microbes and behaviour – Cheap Research Essays

microbes and behaviour
M2D2: Microbes and Behavior

In this activity you have the opportunity to apply your understanding of microbial growth control. Your main post must be at least 150 words. It should include citations and references to support your conclusions. You must also respond to at least two of your classmates’ main posts in a substantial manner, and answer all questions from the instructor.
Compose responses to the following questions incorporating the knowledge you’ve gained from your readings and then post to the discussion. Please respond thoughtfully and in your own words, being sure to back up each statement with cited sources. Your response should be at least 150 words.
Using the information you learned from the articles you reviewed this week by Birch, Willyard, and Sifri, answer the follow questions:
• What do you think are the most important changes in chemotherapeutic management of microbial disease?
• How are these related to new discoveries in intracellular bacterial communication and cooperative behaviors?
• How can biofilm formation impact treatment of infectious disease?

Bugging your bugs.
Authors:
Birch, Hayley
Source:
New Scientist. 3/6/2010, Vol. 205 Issue 2750, p36-39. 4p.
Document Type:
Article
:
*BACTERIOLOGY
*QUORUM sensing (Microbiology)
*CELL interaction (Biology)
*HOST-bacteria relationships
*HUMAN microbiota
t:
The article discusses a process of communication in bacteria called quorum sensing which is the use of chemical signals that release and receive signaling molecules in order keep track of neighboring cells. Topics include an example of how bacteria use quorum sensing to thwart a host’s white blood cells (WBCs) from destroying them, the competition between various bacteria species to colonize human hosts, and ways in which the body manages microbes.
FN

Bugging your bugs
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Section:
Features
Our bodies are chock-full of chatty microbes that are also eavesdropping on us. Time to turn the tables, says Hayley Birch
DEEP in your lungs, there’s a battle raging. It’s a warm, moist environment where the ever-opportunistic bacterium Pseudomonas aeruginosa has taken up residence. If your lungs are healthy, chances are the invader will be quickly dispatched. But in the mucus-clogged lungs of people with cystic fibrosis, the bacterium finds an ideal habitat. First, the microbes quietly multiply and then they suddenly switch their behaviour. A host of biochemical changes sticks the population of cells together, forming a gluey biofilm that even a potent cocktail of antibiotics struggles to shift.
Microbes like P. aeruginosa were once thought of as disorganised renegades, each cell working alone. Microbiologists like Thomas Bjarnsholt, who is battling to understand how P. aeruginosa causes chronic infection in people with cystic fibrosis, now know otherwise. They are up against a highly organised army, using a sophisticated communication system to coordinate its behaviour.
But it’s Bjarnsholt’s latest discovery that reveals microbes’ gift for language: the bacteria aren’t just talking amongst themselves, but also quietly listening in on signals sent by their human host. So when a cavalry of white blood cells arrives to repel the invading bacteria, the entrenched biofilm senses their presence, and launches a coordinated counterattack (Microbiology, vol 155, p 3500). The microbes release deadly compounds called rhamnolipids, which burst the white blood cells, killing them before they can even take aim, says Bjarnsholt, who is at the University of Copenhagen in Denmark.
Examples like this belie the notion that bacteria are simple, silent loners. Over recent decades, many species of bacteria have been shown to be in constant communication with each other. But now an even more sophisticated picture is emerging, one in which bacteria not only receive signals from each other, but also intercept them from the cells of their plant or animal hosts, including us.
These communication skills seem to offer invading bacteria quite an advantage on the battlefield. But they are also drawing the attention of researchers looking for new ways to fight microbes. If these “cross-kingdom” signals are indeed widespread, then intercepting or subverting them would offer a whole new way of tackling infection, not only in cystic fibrosis, but also in a wide range of other diseases. Such an approach would simply block the signals prompting the bacterial army to mobilise, rather than trying to wipe it out as antibiotics do. Bacteria would then no longer be forced to evolve drug resistance to survive, potentially bringing to an end the scourge of the superbug.
Bacteria communicate using chemical signals, releasing and receiving signalling molecules in a process known as quorum sensing. In its simplest form, bacteria use quorum sensing to keep track of their neighbours. Some bioluminescent bacteria, for example, light up when their population exceeds a threshold size (Journal of Bacteriology, vol 104, p 313).
Studies of the phenomenon in 1970 discovered that bioluminescent bacteria were using molecules called N -acyl homoserine lactones (ALHs) to coordinate this behaviour – an early hint that bacteria are a talkative bunch. But it wasn’t until the early 1990s, with the discovery that ALHs are produced by many species of microbe, that it started becoming clear that quorum sensing was common throughout the bacterial kingdom. And this signalling isn’t all friendly chatter, some bacteria intercept and break down the signals from other species, or even release signals to trick others into changing their behaviour.
But in a research review published in August 2009, Steve Atkinson and Paul Williams, microbiologists at the University of Nottingham in the UK, brought home just how widespread these signalling networks are: they reach far beyond the humble bacteria into other kingdoms, including plants, fungi, and our own (Journal of the Royal Society Interface, vol 6, p 959). As Atkinson puts it, “There’s a war going on out there.”
Take Candida albicans, the yeast that causes thrush infections. This organism likes the same warm, moist habitats as P. aeruginosa and the two battle it out in a bid to colonise their human hosts, deploying quorum-sensing signals as weapons against each other. The yeast fires off signals that trick the bacterium into slashing production of one of its armaments – a reactive chemical called pyocyanin, which makes life particularly uncomfortable for the yeast. The bacterium, meanwhile, produces signals that keep the yeast’s growth in check, preventing it from transforming itself from a single-celled yeast into a branching, multicellular fungus.
Then there’s our own immune system’s battle to prevent P. aeruginosa making itself at home in our lungs. Bjarnsholt is hunting for the signal P. aeruginosa uses to “listen out” for white blood cells, and ways to block it. He doesn’t think of the bacteria as being physically aware of their hosts. To them, the signals they detect are just foreign compounds they have to fend off. But it’s certainly a far more sophisticated take on the host-pathogen relationship than we’re used to, notes Atkinson. “Rather than the pathogen just piling into the host cell and taking over its DNA, it’s about signal production, interception – and maybe even coercion of the host to do something that it wouldn’t normally do.”
Microbe management
This coercion might even extend to including bacteria that can modify the way our bodies work, says Vanessa Sperandio, a microbiologist working on quorum sensing at the University of Texas Southwestern Medical Center in Dallas. “It’s a little out there,” she admits, “but there are some good examples. Kids who have certain bacterial infections can be very compulsive about touching their mouths, which helps the spread. I think we’re going to start seeing lots of examples like that.”
Many of the early examples of cross-kingdom communication that Atkinson and Williams catalogued are less than congenial, but there is also good evidence for cooperative interaction between bacteria and their hosts, says Atkinson – particularly between ourselves and our microbiome, the huge population of bacteria that live in us and on us.
These days we’re all well acquainted with the millions of microbes lining our insides. Yogurt adverts have taught us nothing if not to love the friendly bacteria which line our guts, helping to keep nastier bugs at bay. Microbes don’t just make themselves at home in the intestines, however. They’re in your mouth, up your nose, and covering your skin, all the while releasing a cacophony of quorum-sensing signals.
Atkinson thinks our own cells exploit this same signalling system to monitor and cajole our personal population of microbes, just as they eavesdrop on and manipulate us. In other words, we don’t passively host this bacterial colony, but actively engage it in conversation. We’ve evolved together, he says. “We have to consider that we’re intrinsically linked.”
Sperandio, who is studying how bacteria sense and respond to human stress hormones like adrenalin, agrees. “I think that if you consider how much we interact with microbes, it’s not surprising that you’re going to have some chemical signalling. Just consider in your intestine, you have 10 times more bacterial cells than you have your own.”
Picking out these chemical signals from the maelstrom of molecules that swirl in our gut is proving to be a battle, but that’s exactly where some of these cooperative signals have been spotted. Take those friendly gut bacteria, for example, and in particular one that goes by the name of Bacillus subtilis. Not a natural gut bacterium, B. subtilis has long been used as a probiotic agent in food. Though its health-boosting properties were not well understood, some have suggested it gently stimulates the immune system, priming it for action against less friendly bugs.
Then in 2007 a team led by Eugene Chang at the University of Chicago suggested a route by which these bacteria could influence the health of intestinal cells – a route involving quorum-sensing molecules. The team discovered that a certain B. subtilis signalling molecule, known as competence and sporulation factor (CSF), is detected by human gut cells (Cell Host & Microbe, vol 1, p 299). Chang thinks of this signal detection as a kind of “bacteriostat” mechanism: our cells are monitoring CSF as a way of detecting and adapting to important changes in the gut flora.
Cracking the code
“The idea is that when quorum sensing molecules are secreted, it usually signals some change in the balance of the bacterial population,” says Chang. So by listening for signals, our cells can adjust to these changes. In this case, the detection of CSF causes our cells to fire up the production of molecules called heat shock proteins, protective molecules known to help cells maintain crucial machinery during times of stress – from temperature extremes to toxins.
Perhaps the most intriguing evidence for the importance of monitoring our microbiome comes from the gut’s CSF receptor itself. This receptor was previously thought to be a simple nutrient transporter, despite being found in even the furthermost reaches of the intestine, where most nutrients would already have been absorbed. Its blueprint is encoded in a region of the human genome in which mutations are associated with inflammatory bowel disease. This suggests that without these receptors, we’re unable to maintain a normal, healthy gut.
Such examples suggest cross-kingdom signalling has medical implications far beyond infectious diseases. Several other illnesses, including Crohn’s disease and some cancers, have been linked to imbalances in the species of bacteria that live in our guts. Sperandio suggests that any number of illnesses could be associated with your balance of bacteria, and that these illness might be tackled using signal interference.
But with so many bacteria – hundreds of different species can inhabit your skin alone – how can we begin to master this chemical language to examine its medical potential? Is there a better way to spot these signals than to pick them out one by one? Pieter Dorrestein and his team at the University of California, San Diego, and Paul Straight at Texas A&M University in College Station, have been developing a tool that could accelerate efforts to crack the code of microbial communication.
The team is using an imaging system based on mass spectrometry to detect swathes of signals at the same time. They grow their bacteria on a stainless steel plate, and use a laser to vaporise their signalling molecules, feeding these into a mass spectrometer to catalogue the molecules present.
As proof of principle, Dorrestein and Straight have mapped the interactions between two species of soil-dwelling bacteria (Nature Chemical Biology, vol 5, p 885). Even in this simple case, the instrument detected as many as 100 different signalling molecules fired off by the two bacteria, only 10 of which the team managed to match to known molecules. Despite the huge scale of the problem, the team is already starting to translate their work into inter-kingdom studies, probing the interactions between bacteria and cells of the human immune system. By imaging cross-talk between different species, they even hope to identify inhibitors for Staphylococcus aureu s, the hospital superbug that has evolved to defend itself against whole groups of our most effective antibiotics.
The method should provide food for thought for Bjarnsholt, who has yet to find any serious candidate compounds for signal-blocking in P. aeruginosa infections in people with cystic fibrosis. His best bet for a drug lead is an extract of garlic, although the active component that interferes with the signal remains unknown. He thinks it will be a few years yet before quorum sensing inhibitors come into their own. “I don’t think it’s just around the corner – there’s got to be a lot more research,” he says. But when it comes to fighting drug resistance, themore targets we go after the better, he adds. We need to target signalling, biofilm formation and classical biological processes like bacteria cell wall formation, all at once.
Whatever the potential for medical advancement, the growing recognition of cross-kingdom signalling has a more immediate philosophical implication: we’re going to have to start changing the way we think about microbes. Bacteria aren’t just isolated cells, or even isolated populations, but multi-species communities that communicate with each other and, crucially, us. We are, almost certainly, more intimately connected with the bacteria that inhabit us than we ever would have believed. “We’d be naive to believe that we exist in splendid isolation from all other organisms,” says Atkinson. “We’ve thought that way for too long.”
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By Hayley Birch
Hayley Birch is a freelance science writer based in Bristol, UK
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CiteSilent Treatment.
Authors:
WILLYARD, CASSANDRA
Source:
Discover. Jun2014, Vol. 35 Issue 5, p30-33. 4p.
Document Type:
Interview
Subject Terms:
*BACTERIA — Behavior
*QUORUM sensing (Microbiology)
*RESEARCH
*INFECTION — Treatment
*WOMEN molecular biologists
People:
BASSLER, Bonnie — Interviews

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The article presents an interview with molecular biologist Bonnie Bassler about her discovery that by disrupting the process in which bacteria coordinate their actions, also known as quorum sensing, doctors can stop infection. Topics include how bacteria communicate through a chemical language, the work of biologists Woody Hastings and geneticist Mike Silverman, and how many molecules are involved in quorum sensing.Full Text Word Count:Accession Number
Silent Treatment
Contents
1. The Princeton biologist aims to stop infections by interrupting the chatter of bacterial communities
2. * What got you interested in bacteria?
3. Q How did you get interested in bacterial communication?
4. Q Why was this so mind-blowing? Nobody understood that bacteria could work as a team back then?
5. Q What had Silverman and Hastings learned by that point, and where did you come in?
6. Q What was the second one doing?
7. Q 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?
8. Q Did other scientists buy your explanation?
9. Q ln 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?
10. Q 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?
11. Q Why would they need to know that? How does that help them?
12. Q Do you think the language of bacteria is more complex than we realize?
13. Q 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?
14. Q Have you discovered any promising candidates?
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Discover Interview
BONNIE BASSLER
The Princeton biologist aims to stop infections by interrupting the chatter of bacterial communities
* 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.
* What got you interested in bacteria?
A 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.
Q How did you get interested in bacterial communication?
A 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.
Q Why was this so mind-blowing? Nobody understood that bacteria could work as a team back then?
A 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.
Q What had Silverman and Hastings learned by that point, and where did you come in?
A 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.
Q What was the second one doing?
A 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.
Q 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?
A When 1 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.
Q Did other scientists buy your explanation?
A 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.
Q ln 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?
A 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.
Q 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?
A 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.
Q Why would they need to know that? How does that help them?
A Having that information is extremely useful for decisionmaking. 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.
Q Do you think the language of bacteria is more complex than we realize?
A 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.
Q 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?
A 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.
Q Have you discovered any promising candidates?
A 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.
It was in Vibrio fischeri that researchers learned that bacteria could count their neighbors.
Bacteria release a molecule that allows them to recognize each other. When the population reaches a critical mass, these molecules enable the bacteria to sense that, and turn on or off target genes.
Colonies of the bioluminescent bacterium Vibrio harveyi glow in the dark in a petri dish.
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.
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By CASSANDRA WILLYARD
Cassandra Willyard is a freelance writer in Madison, Wis.
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© 2014 Discover Magazine
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• Oxford Journals
• Medicine & Health
• Clinical Infectious Diseases
• Volume 47, Issue 8
• Pp. 1070-1076.
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Quorum Sensing: Bacteria Talk Sense
1. Costi D. Sifri
+Author Affiliations
1. Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville
1. Reprints or correspondence: Dr. Costi D. Sifri, Div. of Infectious Diseases and International Health, University of Virginia Health System, P.O. Box 801361, Charlottesville, VA 22908-1361 (csifri@virginia.edu).

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Abstract
Cell communication in bacteria occurs through a vernacular of small diffusible chemical signals that impact gene regulation during times of high cell density. This form of intercellular signaling, known as quorum sensing, optimizes the metabolic and behavioral activities of a community of bacteria for life in close quarters. Quorum sensing is best characterized as a means of communication within a bacterial species, whereas competitive or cooperative signaling can occur between groups of bacteria or between bacteria and the host. These systems are often integrated into complex, multilayered signal transduction networks that control numerous multicellular behaviors, including biofilm formation and other virulence traits. In addition, quorum signals, sensors, and signaling pathways are increasingly recognized as having biological properties that extend beyond cell communication. The deeper understanding of microbial cell communication promises to shed light on the complexities of the host-microbe relationship and may lead to novel therapeutic applications.
Cell communication and signaling are essential for the proper growth and development of all living multicellular organisms. Because of its universal importance, it is not surprising that many fundamental aspects of cell communication have been evolutionarily conserved between plants, animals, and unicellular eukaryotes, even though these kingdoms diverged more than 1 billion years ago [1, 2]. However, the ability to coordinate cellular behavior was once thought to be restricted to eukaryotic organisms, and bacterial perception of neighboring bacteria was thought to only occur indirectly (e.g., through sensing changes in nutrient availability). Now, work over the past 2 decades has firmly established that sophisticated communication systems are also used by bacteria to coordinate various biological activities, including the production of virulence factors and growth in biofilm communities [3]. Cell communication in bacteria and in some eukaryotic microorganisms occurs in a population-density dependent manner and is based on the production of and response to small pheromone-like biochemical molecules called autoinducers. Differential gene regulation in response to intercellular signaling provides microbes with a means to express particular behaviors only while growing in social communities. This process has been termed quorum sensing to reflect the need for a sufficient population of microbes (and concentration of signal) to activate the system [4].
Quorum-sensing systems have been shown to be key virulence regulators in both gram-negative and gram-positive pathogens. In addition, quorum sensing in Candida albicans has been found to influence filamentation [5,6], which is intimately associated with virulence [7]. Interestingly, quorum sensing-regulated genes encode not only classic virulence factors but also other proteins, including those that are involved in basic metabolic processes. Indeed, a significant portion of a bacterial genome (4%–10%) and proteome (20% or more) can be influenced by quorum signaling [8–11], which suggests that quorum sensing is a mechanism used by pathogenic bacteria not only to modulate virulence factor production but also to adapt to the metabolic demands of living in communities.
It has been suggested that quorum sensing represents a novel target for the development of agents to treat or prevent bacterial infections. Theoretically, development of resistance to these antipathogenic compounds would be minimized, because they would target virulence mechanisms and not growth [12]. Accordingly, significant efforts in both academia and industry have been committed to the development of compounds that inhibit or inactivate cell density-dependent signaling. Recent studies, however, have demonstrated that quorum sensing is significantly more complex than first appreciated. Impeding these signaling pathways may have unexpected and unintended consequences in vivo. In some cases, bacteria with disabled cell communication systems may be particularly well-suited for certain types of infections. In addition, it is becoming increasingly clear that interspecies communication among pathogens and between pathogen and host adds another layer of complexity that likely impacts clinical disease. In this article, I will review the general mechanisms of quorum sensing in bacteria and discuss recent findings that underscore the evolving complexities of these systems.
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Quorum Sensing: An Overview
Quorum sensing can be divided into at least 4 steps: (1) production of small biochemical signal molecules by the bacterial cell; (2) release of the signal molecules, either actively or passively, into the surrounding environment; and (3) recognition of the signal molecules by specific receptors once they exceed a threshold concentration, leading to (4) changes in gene regulation. One common consequence of quorum sensing induction of gene expression is increased synthesis of the proteins involved in signal molecule production. Increased synthesis of the signal molecule creates a positive feedback loop, which is why quorum signals are commonly called autoinducers.
One limitation to this model of microbial intercellular communication is that environmental conditions for autoinducer diffusion are not taken into account. Recognizing this, Redfield [13] has proposed a less anthropomorphic explanation as to why bacteria monitor and respond to changes in the concentration of self-generated signal molecules. Autoinducer concentration, rather than sensing population density, is used by bacteria to gauge the rate of diffusion of extracellular molecules. Presumably, reduced diffusion (as assessed by increased autoinducer concentration) is a marker of favorable conditions for the production of specific bacterial products. Although there is some experimental evidence in support of this hypothesis [14], it remains to be seen whether diffusion sensing will be a generalizable model of small molecule-based signaling in bacteria.
At least a half-dozen types of quorum-sensing systems have been described in pathogenic bacteria, and more are almost certain to exist. A brief mechanistic review of quorum sensing, with a focus on Pseudomonas aeruginosa and Staphylococcus aureus as prototypical pathogens, is presented here.
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Gram-Negative Bacteria
N>-acylhomoserine lactone (AHL) signaling. The lux-type quorum-sensing system is the archetypal and preeminent mechanism for species-specific communication in gram-negative bacteria [15]. First identified in marine Vibrio species, lux-type quorum sensing is based on the production of and responses to AHLs. In general, lux-type systems consist of 2 components, an autoinducer synthase (e.g., LuxI), which synthesizes AHLs from S-adenyosyl methionine, and a transcriptional regulator (e.g., LuxR). Because of its small size and lipophilic character, AHL freely diffuses across cell membranes. As the population density increases, intracellular AHL binds the functionally linked (cognate) LuxR-like receptor at a sufficient concentration within the cytoplasm to induce differential gene expression.
P. aeruginosa virulence traits are controlled by 2 distinct lux-type quorum-sensing systems, termed las and rhl, which are named after their influence on elastase and rhamnolipid production, respectively. The lassystem regulates the rhl system as part of a cascade of virulence regulators (figure 1). This hierarchical circuitry controls multiple virulence traits beyond elastase and rhamnolipid, including exoprotease and toxin production, motility, and biofilm formation [16]. Genetically engineered lasand rhl system mutants have been shown to have significantly reduced virulence in several animal models of infection [17–19], and las and rhlAHLs (N-3-oxododecanoyl homoserine lactone [OdDHL] and N-butanoyl homoserine lactone, respectively) have been detected in sputum samples obtained from individuals with cystic fibrosis who were infected with P. aeruginosa [20, 21]. These studies support a central role for AHL quorum sensing in P. aeruginosa disease.

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Figure 1
The hierarchicalPseudomonas aeruginosaquorum-sensing systems. P. aeruginosa contains 2 LuxR/LuxI-type quorum-sensing systems, termed thelas and rhl. LasI produces the acyl-homoserine lactone autoinducer N-3-oxododecanoyl homoserine lactone (OdDHL; squares), whereas RhlI produces N-butanoyl homoserine lactone (BHL; diamonds). OdDHL and BHL bind to their cognate receptors, LasR and RhlR, respectively. Activated LasR or RhlR induce expression of a complement of genes, including their own loci (completing the autoinducing circuits). Activated LasR also enhances expression of the rhllocus, as well as mvfR, leading to increased production of 4-hydroxy-2-heptylquinoline (HHQ; triangles) and Pseudomonasquinolone signal (PQS; inverted triangles). Thus, the las system is placed squarely above the rhl and mvfR system in a hierarchical cascade of virulence regulators. Trafficking of hydrophobic PQS to neighboring cells occurs through small, extracellular membrane vesicles. HAQ, 4-hydroxy-2-alkylquinoline.
Interestingly, AHLs may have biological functions that extend beyond quorum signaling. For example, OdDHL and other AHLs spontaneously degrade to tetrameric acid derivatives in water [22]. These tetrameric acids and OdDHL itself have bactericidal activity toward gram-positive organisms, including S. aureus, but not toward gram-negative bacteria. Therefore, AHLs may serve to enhance the competitiveness of P. aeruginosa in a consortium of mixed bacteria.
>Quinolone signaling. In addition to the las and rhl systems, a third non-AHL quorum-sensing system has been identified in P. aeruginosa. Cell signaling in this system occurs through members of a family of quinolone compounds termed 4-hydroxy-2-alkylquinolines (HAQs) (figure 1) [23]. Synthesis of HAQs is controlled by the transcriptional regulator MvfR, which modulates expression of several genes involved in the production of anthranilic acid and its conversion to 4-hydroxy-2-heptylquinoline (HHQ) [24, 25]. HHQ is then converted to 3,4-dihydroxy-2-heptylquinoline, also known as Pseudomonas quinolone signal (PQS), through the action of PqsH [25]. LasR controls production of MvfR and PqsH, thereby intertwining the mvfR signaling pathway with AHL quorum sensing [25].
Transcriptional analysis has shown that MvfR regulates expression of a set of quorum sensing-regulated genes that partially overlap with but are distinct from those modulated by AHL autoinducers [26]. Interestingly, both PQS and HHQ have dual roles as MvfR ligands and as intercellular signaling molecules for the mvfR regulon, although there are differences in their biological properties [27, 28]. PQS is markedly hydrophobic. To be trafficked between cells, PQS is packaged into small membrane vesicles that pinch off from the P. aeruginosa outer membrane and disperse through the bacterial population in a manner analogous to vesicular trafficking in eukaryotic cells (figure 1) [29]. Although PQS and HHQ have no known intrinsic antibiotic activity, other HAQs do. Like tetrameric acids, this antibacterial activity may enhance P. aeruginosa fitness in a mixed population of bacteria [25].
Finally, it should be understood that the las, rhl, and mvfR quorum-sensing systems are components of a large network of regulators that control a wide assortment of cellular functions. Why does P. aeruginosapossess such a complex regulatory network? One possibility is that it may facilitate a temporal progression in virulence factor production during infection that is optimized for P. aeruginosa pathogenesis [8]. Another possibility is that different combinations of chemical signals allow P. aeruginosa the flexibility to fine-tune its virulence programs and other social behaviors to adapt to many different environments. This theory of modulated chemical communication using multiple small-molecule messengers is neither new nor unique to bacteria. Over 40 years ago, naturalist Edward O. Wilson noted that changes in the pheromone concentrations and blends expand the repertoire of behavioral responses observed in social insects [30].
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Gram-Positive Bacteria
Intercellular communication is also used by gram-positive pathogens to control virulence. Rather than using AHL or quinolone-based signaling molecules, cell communication in gram-positive bacteria is based on the production and detection of modified oligopeptides called autoinducing peptides (AIPs). The best-studied system is the quorum-sensing system ofS. aureus, which is encoded by the accessory gene regulator (agr) locus. Analogous to the function that quorum sensing plays in P. aeruginosavirulence, the agr system is central to a complex regulatory network that controls the production of a broad array of S. aureus virulence factors [31]. Moreover, agr also has a complex relationship with biofilm formation, which has significant clinical implications.
The agr locus is composed of 2 divergent transcripts, RNAII (encodingagrB, agrD, agrC, and agrA) and RNAIII, which act to suppress cell wall-associated protein production and enhance secreted exoprotein production in response to high cell density (figure 2). Cell wall-associated adhesins facilitate S. aureus colonization, whereas secreted products are necessarily for invasion and dissemination. The 4 genes encoded by RNAII are involved in the production and sensing of the AIP. agrD encodes the precursor of the autoinducing signal peptide, whereas the integral membrane protein AgrB is involved in its processing and secretion as a thiolactone-modified cyclic oligopeptide. AgrA and AgrC constitute a two-component histidine kinase-response regulator pair that respond to the extracellular accumulation of the AIP. Activation of AgrA-AgrC induces transcription of RNAII (completing the autoinducing circuit) and RNAIII [31]. Remarkably, instead of encoding a regulatory protein, the 0.5 kb RNAIII transcript itself acts as the regulatory effector molecule for the agrsystem, largely through translational inhibition of the virulence gene repressor Rot (i.e., repressor of toxins) and perhaps other gene regulators [32, 33]. In addition, RNAIII itself encodes δ-hemolysin. Genetically engineered agr mutants have been show to be attenuated in many animal models of S. aureus disease [34].