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TINY ORGANISMS, TRANSFORMATIVE OUTCOMES
SINGLE-CELLED ORGANISMS SUCH AS YEAST AND BACTERIA PRESENT DIFFERENT ADVANTAGES FOR GENETIC ENGINEERS LIKE NATHAN CROOK.
Dr. Nathan Crook is an Assistant Professor of Chemical and Biomolecular Engineering at North Carolina State University. He received a B.S. in Chemical Engineering from the California Institute of Technology and a Ph.D. in Chemical Engineering from the University of Texas at Austin. He pursued postdoctoral studies in Pathology and Immunology at Washington University in the Saint Louis School of Medicine and came to NCSU in 2018. In 2023, he received an NSF Career Award and a NIH New Innovator Award. Today, we talk to Dr. Crook and several of the graduate students and postdocs he continues to mentor.
INTERVIEWER
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INTERVIEWER
INTRODUCTION
THIS SPACE RESERVED FOR INTERVIEWER OPENING REMARKS
But before we go further, we know that you came to the University as part of its Chancellor’s Faculty Excellence Program. Can you please tell us what this means?
{interviewer name} I think you will find this interesting.
Normally, academic departments foot the bills for bringing in new faculty, including paying their salary for their first few years of research. Later, the new faculty members will apply and compete for their own external research funding.
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But with the Faculty Excellence Program, the initial expenses are paid from a central fund rather than from a department’s budget.
Why is this important?
Because it allows departments to recruit individuals that have expertise in new or emerging research fields that are important to the departments, but for which they may not have the necessary funds to pursue on their own. This program funds these initial new faculty startup costs before they apply for external research funding.
You have spent much of your career working with single-celled organisms such as yeast and bacteria. Generally speaking, why did you decide on this particular career path and what is it that you hoped to understand and accomplish?
When I first learned about biological evolution in high school, I was fascinated! I remember reading a paper in Scientific American about engineers who used an evolutionary algorithm to design better electronic circuits. They found that these innovative designs performed better than the existing circuits at that time, and I found this to be most interesting.
Let’s move on to your experiences in your college years.
In college, I was fortunate to have worked in the lab of Professor Frances Arnold at Caltech, who was using evolutionary methods to design enzymes that can perform chemical reactions that natural ones cannot. I worked in her lab for 3 years.
You should know that she won the Nobel Prize in Chemistry in 2018 for using evolutionary methods to design enzymes. And since January 2021, she served as an external co-chair of President Joe Biden's Council of Advisors on Science and Technology.
You said you wanted to talk about our body’s growing resistance to antibiotics. Where do you want to start?
Let’s cover bacterial infections of the gut first as these pose a growing clinical urgency that affects millions of people each year. While treating these types of infections with antibiotics has been highly effective in the past, increasing resistance to antibiotics is making these types of infections much more difficult to treat.
What are the consequences of this growing resistance to antibiotics?
Just five years ago, it was estimated that this resistance had already cost 35,000 deaths in the US and 1.27 million worldwide.
So what had been done thus far to address this growing issue?
To start with, it is currently thought that one reason why antibiotic resistance is now so prevalent is because these often kill good bacteria as well, including some which are really beneficial to our bodies. The more bacteria that a drug targets, the higher the likelihood that one of these bacteria will stumble upon a way to become resistant.
Therefore, there has been increasing interest in targeting only the bacteria that are causing an infection. The drugs currently being tried for this purpose are called “narrow-spectrum” or “targeted” antimicrobials. But these are not always effective in isolating only the bacteria that may be causing an infection.
Nathan, I had never given much thought to the issue of antibiotic resistance. I hope that your research in this area will prove fruitful. What can you tell us?
{interviewer name}, the approach our lab is taking is to inactivate certain “weapons” that bacteria use to cause disease.
You might be surprised to learn that many bacteria produce special proteins whose only purpose is to cause infections. Studies have indicated that if one could delete these genes, it would prevent the bacteria from causing infections. However, it does not impact their survival otherwise.
In fact, many healthy people harbor disease-causing bacteria which have had their disease-causing genes turned off.
We think that inactivating these genes is a potential way to cure disease, and this is one of the things we are working on.
This would seem to be such an important breakthrough. Can you tell us more?
Of course! But I do want to emphasize that we are not the only people taking this strategy – many more clever and hardworking scientists are also working on drugs like this. Right now,
we are focusing on a specific bacterium which we call “Cdiff” for short.
“Cdiff” likes to cause infections in people who have taken antibiotics recently, so it is a particular problem in hospitals. While It can be successfully treated by antibiotics 80% of the time, in the remaining 20% of cases, patients experience “recurrent” infections where antibiotics are only able to provide temporary relief rather than a permanent cure
Are there any other treatment options at the present time, knowing that your research is intended to provide better treatment alternatives down the road?
Aside from antibiotics, the only drugs available for Cdiff are an antibody therapy, which is rather expensive, and fecal microbiota transplants, which are difficult to manufacture at large scale.
Can you give us just one example of a research approach you are using here?
Yes, but I first want to point out that we work with several University collaborators in this research, and without their assistance, I don’t think our work would be fruitful. I want to recognize just a few and thank them for their efforts - Drs. Carol Hall, Stefano Menegatti, Casey Theriot, and Scott Magness. With them, our goal is to develop lower-cost treatments that inactivate Cdiff’s weapons.
Moving on, while Cdiff produces three different toxins, only one is important to us, because studies have shown that the others do not cause disease. We are trying to find specific molecules that can stick to the harmful toxin and prevent it from working normally. Our effort to find them is truly a collaborative one.
-Dr. Menegatti’s group tested about 10,000 peptides and selects those that stick the most to the toxin.
-Dr. Magness’s lab tests how well these peptides block toxicity when added to human cells.
-Dr. Hall’s group then takes the selected peptides and makes changes to make them that stick to the harmful toxin better.
The results of these tests are used to design better versions of those peptides, learning from what worked and what didn’t.
Thank you Nathan, your research in this area is most commendable.
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Your featured article at NC State News mentioned your study of certain strains of yeast. What do you feel may be short and long term implications of this research?
Actually, yeast engineering was the focus of my PhD research, so it’s sort of like my scientific “home".
I wanted to study yeast because nearly everyone encounters it in daily life. It can also be engineered to produce many different and useful things – anything from ethanol and fuels to medicines. At this time, we are focusing on how to engineer a type of yeast that will reside in our gut and produce medicines there.
Why do you feel this is important enough to warrant your research?
We think this work is important because, while there are a wide range of microbes that you can engineer to produce things in the gut (like yogurt-making bacteria), yeast is rather unique in that it is really good at producing proteins, and it can be manufactured in large amounts. It can even be freeze dried and shipped around the world at room temperature.
If we are able to successfully engineer this yeast to produce medicines in the gut, it should make it promising for delivering medicines to remote places that are difficult to reach.
We certainly wish your lab success in this research, and the sooner the better, as it appears that the results of your efforts will help treat diseases worldwide. Can you share a few more details with us? The NC State News article mentioned a strain of yeast used to treat various gastrointestinal illnesses.
This one is called Saccharomyces boulardi (S.boulardii) and has an interesting history. There is a story here that might prove interesting to your readers.
The story goes that a French microbiologist was in Indonesia in the 1920s to research cholera and found that locals would drink tea made from peels of local fruits during
cholera outbreaks. He hypothesized that a
microbe on the peels was responsible for the tea’s protective effects.
He even isolated a strain of yeast that appeared to resist the harsh environment of the gastrointestinal tract. That was just the beginning!
Later research indicated this yeast can improve digestive health in the gut and is now sold under brand names such as Florastor which can be added to a variety of probiotic foods.
Thanks Nathan, another useful product from your lab to help promote good health! Nathan, you said you wanted me to also talk to Aryan Razdan about S. boulardii as well.
Aryan, in 2023 you won a National Science Foundation prestigious award that enabled you to study why S. boulardii doesn’t stick around as long as other species of yeast. Why is this study important, and what are you looking to accomplish?
Let me start here by stating that S. boulardii is not a good colonizer of our gut. In healthy people, this yeast tends to drop to undetectable levels within one week of consumption.
But because this yeast strain may be a good platform for the delivery of medicines, it would certainly be helpful if we could have it live in the gut for longer periods of time.
And if you succeed in having it live longer in the gut, what does this imply?
Another good question!
Our goal here is to have this strain reside closer to sites of disease and deliver higher overall doses of a medicine.
We are currently looking at the mucus that is produced by our intestinal walls. This mucus serves a variety of important functions, one of which is a food source for good bacteria living in our gut. Unfortunately, S. boulardii cannot eat this mucus.
Our research aims to determine whether S. boulardii will end up residing in the gut for longer periods of time if we engineer it to consume the mucus,
If successful, this could allow us to engineer S. boulardii to produce more complex medicines, such as anticancer or anti-inflammatory drugs.
Aryan, that is so encouraging to hear, and of course this is just one more example of the kinds of research you are doing for the benefit of mankind.
Nathan, in your research you introduce toxins to yeast cells in order to kill the yeast cells. While this may seem counterproductive, this must not be the case. Can you please explain?
{interviewer name}, studying toxins that kill yeast gives us a way to identify drugs that will control these toxins, and enable yeast survival.
We try to make each yeast cell produce the active part of the toxin and produce possible drugs that can be used against these toxins. The yeast cells that make poor drugs die, but cells that produce good drugs will grow and divide. And we can test millions of possible toxin inhibitors in a single test tube – such is the power of yeast!
Thank you, Nathan. In 2023, NC State University was awarded more than $1.3 million from the National Institutes of Health to study whether yeast can be used to develop new drugs faster, as well as aid existing antibiotics — and, ultimately, even help treat antibiotic-resistant infections. How essential to your research, and to the research at other universities, are awards like this?
Research awards such as these are the lifeblood of labs like mine.
Researchers that have previously received grants are constantly scrambling to renew their funding because their awards may only last a couple of years and do not include enough funding to support several people on one project.
Because these awards are so hard to get, one often has to apply for renewed funding several years before their grant expires with the hope that they will receive renewed funding before the existing funds run out.
Our NIH award is for 5 years and for several million dollars, and allows us to hire 2-3 graduate students and postdocs to work together It allows me to focus on research rather than funding.
Congratulations are certainly in order for your getting this prestigious and important NIH award!
Students must be critical to your research efforts. Can you give us a few examples of students that have made significant contributions to your research efforts?
Sometimes you get students who are just as easily excited about crazy, off-the-wall ideas as you are.
Here is an example – we were brainstorming ways to make screening for Cdiff toxin inhibitors faster, and one of our students found that once yeast turned on the production of Cdiff toxin, the yeast died.
Then, as all good students must do, she got her PhD and moved to another lab to do her postdoctoral research.
This is where another student came in. She found that while these toxins certainly killed yeast, they were almost “too toxic”. And none of the currently available drugs seemed to allow them to grow again.
It was the most excited that we had been to see a blank petri dish!
We now have a great platform for identifying toxin-blocking drugs, and Amanda Taylor (a 2nd year Microbiology graduate student in the Crook Lab) is applying this platform to identify new drugs. Together, Amanda and two fellow students are now writing a paper describing this very exciting joint effort.
I understand that your lab tried to determine if a particular strain of yeast might reduce the amount of methane that cattle emit. This is also exciting as some sources say cows and other livestock are responsible for about 40 percent of methane emissions, and 6% of global greenhouse gas emissions world-wide. Can you elaborate?
I think we’ve all heard that livestock are a major source of greenhouse gas emissions via the methane in their “burps.” Livestock use microbes to break down the plants they eat, allowing their body to absorb nutrients. Unfortunately, one by-product of this process is the greenhouse gas methane
Two members of our research team are both involved in a startup company to develop probiotics and natural enzymes that improve cattle health and digestive efficiency while eliminating methane.
Nathan, it is obvious that student participation in your research programs is an important ingredient to your success. In your mind, what makes a good student for your curriculums and research programs, and what are the qualifications?
I look for students who did some type of undergraduate research during college, ideally with microbes and involving cloning of recombinant DNA. Equally important are students who are intensely motivated – the type who get obsessed about things and are willing to tinker and experiment tin order o figure out the right approach.
Students who have graduated from a bachelor’s program, and are pursuing their PhD, make up the majority of my group.
Are financial assistance and grants available to them?
Each student is provided with a stipend to support their studies.
In my case, it comes out of the research grants that I compete for. That said, students who are US citizens are eligible for a wide range of government fellowships. ​
What about industry collaborations?
I understand that you regularly collaborate with industry, and you have performed research for several companies to help bring beneficial therapies to market. Can you give us a few examples of your collaborative efforts?
We previously worked with Novozymes (now Novonesis) on a project to help genetically engineer a microbe with which they were working. The Novozymes project started with an event that NC State University hosted, where companies are invited to attend, and our professors talk about some of their research projects.
We also collaborated with company called Synlogic. In this instance, Synlogic knew of our research on this subject through our publications and reached out to us directly
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