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