A month ago, we reported that the 2022 Wolf Prize in Chemistry was awarded to Bonnie Bassler, a researcher in the field of cell communication research. Professor Bassler has studied a unique language system for communication between microorganisms. How was this system discovered and how was it cracked? What is its practical significance for clinical pharmaceutical applications? Today's article shows us the fascinating charm of this basic research.
Written by | Wei Hong (Ph.D. in history of science and technology)
The well-known French social psychologist Le Pen said in "The Ragtag Crowd":
The sum of the common characteristics acquired by all individuals of a race as a result of their inheritance constitutes the soul of that race. But when a certain number of individuals gather in groups to carry out some kind of action, then, observations show that, as a result of their coming together, new psychological characteristics appear, overlapping with racial characteristics, and sometimes deeply different from racial characteristics.
The emergence of human group behavior is a topic that sociologists have been concerned about. However, group behavior is not unique to humans or higher animals.
In recent years, microbiologists have discovered that bacteria also have their own unique group behaviors. Through a unique system of chemical language, bacteria are able to communicate with each other, perceive each other, and collectively vote to make decisions. Specifically, the physiological and biochemical properties of bacteria vary with the density of the population, showing characteristics and group behaviors that are not available in a small number of bacteria or individual bacteria, in order to cope with changes in the environment. This phenomenon is what microbiologists like to talk about as "quorum sensing."
The bacteria are so small that they cannot be observed by the naked eye and do not make a sound. Without the help of modern technology, we cannot directly observe their individual behavior, let alone monitor their communication process and analyze their group behavior. To human beings, all their actions before they are discovered are like a conspiracy, and scientific experiments are the exploration behavior of researchers. Next, we will make a brief review of the discovery of quorum sensing and the process of cracking microbial language systems.
One
Luminous bacteria leaked secrets
In 1913, E. Harvey, a young teacher at Princeton University who had been hiking and collecting biological specimens since childhood, was an avid hiker. Newton Harvey traveled around the South Pacific with aquatic biologist Alfred G. Mayer for three months on Murray Island in Australia. It was during these three months that Harvey became fascinated by the phenomenon of bioluminescence.
Three years later, Harvey and his newlywed wife, who is engaged in aquatic biology research, went on a honeymoon trip to Japan. On the west coast of Japan, he was attracted by the blue light phantom on the surface of the sea. What fascinated him even more was that the light source sea firefly (Vargula hilgendorfii) was a marine invertebrate and a perfect research material. After years of drying and storage, its luminous system is rejuvenated as long as it is moistened by water. No one knows exactly how many dried seaflies Harvey transported back to the United States that time, but for the next four decades, he has been doing research with these materials.
E. Harvey Newton Harvey) 丨 Image source: Arizona State University, https://embryo.asu.edu/pages/e-newton-harvey
On the coast of Okayama, Japan, sea fireflies form a blue river 丨Picuish source: https://freeyork.org/photography/blue-rivers-sea-fireflies-trickle-oceanside-rocks-japan/
Harvey's research interests are extensive, and bioluminescence is just one of them. By chance, he discovered that microbes actually glow. In 1953, Bernard L. Strehler first found the chemical molecule DPN (Disphosphopyridine nucleotide, nucleotide diphosphate) from the luminescent bacteria (Achromobacter fischeri, also known as Vibrio fischeri, Freudii), and completely extracted the luminescence system from the bacteria, achieving extracellular luminescence. When Harvey discovered that Strler's mentor was his proud protégé, William D. McElroy, he proudly said, "Now, I have the feeling that I am the grandfather of bioluminescence research!" ”
Interestingly, among Harvey's many students, J. Hastings Woodland Hatstings) was both his disciple and his disciple's disciple. In 1948, Hastings studied with Harvey for his Ph.D., and in 1951 he followed McElroy on postdoctoral research. At Harvey's lab, Hastings developed a new technique to measure the quantitative requirements of oxygen in the luminescence response of different species. At McIlroy Lab, Hastings turned to luminescent bacteria after struggling for some time in the traditional field of firefly light-emitting systems.
J. Hastings Woodland Hatstings) 丨 Image source: Harvard University
At that time, it was generally believed that the behavior of bacteria was independent and independent of other individuals. According to this line of thinking, if the number of bacteria doubles, the brightness intensity should also double at the same time. But in 1970, Hastings noticed a peculiar phenomenon in Vibrio and Vibrio harveyi (named after Harvey in 1936). In the newly inoculated medium, the number of bacteria can be doubled every 30 minutes, but the luminescence takes more than 2 hours to begin to increase, and then the brightness doubles every 5 minutes.
At the same time, bacteria are constantly releasing a molecule called homoserine lactone (HSL) into the medium, and only when HSL reaches a certain concentration, the specific gene that is suppressed initiates the transcriptional program, and the bacteria can bloom with a charismatic glow. This is the first time scientists have discovered the phenomenon of quorum induction since the record.
Hastings refers to the signaling molecule HSL as an "autoinducer" (AI) because, unlike the applied inducer, HSL both induces the expression of genes and is produced by bacteria themselves.
For new things or new doctrines, the public always needs a slow process of acceptance, sometimes even full of resistance. Like many people in history who have made major discoveries, this time, Hastings is in a similar situation, and instead of accepting his discoveries, the academic community sneers at him.
Two
The language system is cracked
Michael R. Silverman of the Agouron Institute is an exception. He thought Hastings' discovery was extremely interesting, and in the 1980s he found the core molecular mechanism of quorum sensing. In Vibrio fischerii, Silverman found that the LuxI protein catalyzes the synthesis of AI molecules, and the LuxR protein as a receptor binds to AI, which in turn activates transcription of genes encoding luciferase. Next, he performed an E. coli transformation experiment, which was modified to produce the signaling molecule HSL and can also use it to sense the concentration of the bacteria. When the concentration of the bacteria reaches the threshold, E. coli begins to glow. One by one, he identified genes and proteins that could produce and detect extracellular signaling molecules, and explained to the academic community how these components stimulate crowd sensing.
Silverman rarely speaks publicly and has always existed in the scientific community like a "hermit". By chance, Bonnie Bassler, who was graduating with his Ph.D., listened to a lecture on how bacteria activate light-emitting systems through group induction. Basler felt it was so wonderful that germs could vote on when to turn on the lights! More importantly, she perceives the convenience of luminescent bacteria as a material for genetic research, and as long as she presses the light switch in the laboratory to see if the bacteria emit light, she can know the results of the experiment. As soon as the lecture was over, she went straight to the podium and told Silverman that she wanted to do postdoctoral work with him. Silverman was so enthralled by her passion for research that she gave a promise of work on the spot. This dramatic beginning became the most enjoyable scene in Basler's various public speeches. After harvesting a capable general, the generous Silverman completely handed over the study of group sensing to her and retreated to the countryside again.
Michael R. Silverman and Bonnie Bassler 丨Author: Princeton University Chemistry Department Twitter homepage
Remarkably, they found signaling molecules and associated genes and proteins, and had a rough idea of how the bacterial luminescence system worked, but none of them formally named this group phenomenon of bacteria. In 1978, A postdoc at Hastings, E. Greenberg, was a postdoc. Peter Greenberg began independently leading an experimental team at Cornell University, doing similar work using Vibrio fischerii. Biological research was time-consuming, laborious, obstructive, and long, until 1994, when this group behavior of luminescent bacteria finally took on a formal name in a brainstorming session by Greenberg's research team: "quorum sensing." The field was quite cutting-edge at the time, and in order to gain widespread attention from the academic community, the name had to be slightly distinctive. Sure enough, the name "group sensing" has gradually been widely accepted by the academic community, and more and more scientists have paid attention to and studied it, and gradually formed a larger group. At present, there are hundreds of laboratories around the world engaged in related research.
However, Basler, who arrived at the Agron Institute in 1990, did not focus on Vibrio Philaea and turned to Vibrio Harvey's. Basler found that Vibrio Harvey's also relies on an HSL autoorogene when communicating with its all-species bacteria. The difference is that Vibrio Harvey's also releases another chemical molecule to stimulate interspecific induction. That is, Vibrio Harvey has two different quadrant sensing systems, the first autoorder for intraspecific communication, called AI-1, and the second self-inducer for interspecific communication, called AI-2. It turns out that bacteria are not only not autistic, but more like language geniuses.
Scientists realized that the bacterial world could be as multilingual as humans. Later studies found that bacteria even have a lingua franca. Auto-inducers not only have HSL, the common acyl-homoserine lactones (AHLs), but also other types: for example, Gram-positive bacteria generally use oligopeptide signaling molecules (Autoinducing Peptide, AIP), Vibrio-specific cholera autoinducer (Cholera autoinducer 1, CAI-1), And the interspecific AC signaling molecule is the furanylborate disterster (Autoinducer-2, AI-2) and so on.
The discovery of these signaling molecules condenses the painstaking efforts of many researchers, and it is also the inevitable result of their exploration. Inevitability often hides contingency, and even sometimes there are romantic embellishments, such as the crystal structure analysis of AI-2 is slightly legendary. At the time, Basler had just begun to lead its own lab independently at Princeton University. Soon, she and crystallographer Fred Hughson worked together to resolve the crystal structure of AI-2 produced under experimental conditions. The results stunned all the researchers present, because the AI-2 molecule actually contained boron atoms, and the element "boron" is extremely rare in nature. Even more incredible is that boron comes from a glass test tube . In order to improve the performance of glass, glass companies often involve some boron elements, and this trace amount of boron perfectly restores the living environment of Vibrio in nature. Imagine if they had used a plastic test tube that did not contain boron, perhaps the secret of the ai-2 molecular structure would not have been discovered so early! Interestingly, different bacteria process AI-2 differently, so AI-2 has become the lingua franca of the bacterial world.
What's even more interesting is that some bacteria can also use this common language to trick other populations. For example, Basler found that in a mix of multiple strains of bacteria, E. coli would consume the AI-2 of Vibrio cholerae, making them think that the number of groups did not meet the threshold required for group induction. The key to the pathogenesis of Vibrio cholerae is that it initiates quadrangulation to release toxins, which in turn causes diarrhea in the host. When Vibrio cholerae found itself unable to reach the required number of group induction, it took the opportunity to sneak out of the main body and wait for the opportunity to find the next victim. In this way, E. coli in the intestine may be able to prevent Vibrio cholerae infection and further transmission by interfering with quorum induction.
Bonnie Bassler and her luminous bacteria 丨Education credit: Public Broadcasting Corporation, https://www.pbs.org/wgbh/nova/body/bassler-bacteria-au.html)
After decades of research, scientists now know that quorum sensing can turn hundreds of gene expressions on or off in bacteria. That is, the process of communication between bacteria initiates a huge genetic program. This process is similar to the development of an embryo, which enables bacteria to quickly complete the transition from individual members to group members. Quadrangulation also regulates many other behaviors of bacteria, such as the formation of biofilms, the production of pathogenic factors, the synthesis of antibiotics, the joint transfer of plasmids, and the migration movement of bacteria.
Some of these behaviors are related to the pathogenic mechanisms of bacteria. The aforementioned Greenberg is a leading figure in this regard. Since leading the research team independently, he has studied Vibrio Frediella, but also spirochetes, and has been quite successful and influential in the latter field. In 1988, greenberg returned to his alma mater, the University of Iowa, and began to focus on the study of the LuxR protein in Vibrio Fermatis.
E. Greenberg Peter Greenberg) 丨 Image source: University of Washington
At that time, Silverman had already studied that the LuxR protein was a transcription factor activated by an autoorgener, which in turn bound to the autoorder to participate in the quorum sensing of cells. Later, Greenberg's team found that the C-terminus of the LuxR protein was conservative and the N-terminus was variable. 30% of the C-terminal sequence retains the ability to activate the luminescent gene, but does not require a signaling molecule. The nearest 60% sequence to the N-terminal can bind to signaling molecules, but cannot affect the transcription of genes. That said, they further figured out the molecular mechanism by which the LuxR protein participates in group sensing, which is one step closer to a complete cracking of the bacterial language system. Greenberg immediately stopped the study of the spirochetes and went all out to study the "signal" of quorum induction.
At one meeting, he stumbled upon a discovery of a regulatory gene for pseudomonas aeruginosa (also known as Pseudomonas aeruginosa), in which a gene sequence was highly homologous to the LuxR gene, suggesting that quorum sensing may be closely related to the production of pathogenic factors. After a bout of exchanges, he and Iglevsky immediately decided to cooperate, focusing on the group sensing of Pseudomonas aeruginosa. As the research progressed, they found that quorum sensing controlled many important genes associated with pathogenic factors.
It is worth mentioning that Pseudomonas aeruginosa is widespread, and we have traces in the skin, respiratory tract and intestines of normal people. However, it is also a conditioned pathogen and is one of the main pathogens of infection in hospitals. It is also the fatal killer of cystic fibrosis (CF) patients. CF is an autosomal recessive disease that is highly prevalent in Caucasian populations and affects about 35,000 people in the United States. The patient's mucus is too viscous and the lungs have difficulty excreting bacteria, making infection relatively prone to occur. If Pseudomonas aerugin produces virulent proteins at low cell concentrations, this is undoubtedly a reminder to the host to initiate an immune response as soon as possible. However, Pseudomonas aeruginosa is very clever, and they only turn on group sensing when their cell concentration is quite high, producing toxic proteins, destroying lung tissue, and achieving the purpose of seriously damaging lung function.
During treatment, patients with CF require constant use of antibiotics. However, Pseudomonas aeruginosa is inherently resistant to most antibiotics and can quickly develop resistance mutations, which is really worse! Over the years, CF patients end up trapped in a swamp with no cure, allowing the disease to wither until it withers. If a group-sensing inhibitor can one day be developed, cf patients may be relieved.
Hastings once said in an interview: "Compared with a very practical research field such as cancer, bioluminescence or circadian rhythm is a very basic research, and researchers must love it from the bottom of their hearts." What he did not expect was that it was precisely because of the study of bioluminescence that scientists gradually carried forward his "group sensing", and now unconsciously introduced basic research into practice.
Three
The virus is eavesdropping
In recent years, researchers have begun to join hands with drug development companies to try to develop group induction inhibitors, hoping to bring new technologies to the fight against disease, so that patients will no longer be plagued by bacterial resistance and enter the "post-antibiotic era" as soon as possible. The headache is that many cunning bacteria are patient. They know that, on their own, individual bacteria can be easily cleaned up and eliminated by the host. So they quietly copy, divide, mutate, and wait until the time is ripe, until they think the group has reached a certain size, and then they launch collective action, coordinate operations, and attack the host. It can be seen that they are by no means "ragtag". Because of this, the community of scientists and pharmaceutical companies have not yet developed feasible drugs to deal with the "group induction" of bacteria and break through the bacterial fortress.
Group induction schematic 丨 Image from Twitter
It is such a group of "elite masters" who also have natural enemies. There are already other species in the world that have cracked their secrets one step faster than humans, and that is the bacteriophage , a virus that invades bacteria.
One day, Justin Silpe, a graduate student at Basler, came up with a bold idea that viruses would eavesdrop on bacteria's communication. Basler was skeptical, but he was still free to explore. Silpe used a bacteriophage called VP882 to infect Salmonella. He observed that the phage extracts the molecular signals of the host, but does not participate in the bacterial communication, but uses it to determine the time to attack the bacteria. When the signal gets stronger, the virus knows that the bacterial population is large enough that it's time to produce offspring and lyse the host! The new bacteriophages released will infect other bacteria, and the bacterial army will be wiped out.
Just a year before Silpe published the study, a laboratory at the Weizmann Institute of Science in Israel confirmed that there was also a communication system in the virus. Rotem Sorek and his team had wanted to use phages to infect bacteria, and then see whether the bacteria counterattacked the phages alone or in groups, but they made an unexpected discovery. The bacteria are quiet, the virus is noisy, and it turns out that the virus is transmitting information in its own language. Bacteriophages know when to lurk in host cells and when to attack. As his research progressed, Sorreck discovered that the signaling molecule used by bacteriophages is an oligopeptide class and named this molecule "arbitrium."
Through the study of viruses, scientists have gained a better understanding of the group induction system of microorganisms and found new breakthroughs in dealing with microbial infections. But we must not forget that microbes are everywhere and have been on Earth for billions of years. Scientists' vision is beautiful, but the road is long!
Thank you to Professor Yan Jing of Yale University for his professional guidance and patient review!
Primary references
[1] (French) Gustav Le Pen, translated by Dong Qiang: The Rabble-Rouse: Group Psychology, Zhejiang Literature and Art Publishing House, 2018.
[2] Frank H. Johnson, Edmund Newton Harvey (1887-1959), National Academy of Sciences, 1967。
[3] L. Stephen Coles, Bernard Strehler(university of Southern California, Professor of Biology), Journal of Anti-aging Medicine, Volume 4, Number3,2001,p:233-234。
[4] Tinsley H. Davis，Profile of J. Woodland Hastings，Proceedings of the National Academy of Sciences Jan 2007, 104 (3) 693-695。
[5] Farooq Ahmed，Profile of Bonnie L. Bassler，Proceedings of the National Academy of Sciences Apr 2008, 105 (13) 4969-4971。
[6] Tinsley H. Davis，Biography of E. P. Greenberg，Proceedings of the National Academy of Sciences Nov 2004, 101 (45) 15830-15832。
[7] Introduction to cystic fibrosis on the CDC website. https://www.cdc.gov/genomics/disease/cystic_fibrosis.htm#:~:text=Cystic%20fibrosis%20(CF)%20is%20a,makes%20infections%20more%20likely%3B%20and
[8] Bernard L. Strehler, Milton J. Cormier, Factors Affecting the Luminescence of Cell-Free Extracts of the Luminous Bacterium, Achromobacter fischeri, Archives of Biochemistry and Biophysics，1953: p16-33。
[9] Susan Brink, A Virus Can Eavesdrop On Bacterial Communication, December 13, 2018. https://www.npr.org/sections/goatsandsoda/2018/12/13/676389858/a-virus-can-eavesdrop-on-bacterial-communication
[10] Elie Dolgin, The Secret Social Lives of Viruses, Nature, 18 June 2019。 https://www.nature.com/articles/d41586-019-01880-6
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