Hi, my name’s Bonnie Bassler and I’m from Princeton University and I’m also a Howard Hughes Medical Institute investigator. And for the second part of my talk today what I want to focus in on is how quorum sensing is involved in pathogenesis in a very important pathogen Vibrio cholerae and our efforts to exploit quorum sensing in order to make new kinds of antibiotics. So, hopefully what you remember from the first part of my talk is that quorum sensing is this mechanism that bacteria use to communicate with one another and to act in groups. And so you’ll recall that they make and release small molecules that we call autoinducers, and when these autoinducers build up to a critical amount, they recognize that those autoinducers are there which tells them that they have lots of members of the community around, and then all the bacteria switch gene expression which is, again, behavior in unison. So they carry out tasks as enormous multicellular organisms. And so what you’ll remember from the first part of my talk is that we had focused on, for that talk, this harmless but beautiful bacterium Vibrio harveyi which uses quorum sensing to control bioluminescence. And you’ll remember that Vibrio harveyi has two autoinducers called autoinducer-1 and autoinducer-2. And each of those autoinducers is detected by its own sensor. LuxN detects autoinducer-1 and two proteins work together to detect autoinducer-2. And the information from those extracellular signal molecules comes to a protein called LuxU. It gets transferred to a protein called LuxO. And LuxO’s job is to control luciferase, the light producing enzymes. And so we had studied that and figured this out in this harmless bacterium Vibrio harveyi. But we wanted to try to think about quorum sensing in Vibrios but in pathogenic bacteria. And so what we did is we turned out attention to this very important pathogen Vibrio cholerae that is endemic in under-developed countries and we figured out its quorum sensing circuit. And what we found is that the circuit is incredibly similar to this circuit in Vibrio harveyi. So on this next slide I’m showing you just a very slightly different circuit but this is the quorum sensing circuit for Vibrio cholerae. And most of it should look identical to what I just showed you for Vibrio harveyi. So what we know is that cholera has its own autoinducer-1 and so we call that CAI-1 for cholera autoinducer-1. And that’s made by an enzyme that we called CqsA for Cholera Quorum Sensing Autoinducer. And this gets detected by its own sensor which we call CqsS for Cholera Quorum Sensing Sensor. So this circuit, this one on the bottom of my slide, is the intraspecies communication circuit. So in Vibrio harveyi, on my last slide, this was called AI-1 and LuxN. In Vibrio cholerae it’s called CAI-1 and CqsS and it is cholera’s private language. But then, just like Vibrio harveyi, cholera has this second circuit. It has the LuxS enzyme that makes the generic autoinducer-2 molecule and that gets detected by LuxP and LuxQ. So that part of the circuit, the generic part is identical to Vibrio harveyi. And then the downstream components, LuxU and LuxO, are also identical. So the only point is that each Vibrio harveyi and Vibrio cholerae have their own private autoinducer-1 system. They have the same autoinducer-2 system and the way the information gets relayed into the cell is identical. It’s through LuxU and LuxO. And then, of course, the job of this circuit is to turn on and off genes when Vibrio cholerae needs them. And so now I’ll tell you how…so that’s generically the system and now I want to tell you how cholera uses quorum sensing for pathogenesis because it’s actually, sort of a unique way. So what we’ve learned about cholera is that at low cell density, so when these autoinducers are at low levels, what happens is that information that there are very few cells around comes into the cells and it tells Vibrio cholerae, surprisingly, to turn on all of its virulence genes, all of the biofilm genes and other genes that are required for infection. But then at high cell density…so that’s…I should say this, be more serious… So, remember, at low cell density cholera is turning on virulence. But then at high cell density, when the autoinducers accumulate and there’s lots of Vibrio cholerae cells around, what happens is that the circuit sends this information backwards and what cholera does is to turn off virulence, off biofilms and off all kinds of other genes that are required for infection, and it turns on genes that are involved in escape from the host like an important protease. And so we have to stop for a minute and talk about this. So, cholera, unlike most bacteria that you hear about, causes an acute infection. Most bacteria that we get sick from cause what are called persistent infections. So their goal, if I can say things like that, is to get in you to cause an infection and to stay there. So all of the bacteria that we’ve learned about other than cholera that cause persistent infections use quorum sensing to turn on virulence at high cell number when they know they’re going to be able to make the host succumb. Cholera has this very insidious strategy. What it does is that you get it from eating contaminated water or contaminated food and it gets into your intestine and it immediately turns on this entire battery of virulence genes. So it turns on a gene called Tcp for Toxin Co-regulated pilus that allows it to adhere to your intestinal epithelial cells. It turns on the toxin, which is the thing that makes you sick. And then what happens is it starts growing like crazy. It makes the person very sick. And people get a terrible diarrhea from cholera. But then, when cholera is at high number, it turns all of those genes off, the ones that really make you sick. And it turns on this protease, which is a “detachase” that cuts the bacteria off your intestinal epithelial cells and out they come to infect the next host. So it’s using quorum sensing for virulence but it simply has the mechanism reversed. It turns on the virulence genes at low cell density. It makes you really sick. It grows to the gazillions. It turns off virulence genes. It turns on an escape pathway and out it comes back into the environment to make many more hosts sick. And so it’s a fabulous strategy from cholera’s perspective, but this is why cholera causes an acute disease. If you can actually survive that phase of the disease, you’re cured because it’s self limiting. It gets in and it gets out. And so the important point for today that you need to understand is that the autoinducers shut off virulence. And so as we talked in the first part of my talk, there’s lots of groups now trying to work on strategies to interfere with quorum sensing to work on bacteria that cause persistent infections. If we could make anti-quorum sensing molecules or inhibitors of these enzymes that make theses autoinducers maybe those could be new therapies. And there’s a tremendous amount of work going on about that in the field. But of course, it’s kind of difficult. We have to figure out what molecules interfere with the circuit. We have to try to make inhibitors of these enzymes. And so that’s fun and that’s interesting but it’s taking a little bit of time. And so what we thought about this funny cholera circuit is that because the autoinducers turn off virulence, we could use this as a test case to see if we could manipulate quorum sensing and actually shut down virulence. Because cholera uses this weird circuit to turn off virulence at high cell density what that means is that the autoinducer itself is the drug. If we add the autoinducer, we should be able to shut down virulence. And so that’s backwards of what we’re trying to do in all these other systems where we want to get antagonists of quorum sensing. So we thought, as a proof of principle, to find out is there any merit in the idea of trying to interfere with these quorum sensing systems? Cholera gave us a unique chance because we could just add the autoinducer and see whether or not we could shut down virulence. And so of course to measure these kinds of genes, virulence and biofilm, that’s kind of tricky. And so what we thought we would do to get at that is to use bioluminescence. So you’ll remember from the first part of my talk, that quorum sensing controls bioluminescence in Vibrios. And so what we did is we engineered into Vibrio cholerae a quorum sensing activated luciferase reporter. So now when we add autoinducers bioluminescence turns on. And we thought we could use this as a read out to try to purify this molecule and see if we could control quorum sensing with it. OK, so now we have a Vibrio cholerae that makes light in response to cholera autoinducer-1 and to autoinducer-2. But we just wanted to focus on cholera autoinducer-1 because that would be a test that was simply particular to cholera. And so remember I told you in the first part of my talk that these molecules are on the outside of cells. And so we wanted to find out what this cholera autoinducer was. So the strategy we did was to just grow up a lot of cholera. Spin the cells out of solution. Filter them out. Collect the cell-free supernatants. And we could see that it had a lot out autoinducer activity in it because it turned on luciferase when we added it back to the cells. And so then to purify it what we did was we did a number of extractions of the media and some sort of fancy column chromatography and then finally in the end we put our cleaned up, cell-free supernatants on to an HPLC column and just measured bioluminescence as a measure of activity. And so here you’re looking at bioluminescence and this is just fraction number dripping off that column. And what you can see is that nothing comes off and then all of a sudden a big peak of activity comes off. And so the bacteria make a lot of light in response to the stuff that’s in these tubes. So, sure enough, we could show that all of the cholera autoinducer-1 activity was in this one peak. So we pooled those test tubes full of stuff and there was a lot of activity in it. And then we could just take that and do techniques like mass spectrometry, NMR, ORD, CD, just different sort of techniques that would tell us what the molecule is. And sure enough, this peak had one molecule in it and it was very clean. And so, just from this experiment we could purify the cholera autoinducer-1 activity and identify the molecule. And this is the molecule. It has a funny name, 3-(S)-hydroxy-4-tridecanone which is why we call it cholera autoinducer-1 because that’s simpler. And what you can see, I hope, is that it is a molecule that has 13 carbons in it and only two functional groups. And so the way that this molecule is made by the CqsA enzyme is that it takes a C-10 moiety from fatty acid biosynthesis and connects it to a C-3 moiety to make this C-13 molecule. The only stereochemistry in the molecule is right here at this carbon. And what we did after we purified the molecule was we synthesized this molecule both in the S form, which is shown here and also in the R form. And then using chiral chromatography we showed we could separate those two molecules. Then we took the real thing and what we saw was that, indeed, cholera only makes this S moiety. So this is the only molecule that cholera makes that is cholera autoinducer. OK, so now we had it and we wanted to see if we could start messing around with quorum sensing by having this molecule and being able to synthetically prepare it. So what we did was to test the specificity of the response. And so again, you’re looking at bioluminescence and now we’re using synthetic molecules. So this top one, the C13S, this is the real molecule except that now we’ve made it. So we purified it from cholera, identified it, and then we made it using chemistry. We also, I told you, made the R isomer. And then, going down, we’re simply chopping off one carbon or another. So we have a C12 molecule, a C11 molecule. And we made many more, this is just a sample of the kinds of molecules that we tested. And so what you can see if you look at the activity of the molecule is that the C13S molecule is the most active. The R molecule is slightly less active. And then if you start chopping off carbons the molecules get less and less active. And so, indeed, nature has selected for the most active of the molecules. The C13S molecule, which is the molecule that cholera makes for its autoinducer, is the most active in our activity assays. OK, so that shows you that, indeed, we can find out what cholera autoinducer-1 is. And what I should say is that even though that molecule looks very simple, it’s a brand new molecule to biology. So that molecule has never been seen before and apparently it’s special. It’s just this cholera autoinducer, but cholera, I guess, is the one with CqsA that invented making this particular molecule as a signaling molecule. So now we have it. We know we can make it. We know that it can turn on this engineered luciferase reporter that’s responding to quorum sensing. But the real test and the real goal of this set of experiments was to ask: Can we add this molecule and turn off virulence as a new therapeutic? And so, of course, we wanted to go on to do that and so what we decided to do in our first experiment is to measure production of this pilus that I told you about, which is called TcpA. And so that’s the pilus that lets cholera attach to your intestine and then it delivers the toxin once it’s infected you. And so there’s a Western blot assay for that. Right, so we can do a Western blot for TcpA. And that’s shown on this slide. And so if we just look at wild type cholera, and these black lines, of course, are the TcpA production. And so what we have… and these sort of have fancy names but it doesn’t really matter is that we have mutants that are locked at low cell density. And so you’ll remember, at low cell density, cholera turns on virulence. And so, indeed, in a mutant that’s locked at low cell density you see a lot of TcpA, this virulence factor. We also have a mutant that’s locked at high cell density. And so what you can see is if the mutants are locked at high cell density cholera never turns on virulence factors. So it doesn’t turn on TcpA. But now if we just take the wild type cell and we add increasing amount of our synthetic autoinducer CAI-1 what you can see is as we add more of the synthetic molecule, this virulence factor production turns off. So that was hopeful. And then what we also notice is that if we did this in a mutant that was a CqsA mutant, and so you’ll recall, that’s the enzyme that makes CAI-1, the autoinducer. So if it’s not making its own autoinducer, we get a much more dramatic affect because of course, we’re not fighting against the endogenously produced cholera autoinducer-1. So, indeed, our synthetic molecule can turn down TcpA production. And to show that the molecule is actually working the way we think it should, we tested the exact same experiment but we did it on a mutant that was mutant in CqsS, which you’ll recall is the detector for CAI-1. So what we did was we deleted that detector and now what you see is if we add the cholera autoinducer, nothing happens. And that makes sense. If the bacteria don’t have the detector to transduce that information in, they don’t respond. So, sure enough, CAI-1 can turn down virulence, in vitro and CqsS, the receptor, is required for that. So it’s working exactly the way we think it ought to. And so that’s an in vitro test for whether or not the cholera autoinducer can be used as a therapeutic. But of course the real test is not whether we can turn off TcpA, the pilus, in a test tube of bacteria but can we make bacteria not be infectious? So the next experiment we did was to use a mouse model. So there’s a very well established mouse model for cholera infection. And so what we know is that if we infect wild type Vibrio cholerae into this mouse, the mouse dies. And that’s been used for many years in the cholera field to think about infection. And so the question is: If we infect cholera…excuse me, infect the mouse with wild type cholera but we add cholera autoinducer-1, which we now know is this molecule, can we, in fact, get the mouse to live? And the answer is yes. So, indeed, if you give both these things together, it keeps the mouse alive. You’ll also recall from my slides that there’s another autoinducer involved, autoinducer-2. And it turns out, if we add both CAI-1 and autoinducer-2 together that works even better. And so together, those two autoinducers fully turn off the cholera virulence cascade and they look promising for making a new therapeutic for treating cholera in countries in which it’s endemic. And I should say that, you know, these molecules even though they work, they’re not perfect in terms of what one would like when one thinks about the properties that molecules that are used as drugs have. And so what we’ve begun to do is to make a series of molecules that are related to the real molecule. And so this is just showing you a few of the molecules where we’ve attached different groups on them to see if we can get a molecule that acts even better than the real CAI-1. And so all of these now are being tested in vitro and in vivo to see if we can get a good combination of an autoinducer-2-like molecule and a CAI-1-like molecule to control pathogenesis in Vibrio cholerae which infects a million people a year. And so that’s the state of affairs right now. I hope from my two seminars what you’ve learned is that bacteria talk with a very complicated chemical lexicon. They all, we think now, have at least two molecules. One that says me, one that says other. So, an autoinducer-1 and autoinducer-2. And they use that information to control group activities and act like big multicellular organisms. And in the case of many pathogens, including cholera, what they do is they use those molecules to infect human and animal and plant hosts. And so the goal of the field is to move toward being able to disrupt quorum sensing by making agonists or antagonists of these molecules And the first idea that this could actually work, I’ve show you in this last, short seminar about how we’ve used the cholera autoinducer to shut down virulence in an animal host. And so that’s the state of affairs and of course we’re working on these and other topics right now. And I thought what I would finish my two seminars by doing is to show you my lab because I’m very proud of these people. All of these people are undergraduates, graduate students and post-docs from Princeton. And so I need to make the confession that… So here they are, my gang. and that of course they did all of the work that I showed you today. I didn’t do very much of that at all. I get to give the talks but they did all the pipetting and crystallography and molecular analysis and mutant analysis that you’ve seen. And it’s really wonderful gang of people all between their twenties and thirties years old and they’re just the engine that drives this kind of science. And I’m lucky, here I am over here, I’m really lucky to get to work with them because they’re incredibly creative and have, essentially, figured out all of this idea that bacteria can talk to each other. So again, thanks for listening to me. And I’m Bonnie Bassler from Princeton University and the Howard Hughes Medical Institute.