Small Molecule Modulation of
Vibrio cholerae Virulence through Quorum Sensing
Adviser: Martin F. Semmelhack
“While the worlds of Princeton and the hospitals I was visiting in Bangladesh and India were polar opposites, the information I learned from both came together for me when it was time for me to hand in my thesis at the end of the year.”
I have the overwhelming sensation that the plane is going to descend into water. From my window seat, I can see electrical poles and small patches of green poking out of vast expanses of water. It is monsoon season in Dhaka, the vibrant capital of Bangladesh, whose one claim to fame is that it is the most densely populated country in the world. Upon landing, my ears are immediately assaulted by a deafening cacophony of honking. Cars, taxis, buses, rickshaws, and pedestrians all crowd onto the narrow and pothole-riddled roads with an apparent disregard for safety or rules. Despite my inner disquiet from the racket, everyone surges forward to unknown destinations with a plodding calm. My first night in Dhaka, I am taken to a restaurant located in a suburb on the other side of the city, and enjoy a simple meal accompanied by “clean” water—a mistake I am soon to feel the ramifications of. On the way back, I am soaked through by a sudden torrential downpour; our rickshaw driver is straining to pull us through the pedal-high rivulet, and he turns back to look at us with fear when we nearly tip over as one wheel sinks into an enormous pothole. A few days later, I am hit by a debilitating case of dysentery, from which it will take me almost a week to recover. Somewhere in this span of time, I look up at the incessant fall of raindrops and ask myself: Why am I here?
I am here, in Bangladesh and the neighboring West Bengal region of India, to understand cholera today. As an Adel Mahmoud Global Health Scholar, I had the privilege to travel to these countries during August and September of 2009 as a complement to my laboratory studies of the pathogen that causes the cholera disease, the bacteria Vibrio cholerae. I already had spent two months of the summer in Princeton working in the laboratory of my adviser, Martin Semmelhack, with the guidance of my postdoctoral adviser, Lark Perez. And while the worlds of Princeton and the hospitals I was visiting in Bangladesh and India were polar opposites, the information I learned from both came together for me when it was time for me to hand in my thesis at the end of the year.
In the collective Princeton laboratories of Martin Semmelhack (where I am based), Bonnie Bassler, and Fred Hughson, we are collaboratively working on elucidating the mechanism underlying the phenomenon known as quorum sensing. Quorum sensing is the communication mechanism, the language, used by bacteria. The bacteria produce and secrete small organic chemical molecules called autoinducers, which are detected by other bacteria when the autoinducers bind to an enzymatic receptor protein that is present either on the bacterium’s outer membranes or within the bacterial cell. When the autoinducer is bound to the receptor, it initiates a cascade of reactions in the bacterial cell, known as a signal transduction cascade. The final step is that certain genes become turned on—what genes these are depends on the bacterial species. This allows bacteria to perform an activity collectively, essentially operating together as a multicellular organism. In the case of cholera, quorum sensing modulates virulence in its host—humans.
The collaboration in these three laboratories has served to move our understanding of quorum sensing forward. In the chemistry department, students in the Semmelhack laboratory design and synthesize molecules that can affect the quorum sensing circuits in a variety of bacteria. In the Department of Molecular Biology, the Bassler laboratory tests these analogs in in vitro models of the bacterial quorum sensing circuits with the hope of finding ways to halt quorum sensing. Finally, the Hughson laboratory, also in molecular biology, collaborates to solve the crystal structures of enzymatic quorum sensing receptors; with this knowledge, we in the Semmelhack laboratory can design quorum sensing molecules more rationally by specifically targeting amino acids that are present in the active site of a key enzyme.
Before delving into the laboratory work, I sought to learn as much as I could about the theoretical aspects of quorum sensing by reading previous research on the topic. I had written one of my three fall junior papers with Professor Semmelhack, and the review of the literature afforded me a broad knowledge of the current research on quorum sensing. Going in, I knew that I wanted to be involved in research that would have a clinical aspect, given that I wanted to attend medical school. However, my initial project on quorum sensing, during the spring semester of my junior year when I was working on my second semester of junior independent work, was not what I had originally had in mind since the bacteria I was studying, Chromobacterium violaceum, is harmless to humans. During the spring, I learned to synthesize organic molecule analogs for this Gram-negative bacteria. Emerging antibiotic-resistant strains of Gram-negative bacteria are considered a serious threat to critically ill patients. We therefore planned to use the study of C. violaceum as a model for other Gram-negative bacteria. At the time, an interesting finding in the receptor proteins of two strains of C. violaceum had just come out of the Bassler laboratory. Despite the great similarity in the structure of the two strains’ receptors, the autoinducers that they responded to were different. We also had available the crystal structures of both types of receptor proteins. My task was to synthesize autoinducer analogs that would be tested in the two C. violaceum strains.
During the spring semester, my personal goal was to become adept at the techniques of synthesis in the Semmelhack laboratory. Once I began my thesis work on V. cholerae, I worked on and developed these skills, using more difficult reaction conditions and designing more complicated molecules to synthesize. I was interested in being involved in every aspect of my senior thesis, and so when I began research on cholera during the summer prior to my senior year, I would work in the chemistry department to synthesize the molecules, and I took the initiative to learn testing the analogs I synthesized in vitro with the help of a postdoctoral student in the Bassler laboratory, Wai-Leung Ng.
In the laboratory, we are striving to learn more about the intricate molecular mechanisms underpinning quorum sensing and the elegant pathways bacteria have developed to communicate with one another. In gaining an understanding of quorum sensing, we hope some day to create a novel antimicrobial agent to prevent cholera and other infectious diseases throughout the world. This noble goal was packaged into a manageable project for me as I worked to develop a water-soluble analog of the native V. cholerae autoinducer, which happens to be a nonpolar molecule with a long carbon chain. We identified water solubility as a key physical property of our desired molecule due to our understanding that a cholera anti-quorum sensing molecule would need to remain in the intestine, where the cholera bacteria are located, to exert its effect. Another important consideration we took into account is that, in most cases of bacterial quorum sensing circuits, we wish to antagonize or halt the quorum sensing circuit so that the bacteria cannot exert its harmful behavior. Interestingly, the V. cholerae quorum sensing pathway is different in that, to stop its virulent activity, we want to agonize quorum sensing in the bacteria.
Therefore, I based the design of my molecules on the native (agonist) autoinducer, incorporating polar oxygen molecules in the long carbon chain of the native autoinducer to increase the water solubility. Currently, the crystal structure of the autoinducer’s receptor protein has not been solved, and so any manipulation to the structure of the autoinducer had the potential to give us insight into the nature of the binding site of the receptor. Additionally, because there was no guarantee that any change in the molecule would allow it to remain acting as an agonist, each molecule had to be tested in vitro to assay for agonist activity. The exciting outcome of my thesis is that we found several key agonist molecules that we predict to be water soluble; the next step and final test will be to test the molecules in vivo.
As I reflect on the thesis experience, I would urge seniors to begin early: Write that introduction over the summer. And most importantly, find a captivating topic. My thesis topic was one I found pertinent to my future profession as a doctor. The fact that I could combine my lab-based work with observations on the field made my research feel real to me and made me want to do everything I could to ensure my reactions could succeed. I would urge any senior to find a topic they are passionate about; the long hours, the painful write-ups, the failures, and the successes will all be so much more meaningful.
Small Molecule Modulation of
Vibrio cholerae Virulence through Quorum Sensing
Martin F. Semmelhack
Professor of Chemistry
“Karolina was an exceptional student and her thesis was exceptionally successful. It was the result of her own initiative, dedication, and intelligence, but it also depended on a community of people around her, her advisers, co-workers, and sponsors.”
The senior thesis is a special part of the teaching and learning experience at Princeton. It is my best chance to work closely with an undergraduate and watch her develop as a scholar and scientist. In many ways, it is a stronger experience compared to the relationship with a graduate student, as the change from naïve to sophisticated and from unpracticed to skillful is much larger.
In chemistry, the research is done as part of a group, with a strong group identity and organization. This extends from the day-to-day work in the laboratory, where each group member can consult and advise others in the group, to weekly research discussion meetings as well as occasional social events outside of the lab, such as birthday lunches and a pool party in my backyard. Typically there is a senior person in the group, the “postdoc,” who also helps guide the undergraduate progress.
Working with Karolina Brook was an example of the best of this system. Our work together started with a junior paper, from the fall semester of her junior year. She already was interested in the sort of research my group was doing in collaboration with groups in molecular biology, so we decided on a specific topic that could be an entry into a project that would bridge chemistry and biology. She analyzed the literature regarding chemical communication among cholera bacteria, and was intrigued by the idea that this communication could be controlled by chemistry in a way to control the terrible effects of this pathogen on human health.
With the idea that she could learn the techniques of organic synthesis in order to create new molecular structures as signals, and then move into the biological evaluation with the Bassler and Hughson groups in molecular biology, she joined my group for a junior project in the spring of her junior year. The junior spring semester is a useful training time for people thinking of a senior thesis in chemistry. It is a chance to move from the fairly primitive lab experience in Orgo, or organic chemistry, to the sophisticated techniques used by graduate students and postdocs in frontier research. Karolina rapidly became accomplished in the tools of organic synthesis by doing some fairly straightforward experiments closely related to previous work in my group, and she began to look for a really independent project.
We were aware of molecules that would prevent cholera bacteria from entering the dangerous phase, where the bacteria induce debilitating diarrhea that is sometimes fatal. However, those molecules were highly insoluble in water and therefore did not stand much chance as a drug, as that would require moving through the blood stream and cellular fluids to intersect the bacteria in the gut. They were not working in controlling the cholera infection in animals. Karolina began designing and synthesizing a new structure type that would be much easier to synthesize, and quite likely would solve the water solubility problems. The big question was whether the new structures would still be highly active in inhibiting the cholera bacteria. She synthesized a few examples, and then moved over to the molecular biology department to work with the Bassler group in order to test the biological activity. She learned the new techniques of bioassay work, and brought back the very exciting result that her molecules were equally as active as the best previous examples. This opened up an entirely new direction for our work on the cholera project.
The depth of commitment that Karolina brought to this project became clear when she applied for and was awarded an Adel Mahmoud Global Health Scholarship to have an “up front and personal” look at cholera in hospitals in Bangladesh and India. This was independent of the chemistry and biology she was doing, but obviously related. Everyone in our group followed her adventures that August as she toured and talked with people directly involved with treating this disease, and it was a special experience for all of us, if not as direct as for Karolina.
She polished up her experimental work and began putting together her thesis. Typically, the thesis can be quite thin as the critical part of a chemistry project is carrying out the experimental work and not the expression of it. The experimental work can be described with figures and presentation of factual information in a fairly standard format. Karolina did this, but also took the opportunity to weave together a complete story, from the chemistry and biology that she did in the lab to the descriptions of the human health issues that she observed directly. It was nearly at the level of completeness of a Ph.D. thesis, and will certainly be the basis for a significant publication in the original literature.
Karolina was an exceptional student and her thesis was exceptionally successful. It was the result of her own initiative, dedication, and intelligence, but it also depended on a community of people around her, her advisers, co-workers, and sponsors. That team feeling is common in chemistry and biology at Princeton, for all of our students to take advantage of.