00:13:09.04		So sometimes its just the naked peptide or occasionally they'll have moieties stuck on them.
00:13:13.23		That's what this asterisk is supposed to mean.
00:13:15.12		Or they can be cyclized. So they put a few bells and whistles on these signals.
00:13:18.18		But the bottom line is they're peptides and again, in every case,
00:13:22.26		each species of gram-positive bacterium has its own peptide.
00:13:27.07		So these are private languages that are species specific.
00:13:30.15		And so, gram-positive bacteria use this to count their own cell number for intra-species communication.
00:13:37.07		And so that's how these systems work.
00:13:39.10		And now I want to just tell you a few of the behaviors
00:13:41.19		that are controlled by quorum sensing in different species of bacteria.
00:13:45.13		So I already told you how it works for Vibrio fischeri,
00:13:48.08		this bioluminescent bacteria that lives inside this squid.
00:13:51.10		So that story you've already gotten.
00:13:53.21		There's another bacterium, Pseudomonas aeruginosa,
00:13:56.06		which is a terrible opportunistic human pathogen.
00:13:59.10		This bacterium lives in the soil and it's relatively harmless unless one has cystic fibrosis.
00:14:05.06		And so probably you know that people who have CF have a genetic mutation in their lungs
00:14:10.12		so that they can't clear or sterilize their lungs.
00:14:13.13		So you and I breathe in all kinds of bacteria but we have mechanisms to keep our lungs clean.
00:14:18.06		People who have cystic fibrosis can't do that.
00:14:21.06		And so what happens is they have an infection in their lungs
00:14:24.28		that has all kinds of different bacteria in it.
00:14:27.28		And for reasons that we don't really understand, typically a person who has CF
00:14:33.02		in his or her teens will become permanently colonized by Pseudomonas aeruginosa.
00:14:37.29		And this is the bacterium that kills them.
00:14:40.10		And the reason is because Pseudomonas has about a hundred genes
00:14:44.11		that are controlled by quorum sensing, all of which are important for virulence.
00:14:48.18		And so what happens is that Pseudomonas gets into the lungs
00:14:51.24		and it makes what's called a biofilm. That's how bacteria stick in us or on us.
00:14:56.26		So it sits down, it adheres to the lungs, and it secretes all kinds of terrible virulence factors:
00:15:02.03		proteases and hydrolases that damage the person's lung tissue.
00:15:06.29		And all of these genes are controlled by quorum sensing.
00:15:10.10		And so even though this is a terrible disease,
00:15:12.26		if you actually think about this from the bacterium's point of view,
00:15:16.01		it's a fabulous strategy for being a pathogen.
00:15:19.07		The last thing a bacterium wants to do is to get in and when it's at only a few cells
00:15:24.14		start secreting all of these toxins and virulence factors.
00:15:28.01		That's why your immune system evolved.
00:15:29.17		It evolved to do surveillance and get rid of pathogenic bacteria.
00:15:32.28		So the strategy that Pseudomonas uses
00:15:35.09		is to get in, to wait, and to count itself with these small auto-inducer molecules
00:15:40.16		and to recognize when it has the right number
00:15:43.13		that if all of the bacteria secrete these virulence factors
00:15:46.24		in unison they'll be able to infect an enormous host.
00:15:50.24		And so what I hope you can see is that even though the disease is devastating
00:15:54.08		from the bacterium's point of survival, it's a really good strategy
00:15:58.00		to wait until you know there's enough of your friends around, if I can say that,
00:16:02.16		that if you do this thing together, you will be successful.
00:16:05.22		And so that's basically how we understand quorum sensing.
00:16:08.21		Another couple of examples, Agrobacterium tumefaciens is a plant pathogen.
00:16:14.03		And this pathogen causes crown gall tumors on plants
00:16:17.03		and it's a problem in the agricultural industry.
00:16:19.04		And what it uses for...with quorum sensing is mating.
00:16:22.27		So Agrobacterium has the virulence genes on a mobile piece of DNA.
00:16:27.12		And so what happens is when some of them start infecting the plant
00:16:30.17		what they do in response to quorum sensing is to start transferring this plasmid around.
00:16:35.17		So they make the entire community more infective than it was originally.
00:16:40.04		And so again if you want to give your DNA to somebody
00:16:42.25		you want there to be a recipient there. So that should be a behavior that's social:
00:16:47.11		that lots of cells need to do it together.
00:16:49.17		And then finally, I just put one more on here.
00:16:51.23		This one is called Erwinia carotovora. This is a plant pathogen.
00:16:55.17		You've seen this one. It's the one that turns your lettuce
00:16:57.11		and your potatoes brown in your refrigerator.
00:16:59.13		And it's a lot like Pseudomonas in that what it does is it waits and it counts its cells
00:17:04.11		and it recognizes when there's lots of cells there
00:17:06.28		and then they secrete together all of their virulence factor to make
00:17:10.16		a wound in the plant. But then what it does, it also has this very insidious strategy,
00:17:15.28		because simultaneous to that, what Erwinia also does, is to secrete all kinds of antibiotics
00:17:22.04		that it has immunity to but that other competitor bacteria
00:17:25.27		in the environment won't be immune to.
00:17:28.13		So it keeps the wound for itself and its siblings
00:17:31.06		and it fights off all the other competitors by killing them with these antibiotics.
00:17:35.18		And so this list goes on and on and on.
00:17:38.07		And I hope that what you can sort of see from this list
00:17:40.21		is that all of these kinds of behaviors
00:17:42.28		are behaviors that cells need many, many cells
00:17:45.29		acting in synchrony to make the behavior effective.
00:17:49.23		And so that's what we understand about quorum sensing,
00:17:52.02		that this is the beginning of multicellularity and these bacteria are carrying out tasks
00:17:57.17		that they could never be successful at if they simply acted as individuals
00:18:03.02		because they're too small, individually, to have an impact on the environment.
00:18:07.15		So, if you remember from the beginning of my talk, I told you that Woody Hastings
00:18:12.03		discovered this business of these bacteria communicating with molecules
00:18:16.05		in two different luminescent bacteria. Vibrio fischeri, that I've told you about
00:18:20.05		and also its relative Vibrio harveyi.
00:18:22.14		And so you might recall that I told you that Vibrio harveyi is a bioluminescent marine bacterium.
00:18:27.09		It has quorum sensing, but it lives a different lifestyle than Vibrio fischeri.
00:18:31.05		It's a free-living bacteria. And so we were interested in understanding
00:18:34.11		"How does a free-living bacterium in the ocean achieve quorum sensing?"
00:18:38.16		And so we did what I told you already,
00:18:40.28		which is to simply make mutants in Vibrio harveyi,
00:18:43.28		put them on Petri plates and look for bacteria
00:18:46.23		that weren't making light under conditions they should be
00:18:50.06		or were making light under conditions that they shouldn't be.
00:18:53.08		And just by doing that very simple experiment we were able to find the genes
00:18:58.09		involved in the quorum sensing system for Vibrio harveyi.
00:19:01.05		And so this is the model we've come up with for Vibrio harveyi.
00:19:05.25		And this is supposed to be the inside of the bacterium.
00:19:09.00		This over here is the periplasmic space.
00:19:11.15		And then way out there would be the outside.
00:19:14.03		Right, so this is supposed to be the bacterial membrane.
00:19:17.09		And much to our surprise, when we started working on Vibrio harveyi,
00:19:21.20		what we noticed was that the bacterium had a completely different quorum sensing system
00:19:26.25		than Vibrio fischeri and all these other LuxIR bacteria
00:19:30.16		that I told you about in the beginning of the talk.
00:19:32.26		What we realized right away is that the bacteria made two different autoinducers.
00:19:37.26		And so we named them in the order we found them
00:19:40.01		Autoinducer-1 and Autoinducer-2.
00:19:42.21		Autoinducer-1 is an acyl homoserine lactone.
00:19:46.16		So it's one of these run of the mill quorum sensing autoinducers
00:19:48.28		that's typical of gram-negative bacteria.
00:19:51.07		And actually there's a picture of it on my last slide.
00:19:53.16		The second autoinducer, autoinducer-2, was clearly not a homoserine lactone.
00:19:58.29		So homoserine lactones have very standard biochemical properties.
00:20:02.14		They all sort of behave the same biochemically.
00:20:05.00		And it was very clear to us that autoinducer-2 was something different.
00:20:08.18		And I'm going to spend a lot of time telling you about autoinducer-2 as we go along.
00:20:12.11		Each of these autoinducers was detected by its own sensory system.
00:20:16.25		So autoinducer-1 was detected by a protein that we named LuxN
00:20:20.24		and two proteins called LuxP and LuxQ work together
00:20:24.17		to detect and send the information in from autoinducer-2.
00:20:28.03		So LuxN and LuxQ and all of the other colored proteins that I have in this slide
00:20:33.15		are what are called bacterial two-component signaling proteins.
00:20:36.27		And these are highly conserved proteins in all kinds of species of bacteria.
00:20:41.15		And they're all set up the way that I have on this slide,
00:20:44.15		which is that they have a transmembrane domain that connects the inside of the cell
00:20:49.02		to the outside of the environment.
00:20:50.26		And so these transmembrane domains are responsible for detecting
00:20:54.12		things that are happening on the outside.
00:20:56.22		In the case of today's talk what they're detecting are these autoinducers.
00:21:00.08		And then that information gets sent into the inside of the cell
00:21:03.27		by a phosphorylation cascade that always involves histidines and aspartates.
00:21:08.28		The way that this signaling system works is that at low cell density,
00:21:13.06		so when the autoinducers aren't there, these sensors, LuxN and LuxQ, are kinases.
00:21:18.28		They auto-phosphorylate and the phosphate gets transferred
00:21:22.03		through this circuit to a protein called LuxU and then finally to LuxO.
00:21:26.16		And LuxO's job is to turn off luciferase.
00:21:30.10		And luciferase, which are the genes and the enzymes that make light,
00:21:33.29		is down here. It's called LuxCDABE.
00:21:36.29		So there are five enzymes, C, D, A, B, and E, that are involved in making light.
00:21:41.07		So at low cell density, the system shuts light off.
00:21:44.26		But then at high cell density, so when these autoinducers accumulate,
00:21:48.27		and they get bound by their sensors, it flips a switch.
00:21:51.29		And the sensors change from being kinases to being phosphatases.
00:21:56.15		So phosphate flows backwards through the system
00:21:59.09		and that dephosphorylates LuxO and releases it to allow luciferase to be expressed.
00:22:06.08		And so the bottom line is that at low cell density light is turned off
00:22:10.06		and at high cell density light is turned on.
00:22:13.00		And that matter and the way the bacteria detect those two situations
00:22:16.14		is by whether or not these autoinducer molecules have been built up.
00:22:20.12		And then I want you to also notice that there's another protein
00:22:23.16		which I refer to, named LuxP, that's involved in autoinducer-2 detection.
00:22:28.17		So I just want to remind you that LuxP is the primary binding protein for autoinducer-2
00:22:34.10		and it interacts with LuxQ to send the signal.
00:22:37.12		And LuxP looks like a family of proteins that are typical in bacterial periplasms
00:22:43.07		that all look like sugar-binding proteins.
00:22:46.08		This protein looks like the ribose binding protein of E. coli.
00:22:49.25		And we're going to come back to that in a few minutes.
00:22:51.26		OK, so, by this complicated system information comes in to turn on or off light.
00:22:57.24		And, so, we were curious at this point why Vibrio harveyi would have two autoinducers.
00:23:03.13		So I spent the beginning of my talk telling you about Vibrio fischeri
00:23:06.20		that has this one autoinducer and uses this LuxIR system.
00:23:10.00		What's the point of having two autoinducers feeding into the system?
00:23:14.15		And of course we didn't know the answer to that.
00:23:16.12		But, what we thought was the only way the bacterium can get more out of this kind of circuitry
00:23:21.05		is if those autoinducers encode different pieces of information.
00:23:26.00		Right, if they encode the same kind of information, then two isn't better than one.
00:23:30.21		And, so, we wanted to explore that idea.
00:23:32.26		And, so, what you know already is that these autoinducers are on the outside of cells.
00:23:37.28		And, so, what one can do is just grow up cells, spin the bacteria out of the culture,
00:23:42.28		collect the cell free culture fluids and test them for autoinducers.
00:23:47.00		And so what we did is we just got every bacterium we could get our hands on.
00:23:50.26		We grew cultures of them up, spun them out, and we collected the supernatants.
00:23:54.20		And then we put them onto Vibrio harveyi strains
00:23:57.25		that could only turn on light in response to autoinducer-1
00:24:00.22		or could only turn on light in response to autoinducer-2.
00:24:04.10		So these were engineered strains that could respond to one or the other autoinducer.
00:24:08.19		And what we found in that experiment is that there was never another bacterium
00:24:13.14		that made an activity that turned on light through this first system.
00:24:17.15		But nearly every bacterium that we tested made an activity
00:24:22.14		that turned on light through the second system.
00:24:24.29		And, so, our interpretation of that result is that
00:24:27.13		these two autoinducers are two different languages.
00:24:30.16		Autoinducer-1 is the species specific language for Vibrio harveyi.
00:24:35.00		You'll recall that that's a homoserine lactone.
00:24:37.00		Only Vibrio harveyi makes that.
00:24:39.10		This second autoinducer we could show was broadly made in the bacterial kingdom.
00:24:44.16		We could find hundreds of species that made this activity.
00:24:47.20		And, so, what we thought then is that maybe this is the language of inter-species communication.
00:24:53.02		This is the bacterial trade language.
00:24:55.14		Because if you think about how bacteria really live in the wild,
00:24:59.02		they live in complicated mixtures with hundreds or thousands of other species of bacteria.
00:25:04.02		And if we end up being right in the quorum sensing field that this is about counting,
00:25:08.25		it's not good enough to only be able to count your own siblings.
00:25:12.24		There has to be a mechanism for taking into account other species of bacteria in the environment.
00:25:18.10		And that's what I'm going to try to convince you that autoinducer-2 is about.
00:25:22.03		So to get proof of that, what we did was made mutants that couldn't produce autoinducer-2
00:25:28.02		in several different species of bacteria,
00:25:30.09		including Vibrio harveyi, E. coli, Salmonella, lots of different bacteria.
00:25:34.14		And then we cloned the gene that was involved.
00:25:36.27		And what we found was that indeed, it was the same gene in every case.
00:25:41.16		And so we named that gene luxS.
00:25:45.04		And so what you can do now...you probably know that all these bacterial genomes are sequenced
00:25:50.08		so you can take your gene and plug it into these public databases
00:25:52.27		and then find out who has that gene.
00:25:55.23		And what we found is that, sure enough, at last count there were about
00:26:00.11		500 bacterial genomes that are sequenced
00:26:02.19		and more than half of those genomes contain a highly conserved luxS enzyme.
00:26:07.24		And so what I did was to simply put a smattering
00:26:10.28		of a few of the bacteria that are in the genome database
00:26:13.25		that have this luxS gene. And so what's important about this list
00:26:17.20		is that it is a who's who of clinical pathogens.
00:26:20.24		And so you remember that I told you with autoinducer-1 signaling
00:26:24.09		many bacteria use quorum sensing to turn on virulence.
00:26:28.27		And so what we wondered was that if autoinducer-2 and luxS end up being this generic bacterial language
00:26:35.08		and bacteria are using this to control virulence, one can start to think of making
00:26:40.21		anti-quorum sensing strategies that could be used in a broad range of different bacteria.
00:26:46.02		And so the field now is trying to do that in an effort to make new kinds of antibiotics
00:26:51.27		that no longer kill bacteria but just make them so that they can't count one another
00:26:56.25		and can't turn on virulence. And so what's interesting in that realm is that all of these pathogens
00:27:01.11		and if you look at that list its many, many more pathogens,
00:27:04.09		have luxS. They all make autoinducer-2; we showed that.
00:27:08.00		And what's amazing about the list is that this list
00:27:10.20		is both gram-negative and gram-positive bacteria.
00:27:13.26		So unlike in the acyl homoserine lactone and the peptide quorum sensing systems,
00:27:18.07		we think that autoinducer-2 is a much more ancient language
00:27:21.23		that arose before the split
00:27:23.19		between gram-negatives and gram-positives.
00:27:25.27		So now we knew that all these bacteria made autoinducer-2,
00:27:29.19		they all had this highly conserved gene and enzyme LuxS.
00:27:33.08		And so the questions for us at this point was: What does luxS do in producing autoinducer-2?
00:27:39.29		What is the autoinducer-2 molecule?
00:27:41.12		And then, what do all these other bacteria do in response to autoinducer-2?
00:27:46.07		Obviously they don't turn on bioluminescence.  These aren't bioluminescent bacteria.
00:27:50.00		And, so we wondered, do they use this molecule as information?
00:27:53.18		And so that's what I'm going to finish this part of the talk by telling you is first:
00:27:57.22		what is autoinducer-2 and what is luxS's job?
00:28:00.19		And then what these other bacteria are doing with this new molecule?
00:28:04.17		OK, so, the first thing: What's the autoinducer and what's LuxS?
00:28:08.14		So, remember, I've already told you that the bacteria give you this gift.
00:28:11.13		They put these molecules on the outside and so lots of people, you know,
00:28:15.01		we included, have been purifying autoinducers from cell free culture supernatants of bacteria.
00:28:20.07		And so we had been trying to do that with autoinducer-2, sort of doing traditional biochemistry
00:28:25.08		and we could never purify this molecule.
00:28:27.12		And we didn't understand at the time why we couldn't get it
00:28:31.10		but the point was, we couldn't purify it.
00:28:33.05		And so we thought, "OK, we can't use traditional biochemistry to get it
00:28:36.26		but can we use what we've learned from these genomes to just figure out,
00:28:40.15		to infer what luxS does just by looking at where it's placed in these genomes?"
00:28:45.12		So probably you know that bacteria put genes that work in pathways
00:28:50.05		nearby one another, usually in operons.
00:28:52.10		And so what we did was to just look over these genomes and ask:
00:28:56.01		"Could we find any pattern to where luxS was in these different genomes?"
00:29:00.21		And in a couple of cases, indeed, we could.
00:29:03.03		And so I've just put on this next slide the case that we've found for Borrelia burgdorferi,
00:29:03.17		which is the bacterium that causes Lyme disease.
00:29:09.20		What we noticed was that LuxS was in a three gene operon in the Borrelia genome
00:29:15.25		and in a few other genomes.
00:29:17.15		And the operons were always set up like this. There were three genes.
00:29:20.24		One was called pfs, the second one was called metK and then luxS was right next door.
00:29:26.16		And so we didn't know what luxS does, but in fact, pfs and metK have known functions.
00:29:33.02		And they work together in a biochemical pathway.
00:29:35.28		And so we wondered, "Couldn't luxS work in that pathway with pfs and metK?"
00:29:40.29		And so what has been known about pfs and metK for almost 50 years now
00:29:45.19		is the following: those two enzymes work in S-adenosyl methionine utilization.
00:29:51.21		So maybe you know that S-adenosyl methionine or SAM is a very important substrate in cells.
00:29:57.23		It's involved in methylating different substrates.
00:30:00.04		So SAM is involved in putting methyl groups on DNA and RNA and proteins.
00:30:04.25		So you can't make any of those things unless you have SAM.
00:30:07.17		So it's a really important, ancient molecule.
00:30:10.06		And so the way that that works is that methionine is converted into S-adenosyl methionine
00:30:16.06		by that first enzyme we noticed which is called metK.
00:30:19.10		And then S-adenosyl methionine or SAM does its job.
00:30:23.08		It puts a methyl group on all these different substrates.
00:30:25.19		And so what the cell gets from that is the thing it wants,
00:30:28.18		like DNA or RNA, but it gets, unfortunately, a bi-product which is called S-adenosyl homocysteine.
00:30:35.27		And this is incredibly toxic because it feed back inhibits all of these methyltransferases
00:30:41.10		that are putting the methyl groups on the different substrates.
00:30:44.17		So what happens is that every time SAM methylates something a molecule of SAH is produced
00:30:50.06		and the cells have to get rid of that because of its toxicity.
00:30:53.25		So the way that they decrease toxicity is with this second enzyme I told you about, pfs.
00:30:59.24		So pfs is a nucleosidinase that takes adenine off of SAH and makes S-ribosyl homocysteine.
00:31:07.15		And so that relieves toxicity.
00:31:09.25		And it's been known since the early 60's that S-ribosyl homocysteine gets converted by
00:31:15.14		some unknown enzyme into homocysteine
00:31:18.02		and this funny molecule, 4,5-dihydroxy-2,3-pentanedione.
00:31:22.01		that nobody knows what it does.
00:31:23.15		And so what we wondered when we saw that metK and pfs were involved in this pathway
00:31:28.12		and there was a step that didn't have an enzyme associated with it
00:31:31.27		we wondered, "Well, couldn't that be luxS?
00:31:35.01		That what luxS's job is to do is to carry out this last step in this pathway."
00:31:40.10		And so to prove that was the case, what we did is we cloned and purified the luxS protein.
00:31:45.26		We put that protein in a test tube with the molecule S-ribosyl homocysteine,
00:31:50.27		let it react and then we filtered out the products.
00:31:53.16		And what we got from that was homocysteine and autoinducer-2.
00:31:58.07		And so indeed this is the pathway for making autoinducer-2.
00:32:02.03		And this final product that nobody had ever known what it was, is in fact, autoinducer-2.
00:32:07.16		And so this has always been considered a salvage pathway in bacteria.
00:32:12.16		They have to detoxify to get rid of this SAH
00:32:15.22		and doing it this way they get two useful things: they get adenine at the first step
00:32:20.11		and they get homocysteine at the second step.
00:32:22.23		But we think the real reason this pathway has evolved is because what they're trying to make
00:32:27.09		is this funny molecule which I'm trying to tell you
00:32:29.15		is a molecule involved in interspecies communication.
00:32:32.20		OK. So now I'm going to show you the biochemistry that luxS does.
00:32:37.21		So this is the reaction. So this is the molecule S-ribosyl homocysteine.
00:32:42.06		And the enzyme luxS's job is to just cut that in half
00:32:45.24		into homocysteine, which gets recycled, and then this molecule that I told you about
00:32:50.16		which is called 4,5-dihydroxy-2,3-pentanedione.
00:32:54.09		So we knew we could do this experiment and we knew we had autoinducer-2 activity in it.
00:33:01.00		So the guess was that this is autoinducer-2.
00:33:03.26		And so what we did to prove that was we carried out big reactions.
00:33:08.01		Right? Then we filtered out the protein
00:33:09.25		and then we took what was on the bottom of those reaction mixtures to the chemistry department
00:33:14.20		and did mass spec and NMR  and techniques like that to try to find this molecule.
00:33:19.11		And what was interesting during this experiment is that we could find lots of homocysteine
00:33:24.12		in these reactions but we could never find this molecule.
00:33:27.21		So even though we knew the biochemistry,
00:33:29.23		we had one enzyme and one substrate in a test tube,
00:33:32.18		we couldn't find the product.
00:33:34.01		And the reason is because this molecule, this pentanedione, is incredibly reactive.
00:33:39.15		What happens is that as soon as it gets made, it starts cyclizing
00:33:43.09		into family of molecules that are in a fast equilibrium, all of which are derived from this precursor.
00:33:49.22		So you get many molecules and they're all sort of the same thing
00:33:52.17		because these rings are just opening and closing.
00:33:54.24		And so what we thought then is that one or some of those molecules
00:33:58.26		must have the autoinducer-2 activity,
00:34:01.15		but we still didn't know the structure.
00:34:04.02		And so actually, I have to say that we were a little discouraged by that because
00:34:07.05		here, we could do the reaction in one step, but we still didn't know what this elusive molecule's structure was.
00:34:12.13		And so we knew we couldn't get it from this because these molecules are opening and closing
00:34:16.25		really fast and they're all turning into one another.
00:34:19.10		And so the question was: how could we figure out which was the molecule that carries the activity?
00:34:24.09		And so what we decided to do is to just go back to what we knew about how
00:34:29.03		autoinducer-2 activity was detected. And you'll remember that I showed you
00:34:33.05		this cascade for how the information from these autoinducers comes into the cells.
00:34:37.25		And you might recall that I pointed out that there were two proteins
00:34:40.25		involved in autoinducer-2 detection, LuxP and LuxQ.
00:34:45.15		And what I slipped by you earlier in my seminar
00:34:48.07		is that this protein, LuxP, is a periplasmic protein that looks like the ribose binding protein.
00:34:54.06		And what I just showed you on this slide,
00:34:57.05		is that LuxS actually takes the ribose moiety of S-ribosyl homocysteine
00:35:04.13		and opens it up. But when this molecule closes back into a ring
00:35:08.09		it looks a little bit like ribose because it was made from ribose.
00:35:11.28		And so that started to make sense to us, that there should be
00:35:15.05		a protein that looks like it can bind a ribose-like molecule involved in autoinducer-2 detection.
00:35:20.20		So what we decided to do then was to take this protein, and purify it.
00:35:25.28		And get it to reach into this in vitro mixture that has all of these molecules
00:35:29.29		that are made from the pentanedione. We could get it to reach in there
00:35:34.19		and pull out the rearranged moiety that had the autoinducer-2 activity.
00:35:39.04		So sort of like an affinity purification.
00:35:41.20		Of all this mix of molecules that were rearranging in solution, just let LuxP
00:35:46.16		reach in there and grab the one that had...that is autoinducer-2.
00:35:51.06		And so we did that experiment in collaboration with a colleague
00:35:54.26		at Princeton University; a professor names Fred Houston, who's a crystallographer.
00:35:59.10		And so what we did was to crystallize LuxP
00:36:01.21		in the presence of autoinducer-2 and solve the structure.
00:36:04.26		And in fact that turned out to be pretty easy to do
00:36:07.16		because these periplasmic binding proteins are very well studied
00:36:13.05		and they all look sort of the same. So this is our crystal structure of LuxP.
00:36:17.25		And all of these proteins, including LuxP, look sort of like clam shells.
00:36:22.03		They lock down on the middle to a sugar that's at the interface between the two domains.
00:36:28.05		And our protein was no different. It looked like this clam shell
00:36:31.09		and there was a big space in the center where a molecule could bind.
00:36:35.11		And when we did a little closer up version of this,
00:36:38.11		where we just get rid of all the electron density from the amino acids,
00:36:42.28		what you're left with is more or less a skeleton of the crystal structure
00:36:47.21		and right at the ligand binding domain, we could see a lot of electron density
00:36:52.13		indicating that the autoinducer-2 molecule was there.
00:36:55.13		And so to prove that that was correct what we did was we just heated the protein up to
00:36:59.19		about 50 degrees and let this stuff fall out
00:37:02.25		and if you put it in a tube with Vibrio harveyi, they turn on light.
00:37:06.08		So indeed, this was the autoinducer.
00:37:09.05		So we could zoom in a little bit more to try to figure out
00:37:11.29		what was the molecule that was sitting there in the active site.
00:37:15.10		And so in this slide what you're looking at is the amino acids from LuxP
00:37:21.00		that coordinate the ligand.  And I hope what you can see are two rings.
00:37:25.25		And this was a surprise to us because from the biochemistry
00:37:28.21		I showed you a couple of slides ago, when that pentanedione molecule folds up
00:37:33.18		there's only five carbons in that.
00:37:35.24		And we could account for those five carbons from this bottom ring.
00:37:39.15		So this was the folded up pentanedione that we knew about.
00:37:42.19		The problem was there were all these other atoms on the top.
00:37:45.29		And we were confused by that because we didn't know where they came from.
00:37:50.08		So to prove in fact that this was the correct molecule what we did was a mass spec of the protein
00:37:56.08		bound to the ligand and then we did this gentle heat denaturation so that the ligand falls out
00:38:01.12		and we took the mass of the protein again.
00:38:03.29		And the difference in mass is 194 which is exactly what this molecule weighs.
00:38:09.09		And so what we thought then is that LuxS, and we know this now,
00:38:13.06		every LuxS makes that linear pentanedione and then it folds up into this ring structure
00:38:19.12		but they're still very reactive because these rings have a lot of oxygens on them
00:38:24.00		which are highly reactive. So we thought, "OK, something must add to the top across those oxygens
00:38:30.08		to make this double ringed molecule that has a molecular weight of 194.
00:38:35.20		And the surprise was this atom right here.
00:38:39.13		We thought that should be carbon because that would connect the ring
00:38:42.15		and that would make a molecule of 194.
00:38:44.21		The problem was that chemists didn't like that idea because it's very well known
00:38:48.21		that carbons are very unstable if they have four oxygens on them.
00:38:53.10		And so what we found out later was, in fact, that's not a carbon atom.
00:38:57.01		What it is is a boron atom.
00:38:59.10		So it turns out that boron is very abundant in the ocean.
00:39:04.25		Right? And remember, this is a marine bacterium.
00:39:07.20		And boron also loves to add across cis-hydroxyl groups that are on rings.
00:39:13.12		And so what we found out, in fact, was that the real signaling molecule
00:39:17.05		is this one that I'm showing you here,
00:39:19.09		that has a boron on the top of that ring.
00:39:22.00		And to prove that that was the case we did boron NMR. You can look for boron 11.
00:39:27.05		And showed that, sure enough, this is the Vibrio harveyi molecule and it has boron in it.
00:39:32.12		And so that was actually a surprise because boron,
00:39:34.23		even though it has a very well studied role in chemistry,
00:39:37.13		it doesn't have a very well defined role in biology.
00:39:40.17		But in fact there's lots of it the ocean
00:39:43.01		and so this molecule could be made spontaneously
00:39:46.00		when that pentanedione gets released from the bacteria.
00:39:48.25		And so now, in fact, even though this molecule doesn't look like the biochemistry
00:39:52.21		that I showed you from the LuxS reaction, in fact, we can account for everything that's happening.
00:39:57.14		What we know now is that every LuxS protein makes this molecule, which we call for short DPD,
00:40:04.28		for dihydroxy pentanedione. Every LuxS makes that linear pentanedione.
00:40:10.22		But that molecule, as I told you is very reactive, and so it starts cyclizing.
00:40:14.19		And in the case of Vibrio harveyi, this important ring is formed,
00:40:18.13		and you have to notice that this is a chiral carbon with the oxygen up and the methyl down.
00:40:23.01		So this ring gets formed. That gets hydrated spontaneously.  There's a lot of borate in the ocean
00:40:28.19		which adds across these two oxygens to make the final signaling molecule
00:40:32.28		that we found in the crystal structure.
00:40:35.03		And so what we know is that all of these reactions happen spontaneously
00:40:39.04		based on the precursor molecule that the bacteria make.
00:40:42.05		And, in fact, we can now make this molecule DPD.
00:40:45.15		We can put into a test tube and we can watch all of this happen.
00:40:49.01		And so we're very confident now about how
00:40:51.13		the Vibrio harveyi...what molecule Vibrio harveyi
00:40:54.12		uses for quorum sensing, autoinducer-2 quorum sensing.
00:40:58.24		But once we figured this out it actually gave us another puzzle.
00:41:03.01		Which is, as I've told you, there's a lot of boron in the ocean,
00:41:05.28		so maybe this kind of a molecule makes sense for Vibrio harveyi.
00:41:09.04		But I've also told you that hundreds of species of bacteria make and use autoinducer-2
00:41:14.00		and there's very little boron in terrestrial environments.
00:41:16.23		And so what we wondered,  "Is autoinducer-2 one word, this one,
00:41:22.00		or is it a family of words that are all made from this precursor?"
00:41:25.11		So for example, this could be someone's autoinducer,
00:41:28.01		or this could be another bacteria's autoinducer.
00:41:30.15		And the other thing that I alluded to a minute ago is that there's this chiral carbon.
00:41:34.19		So when this molecule folds up, oxygen up, methyl down, there's another pathway
00:41:39.24		which is the opposite stereochemistry that could be across the bottom of this chart.
00:41:44.16		So there's many molecules that are possibilities.
00:41:46.28		And we wondered, "Are bacteria using all of these other molecules as autoinducers?"
00:41:51.26		And so we wanted to try to do experiments to get at that
00:41:54.22		and of course the one thing that we had learned from this
00:41:57.17		is we couldn't just purify molecules from cell free supernatants
00:42:00.18		because all of these are present. And so what we had to do is a trick similar to one
00:42:05.03		that I've shown you which is to get a sensor from some bacteria,
00:42:09.00		get it to reach in and pick out the molecule it likes.
00:42:11.28		And so, what was lucky was at the time that we were working on the chemistry of autoinducer-2
00:42:16.22		we were also trying to answer another question that I alluded to earlier
00:42:20.16		which is: What do other bacteria use autoinducer-2 to do?
00:42:25.16		What genes do they turn on and off?
00:42:27.07		And so we had started to work on E. coli and Salmonella, enteric bacteria.
00:42:31.27		And simply, we were doing genetic screens to ask, "What are the genes
00:42:35.15		that are controlled by autoinducer-2 in E. coli and Salmonella?"
00:42:39.05		And we found a number of genes that autoinducer-2 regulates
00:42:42.15		but what's important for today's talk is that we found this operon
00:42:46.07		that we named Lsr for LuxS Regulated operon.
00:42:50.07		And this operon is in the E. coli and the Salmonella genome
00:42:54.17		and it's annotated as the ribose-like transporter operon.
00:42:58.06		So what the Lsr operon encodes is what's called an ABC transporter.
00:43:03.07		And ABC transporters are widespread in different organisms
00:43:07.03		and their job are to bring molecules from the outside in.
00:43:10.18		And so they all sort of look like this one.
00:43:13.07		What they have is a B component which is a binding protein
00:43:16.26		that binds some ligand and takes it to the channel. Right?
00:43:20.07		And delivers it and it comes across into the cell and the energy is supplied by the A component
00:43:26.01		which is an ATPase. And so we found that autoinducer-2 induces the genes
00:43:32.05		that make this transporter operon and this protein LsrB
00:43:36.16		which is a binding protein, looks just like LuxP from Vibrio harveyi.
00:43:40.23		It looks like a ribose-like binding protein.
00:43:43.17		And so, of course, what molecule do we know that looks like ribose?
00:43:46.13		Well, it's autoinducer-2.
00:43:48.07		And we had already shown that the way E. coli and Salmonella respond
00:43:51.10		to autoinducer-2 is that they put it out. Then they bring it in with this transporter
00:43:56.23		and then they respond to it on the inside.
00:43:59.01		And so, of course then the guess was that the molecule that LsrB
00:44:02.28		would be binding in E. coli and Salmonella was their autoinducer-2.
00:44:07.07		And so, exactly analogous to what I told you about a few slides ago,
00:44:10.21		We crystallized this protein, the Salmonella LsrB protein, with the ligand
00:44:15.07		and solved the structure. And what we found was that
00:44:19.03		as expected, the protein looked very similar to LuxP and again,
00:44:23.09		the ligand was in the ligand binding site.
00:44:26.03		And so we could just solve the structure and what we found was that, indeed,
00:44:29.15		the Salmonella molecule and the Vibrio harveyi molecule are different molecules.
00:44:36.07		So now what I have done is to put the two molecules side by side.
00:44:41.00		And so you'll remember that the Vibrio harveyi molecule is this double ringed molecule
00:44:45.15		that has this boron moiety at the top of the ring.
00:44:48.18		The Salmonella molecule, what you can see is that it doesn't have that boron ring.
00:44:53.16		And that makes sense to us because there is no boron in your gut.
00:44:57.03		There's hardly any boron there. Salmonella would never find the molecule that looked like that.
00:45:01.04		So the first thing that's different is that Salmonella's autoinducer-2 doesn't have boron,
00:45:06.09		which makes sense given it's a terrestrial environment
00:45:08.12		and Vibrio harveyi is a marine environment.
00:45:10.20		And the second thing, which is a little more subtle, that you have to notice
00:45:13.20		is that this is that chiral carbon; oxygen up, methyl down in the case of Vibrio harveyi.
00:45:18.26		In the case of Salmonella, the stereochemistry is the opposite; methyl up, oxygen down.
00:45:24.10		But in fact, even though these molecules look different, we can connect them really easily.
00:45:29.01		And so, you'll remember the chemistry that I showed you about Vibrio harveyi
00:45:33.28		is that all the LuxS make this pentanedione and then by these reactions I showed you
00:45:38.19		and borate addition, you get the Vibrio harveyi signal.
00:45:41.23		In the case of Salmonella, remember the other 50% of the time
00:45:45.24		that the DPD molecules cyclize,
00:45:52.22		remember 50% of the time they're going to cyclize with the methyl up and the oxygen down.
00:45:57.07		That's important for Salmonella.  That molecule gets hydrated
00:46:01.10		and this is the molecule that we find in the Salmonella ligand binding pocket.
00:46:05.15		And so now we know only two autoinducers that are made
00:46:09.16		from this precursor molecule, autoinducer-2.
00:46:12.07		And so of course, we're still looking to find if
00:46:14.04		this one's an autoinducer or this one or this one.
00:46:16.11		Right? But that we haven't done yet. But even though we don't know
00:46:19.13		the complete lexicon of how these bacteria use the information in that molecule to talk,
00:46:24.02		we do understand the principle for how interspecies communication works.
00:46:28.13		Because all of these molecules are freely rearranging in a very fast equilibrium
00:46:33.27		Salmonella can talk to Vibrio harveyi and likewise Vibrio harveyi can talk to Salmonella
00:46:38.17		because their molecules rearrange in between the sender and the receiver.
00:46:42.24		And so for example, we have done experiments to show if you put a lot of boron
00:46:48.10		in this reaction mix, all of the equilibrium goes to the Vibrio harveyi molecule.
00:46:52.23		Vibrio harveyi's quorum sensing response turns on and Salmonella is inhibited for quorum sensing.
00:46:58.07		Likewise, one can chelate all of the boron out of the medium and then the equilibrium
00:47:03.00		shifts to this way and Salmonella starts chit chatting and Vibrio harveyi can't talk.
00:47:07.26		So even though we don't know all of the molecules involved,
00:47:11.14		we understand that the reason interspecies works through this interconverting molecular system
00:47:16.06		is because this precursor is common for all of these bacteria and so, in fact,
00:47:21.11		I could talk to you and you can talk to me because they depend on these fast equilibrium reactions.
00:47:27.15		And so that's the state of affairs.
00:47:29.13		We're still trying to find more of these autoinducers that different bacteria use.
00:47:33.27		And so now I've told you two of the three things that I said.
00:47:36.23		What LuxS does: LuxS makes this pentanedione.
00:47:40.13		What is autoinducer-2? Well, we've started to understand that for
00:47:43.28		a few of these species and we think that there are more molecules involved.
00:47:47.01		But we understand how they talk between species.
00:47:50.00		And then the last thing that I said I would tell you is: What are other bacteria doing with autoinducer-2?
00:47:55.19		And so this is just a list of some of the bacteria
00:47:58.01		that different people in the field have started to study.
00:48:00.26		And so what you can see, this is just the name of the different species of bacteria
00:48:04.27		and then on this side are the genes that different colleagues of mine
00:48:09.01		have found that are controlled by autoinducer-2.
00:48:11.25		And so if you actually tried to read this list, what you can see
00:48:15.01		is that in most every case that's been studied, autoinducer-2,
00:48:19.16		just like peptides and homoserine lactones, controls biofilm formation and virulence.
00:48:25.09		And so it appears that the quorum sensing really is a big player.
00:48:29.11		Both intra-species and interspecies quorum sensing
00:48:32.15		are big players in controlling virulence and biofilms.
00:48:36.16		And so where the field is moving now is for people to either take their favorite autoinducer-1
00:48:41.29		or generically, to take autoinducer-2 and try to inhibit the synthases or
00:48:46.29		inhibit reception of those molecules to get new kinds of antibiotics.
00:48:51.11		So, when one works on an autoinducer-1,
00:48:53.20		the hope is to make a species specific antibacterial therapy.
00:48:57.27		And of course the hope for autoinducer-2 is that we could get an inhibitor that controls virulence
00:49:04.13		in all kinds of bacteria because you'll remember, again, both gram negative bacteria
00:49:08.07		and gram positive bacteria use autoinducer-2 quorum sensing to communicate.
00:49:13.15		And so that's sort of the practical way the field is going.
00:49:16.17		But I think we've learned a lot about chemical communication and also about
00:49:20.27		how bacteria have developed multicellularity.
00:49:24.05		And so to sum up, I hope what you've learned through this first part of the talk
00:49:27.27		is that bacteria can talk to each other and their languages are chemical.
00:49:32.08		They don't use words and sounds the way we do, they use chemicals.
00:49:36.05		We know that beyond being able to talk to one another,
00:49:39.20		that they can have multiple languages. And so at a minimum,
00:49:46.14		by having an autoinducer-1 and an autoinducer-2, what we now understand is
00:49:51.05		that bacteria can speak both within and between species.
00:49:56.06		So they have intra- and inter-species communication.
00:49:59.16		We also now know that by just having those two molecules,
00:50:04.09		what that means, we would argue, is that bacteria can distinguish self from other
00:50:09.17		by using a private language and a community language.
00:50:12.26		And what we think, then, is, of course, that's what happens in your body.
00:50:15.29		Its not like your kidney cells get all confused with your heart cells every day.
00:50:19.11		And that's because they use different hormones or different chemicals
00:50:22.14		to communicate and let these different organs in your body carry out different functions.
00:50:27.20		Again, bacteria have been on this earth for billions of years.
00:50:30.20		We think that they invented the idea of distinguishing self from other.
00:50:34.24		The other thing that we know is that there's many more molecules to be discovered.
00:50:40.21		This field is only 10 or 15 years old. We're at the very beginning of it.
00:50:44.27		It's a really exciting time. What I hope you've learned from today's talk
00:50:48.10		is that, indeed, bacteria can distinguish self from other,
00:50:51.17		but autoinducer-2 is a generic molecule.
00:50:54.13		So it doesn't say who the other is. And we know when bacteria make
00:50:58.16		complicated, architected communities like in biofilms
00:51:02.00		in fact, these are very ordered structures, you know, with one species next to another
00:51:06.17		next to a third. They're not willy-nilly.
00:51:08.12		And so we believe, the only way bacteria can get that is to have molecules that say
00:51:13.07		who the other guy is. And that's what this field is hunting for now.
00:51:17.05		And I think that's what you'll be hearing about in the next few years.
00:51:20.07		And then what I've argued and I hope that you think is an interesting idea
00:51:25.12		is that quorum sensing let's bacteria be very similar to higher organisms.
00:51:30.21		Bacteria don't act as individuals. Often times they need to act as a community, in synchrony.
00:51:37.14		And by communicating with chemicals, this allows bacteria to carry out
00:51:41.15		traits that are very similar to those carried out by higher organisms
00:51:45.02		because they've established this way to control gene expression instead of over a single cell,
00:51:50.14		over the entire population. And, of course, along with learning about these first things,
00:51:56.02		we now understand that there's a lot of opportunities for novel biotechnological applications.
00:52:01.02		For example, to make, as we've discussed, to make new therapies, new antimicrobial therapies.
00:52:06.17		Also, people would like to put these anti-quorum sensing molecules in plastics.
00:52:11.08		If one knew one was going to go to the hospital and get a catheter
00:52:13.28		that they'd get infections. If you could embed these in paints or plastics or implants
00:52:18.13		or the saran wrap that wraps up your meat at the grocery store it would be really great if
00:52:23.04		we could just use these anti-quorum sensing molecules to fight against pathogenic bacteria.
00:52:28.17		And at the same time, of course, we now know that we are covered with bacteria.
00:52:32.24		Bacteria live in us and on us all the time and they're playing an active role
00:52:36.06		in keeping us healthy. So we'd also like to look for pro-quorum sensing molecules,
00:52:40.27		molecules that make quorum sensing better in beneficial bacteria...
00:52:46.16		that help to fight off invaders.
00:52:48.00		And so we're looking for both agonists and antagonists of quorum sensing molecules
00:52:53.04		for novel biotechnological purposes.
00:52:55.23		And the final thing to do is to make a confession
00:52:58.14		that I'm not so smart. These bacteria have already figured this out.
00:53:01.15		They have a billion year head start on us.
00:53:03.02		What the field has been learning which is really exciting in the last couple of years
00:53:06.28		is that there's all kinds of natural anti-quorum sensing strategies
00:53:11.00		happening within the bacterial populations themselves.
00:53:14.00		So there's bacteria that eat each other's auotinducers.
00:53:16.07		There's bacteria that cut each other's autoinducers in half.
00:53:18.26		They eavesdrop. They cheat. They free-ride. They're doing all the kind of things that
00:53:22.26		we'd like to do and so we think that we can get a lot of the hints for how
00:53:26.05		to make these strategies just from the bacteria themselves.
00:53:29.12		So, thank you for listening to this talk.
00:53:31.25		The second half will be a much more practical talk but that's the beginning
00:53:35.28		and I hope you think, by now, that bacteria can talk to each other.
00:53:38.27		And, again, I'm Bonnie Bassler from Princeton University.

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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