Cell-Cell Communication in Bacteria via Quorum Sensing

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.

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.

Videos in this Talk

Part 1: Bacterial Communication via Quorum Sensing
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Vibrio Cholerae Quorum Sensing and Developing Novel Antibiotics
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad

Speaker: Bonnie Bassler

Total Duration: 1:13:36

Recorded: June 2009

Educator Resources for this Talk

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Talk Overview

Bacteria, primitive single-celled organisms, communicate with chemical languages that allow them to synchronize their behavior and thereby act as enormous multi-cellular organisms. This process is called quorum sensing and it enables bacteria to successfully infect and cause disease in plants, animals, and humans. Investigations of the molecular mechanisms underlying quorum sensing are leading to the development of novel strategies to interfere with quorum sensing. These strategies form the basis of new therapies to be used as antibiotics.

Speaker Bio

Bonnie Bassler

Bonnie Bassler is a member of the National Academy of Sciences and the American Academy of Arts and Sciences. She is a Howard Hughes Medical Institute Investigator and the Squibb Professor of Molecular Biology at Princeton University. Bassler received a B.S. in Biochemistry from the University of California at Davis, and a Ph.D. in Biochemistry… Continue Reading

Comments

richard schaffner says

October 7, 2018 at 8:37 am

I am a hydrogeologist and have worked for more than 30 years on the investigation and remediation of hazardous waste sites. My specialty is in situ bioremediation, stimulating mostly native degrader communities to transform/degrade contaminants of concern (e.g., petroleum hydrocarbons, halogenated solvents, and heavy metals). We have been adding inorganic nutrients to biological stimulants (largely electron acceptors or donors) for years and have been seeing certain enhanced performance attributes that differentiate the stimulants we have been using from those used by many others and for nearly two decades we could not explain why. After reading about quorum sensing/signaling (QSS), first about a year ago, I now believe that the inorganic nutrients we add are helping to increase population densities of degrader communities in situ and triggering QSS, which enhances certain performance attributes well beyond those of additives lacking these nutrient cocktails. Hence, I am reading nearly everything I can find find about QSS, and related microbial “tools”, and that was the purpose for viewing Part 1: Bacterial Communication via Quorum Sensing. The video was simply outstanding and it helped me pull together a lot of what I have been reading without having the background in biochemistry/molecular biology to really drill deeply into this subject. (I augmented my MS programs in contaminant hydrogeology and environmental engineering with soil microbiology and basic biology, but that was nearly 30 years ago.) Anyway, I just wanted to say thank you and let you know that your video was much appreciated! I cannot wait to watch Part 2 and “mine” iBiology for more on this and related topics!

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Part 1: Bacterial Communication via Quorum Sensing

Part 2: Vibrio Cholerae Quorum Sensing and Developing Novel Antibiotics

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