Battling Infections By Silencing Bacterial Chatter

Princeton biologist Bonnie Bassler studies bacterial "quorum sensing" — the chemical communication that makes pathogens so dangerous.

By Cassandra Willyard|Wednesday, May 21, 2014
bonnie-bassler
bonnie-bassler
Zach Donnell Photography

Bacteria may be primitive, but they’re far from simple. They lead complex lives and communicate through a chemical language that allows them to coordinate their actions, a process known as quorum sensing. Using this chemical communication system, bacteria can sense when they have reached a critical number, or quorum. Then they act en masse. Some might begin producing light, others a deadly toxin. This capability is part of what makes bacteria so terrifying: A single cell can’t manufacture enough toxins to make us sick, but when millions simultaneously churn out noxious molecules, the results can be lethal.

No one has done more to decipher this bacterial lingo than Bonnie Bassler, a molecular biologist at Princeton University. In the 1990s, Bassler discovered that bacteria can converse not only with members of their own species, but also with other species, allowing them to sense their environment more comprehensively and decide when to launch an attack. Today her lab is hunting for compounds that can block this bacterial chatter to shut down infections such as cholera. 

Bassler’s influential work, along with that of other pioneering researchers, has transformed how people view bacteria. But much about quorum sensing remains to be discovered; scientists are still trying to uncover the molecules and mechanisms involved, and figure out how they operate outside the laboratory. And that’s what keeps Bassler captivated. “It’s a 4-billion-year-old mystery, and it’s going to take more than 20 years’ work from lots of labs to figure it all out.” Discover caught up with Bassler on a balmy summer day in her office at Princeton to talk about the history of her discoveries and what they might mean for the future of medicine. 


bassler-lab
bassler-lab

Graduate student Yi Shao works in Bassler's lab. Half the team is figuring out quorum-sensing mechanisms; the other half aims to control the chatter.

Zach Donnell Photography

Discover: What got you interested in bacteria?

Bassler: I went to UC Davis because I wanted to be a vet. It’s a great profession if it’s right for you, but it’s memorizing the bones and the muscles, and I am terrible at stuff like that. Also, there’s a lot of blood and gore involved. We started to dissect these animals — god, ugh! I left the pig and walked outside and puked in the grass. I’m like, “OK, I hate this. I just hate this.” So I was basically lost.

Then my mother died of cancer in August of my junior year. I didn’t know if I wanted to be a doctor or a scientist, but I was going to do something about cancer. I went to see a biochemistry professor named Frederic Troy, who was working on a cancer project, and he agreed to let me work in his lab. But he put me on this bacterial project, and I’m like, “No way! I want to work on the important project.” But, of course, I had no skills. I couldn’t even pipette. So I thought, “Well, if I show him I’m really earnest and hardworking, then he’ll transfer me.”

I fell in love with these bacteria. I just love how sophisticated they are. They do everything that eukaryotic cells do: The way you metabolize glucose is the way a bacterium does. But they don’t have the slop of higher cells and higher organisms. They’re stripped-down versions. They’re a treasure trove of the most basic mysteries, and what we find in bacteria applies all the way up the food chain. 

How did you get interested in bacterial communication?

B: I went to graduate school at Johns Hopkins. Saul Roseman was my Ph.D. adviser. He worked on how bacteria sense their environment, and he had gotten this little grant from the Office of Naval Research for studying how bacteria stick to surfaces, like boats or submarines. In the late ’80s, the Navy put on this symposium for everybody who had one of these grants, and I begged Roseman to let me go. I wanted to learn what other people in this area of marine microbiology were doing. 

One of the speakers was Mike Silverman, a geneticist at the Agouron Institute in La Jolla, Calif. He talked about genetic work he was doing trying to figure out how bioluminescent, or “glow-in-the-dark,” bacteria turn on light when their cell counts reach a certain level. He was saying things like, “Don’t you see? This allows them to coordinate their behavior.” That went against everything anyone had ever learned. That idea that they were doing something as a collective was totally mind-blowing, at least to me. I literally ran up to the podium after his talk and begged him to let me be his postdoc.

Why was this so mind-blowing? Nobody understood that bacteria could work as a team back then?

B: Now we have hundreds and hundreds of examples of this. But at that time it had never occurred to me to think about bacteria acting in groups. Back then it was assumed by scientists, including most microbiologists, that bacteria were extraordinarily primitive, and that they didn’t have the capacity to do fancy behaviors. They consume nutrients from their environment, they grow twice as big, they cut themselves in the middle and they make clones. So one becomes two becomes four. And it was always thought that when that happens, bacteria have no knowledge of their sister cells.

In the ’70s, Woody Hastings at Harvard discovered that if you took a certain bioluminescent species of bacteria and you grew them, they made basically no light. And then when they hit a certain cell number, they would all turn on light together.

To figure out how this worked, he took bacterial cell populations of different sizes and spun each down in a centrifuge. Then he collected the liquid that the cells had been grown in to see whether there was some difference in the chemistry of the culture fluid. What he showed is if he took the fluids from cultures of cells that lit up and squirted them on cells that were at a low cell density, they all turned on light. You could trick the bacteria into thinking that they were at a high cell number. So it was clear there was something outside the cells, some molecule in the fluid. That was an amazing experiment. Hastings named the molecule an autoinducer: The bacteria “autoinduce” themselves to make this molecule and turn on light. But at the time many people considered that kind of coordinated behavior an anomaly of this sort of goofy glow-in-the-dark bacteria. Some people still doubted Hastings’ conclusions, and even Silverman, when I met him, assumed the phenomenon was limited to just a few weird bacteria.

vibrio-fischeri
vibrio-fischeri

It was in Vibrio fischeri that researchers learned that bacteria could count their neighbors.

Dennis Kunkel Microscopy

What had Silverman and Hastings learned by that point, and where did you come in?

B: Hastings and then Silverman worked on this bacterium called Vibrio fischeri, a bioluminescent marine bacterium that lives inside a squid. Hastings isolated the autoinducer, and then Silverman found some of the genes that control this system. They found that the autoinducer allows bacteria to count their neighbors. When a bacterium is alone, it dribbles out a couple of molecules and they just diffuse away. But if you have lots of bacteria together, the autoinducer will begin to build up. And when there’s enough, it turns on a whole host of genes, including those involved in bioluminescence. The first investigators called this process “density sensing,” and we now call it quorum sensing.

I worked on another bacterium, Vibrio harveyi. It’s bioluminescent, too, but it is a free-living bacterium, so it encounters all kinds of different environments. We knew it made a similar autoinducing molecule, so we decided to try and unravel its quorum-sensing machinery. I just assumed that it would work the same as it does in Vibrio fischeri. I did the same experiment a billion times — looked for mutants that didn’t make light, hoping I would have knocked out the quorum-sensing machinery. It should have been super simple. But the strategy of looking for dark mutants didn’t help me find the right genes. I had this crisis of confidence. You think there’s something wrong with you. And then, eventually, you say to yourself, “Perhaps I’m not thinking about this correctly.”

It occurred to me that maybe the reason I couldn’t get the correct genes is because there are two quorum-sensing molecules. It could be that when I knock out the genes for one, the other still works. That was an epiphany. If there were two molecules and you knocked one out, they’d still make some light. So I tweaked the experiment to look for mutants that were dim, not dark. My dim mutants were clearly lacking one molecule, but they still made something else. They were impaired, not broken. There had to be another molecule.

What was the second one doing?

B: I didn’t have a clue. Why do you need two? I knew there was a second molecule out there. The genetics told us that molecule existed. But I didn’t have that gene, and I didn’t know the chemical structure of the second molecule. We took a lot of flack for that. The idea that bacteria could talk with one molecule was only starting to percolate through the community. So the idea that there were multiple molecules involved in bacterial coordination was so out there. 

Your work showing that there had to be a second quorum-sensing molecule helped you get a job at Princeton, and in 1994 you started your own lab there. Did you figure out the purpose of the second molecule?

B: When I got here, we finally found the gene that made the enzyme that made the second molecule. But it wasn’t just about finding the gene. We wanted to understand why Vibrio harveyi has this second molecule. So we sequenced the DNA of that gene. Our real hope was that our gene would look like some other known gene and give us a clue about what it did. There were about 40 genomes of bacteria sequenced at that time, and what you could do was compare your gene of interest to other genomes to see if they contained something similar. The computer would scan through all the known bacterial genomes and say, “Do any of them have that gene?”

That process took a long time back then. You type the DNA sequence of your gene into a database, and then you sit and you wait. I remember the screen filling up one by one. The database told us every bacterium that had been sequenced — not just the bioluminescent bacteria, but every bacterium — has it; they all make an identical molecule. And I said, “They’re talking to each other. That’s how they talk across species.” That idea had not occurred to us. So that was an amazing moment. I still get goose pimples.

Did other scientists buy your explanation?

B: At first, the quorum scientists had trouble getting others to believe that bacteria could speak within their species. And then we came forward with this idea that they could talk across species — lots of people thought I was nutty. The dogma had been that bacteria can’t communicate, so it was hard to accept that they could talk using two molecules, and even more difficult to imagine they could talk across species. 

It takes a long time to change dogma. And there were still problems with the story. We had the gene, and the gene was in all these different bacteria, but we still did not have the identity of the molecule. So there was a chink in our armor.

In 2002, you finally identified the second molecule. And then you won the MacArthur “Genius” Fellowship, a grant awarded to individuals who show exceptional creativity. Did you feel vindicated?

B: That put me on the map. This prize is very coveted, and it also brands you as creative, not crazy. I’d been here at Princeton eight years, and I had never been able to adequately fund this lab. I was struggling to get money. I had been trying, trying, trying — writing five, six, seven grants a year. Most of my grants were getting rejected. And to get this thing that you didn’t try for — that was so important for my confidence. And it branded quorum sensing as the hottest, coolest, most creative science.

You’ve now found that there are actually three molecules involved in quorum sensing. The first, the one Hastings found in the ’70s, allows bacteria to count their siblings. The second allows them to detect other species. And the third, which is made by all bacteria in the Vibrio genus, allows bacteria to identify their “cousins,” or extended family, giving them even more information. How do bacteria use these molecules to communicate?

B: What they first do is they scan the environment. And they’re asking the simplest question: “Am I alone or am I in a group?” They just look for any quorum-sensing molecule. Then, the more sophisticated question that I think they ask is, “Who is that?” 

They can say, “You are my absolute identical twin.” They can say, “You’re my extended family.” And then they say, “You’re some other species.” They’re not just counting. There’s information encoded in these molecules that tells a bacterium who that neighbor is — how related they are. And depending on the ratio of those three molecules, they understand whether their family is winning or losing.

Why would they need to know that? How does that help them?

B: Having that information is extremely useful for decision-making. Bacteria aren’t just swimming around. They live adhered to surfaces. Your skin, your scalp, your intestines — they’re all covered in communities of bacteria, called biofilms. In order to make a biofilm, they have to secrete this substance that glues them all together, which acts like a shield. That’s controlled by quorum sensing. 

For these communities to be maximally productive, they can’t be willy-nilly. They have to use multiple molecules to discern who their neighbor is — self or other — and to direct what job each participant in the community will take on.

Do you think the language of bacteria is more complex than we realize?

B: In 20 years, my field has gone from thinking of bacteria as asocial recluses to seeing them as at least being trilingual. And there’s mounting evidence that this is going to be an inter-kingdom dialog. Humans and all higher organisms live in fantastic association with many species of bacteria. We speculate that the host makes molecules that tell the bacteria what to do, and the bacteria make molecules that the host is tuned into. It has got to be like that.

This field is only really 20 years old. And we just haven’t found all the molecules yet. In the lab, we shake the bacteria around in a flask, and each bacterium perceives an identical environment. It could be that there’s a whole set of molecules that they never deploy there. To find those, you have to put them in a much more realistic environment. 

glowing-bacteria
glowing-bacteria

Colonies of the bioluminescent bacterium Vibrio harveyi glow in the dark in a petri dish.

Zach Donnell Photography

Quorum sensing controls bacteria’s ability to cause deadly infections, allowing bacteria to secrete poisons, swarm and adhere to human tissue. Could you disrupt this communication to develop new ways to fight infections? 

B: Pretty early on, quorum researchers started to think that maybe we could manipulate quorum sensing on purpose. The way molecules and receptors work, it’s like keys fitting into locks. The molecules that are the real autoinducers turn quorum sensing on. But you could have another key that looks the same, but blocks the receptor. It binds, but doesn’t send the signal. You can screen for or synthesize molecules that act as anti-quorum-sensing molecules. We’ve worked on three or four pathogens. 

Have you discovered any promising candidates?

B: We can shut down quorum sensing in Pseudomonas, a notorious human pathogen, using a nematode, a worm model for infection. If you give our anti-quorum-sensing molecule to the worms, they live just fine. Pseudomonas also will kill human lung cells in tissue culture dishes. It’s usually not very dangerous, except when it infects people whose lungs are already compromised — it can kill a person with cystic fibrosis. We’ve also found that in a petri dish, the anti-quorum-sensing molecule prevents Pseudomonas from killing human lung cells and from making a biofilm that would enable it to mount an attack.

In lab experiments it works. But by the time you’re sick, quorum sensing has already happened. Can we really make a molecule that goes where you want it to go to stop an ongoing infection?

I don’t know yet.

When you’re doing this right, there are always more questions than answers. That’s the fun and the torture of it. Bacteria had 4 billion years to evolve this capability. Scientists have been working on it for 20. This field is young. We are still pioneers.

[This article originally appeared in print as "Silent Treatment."]

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