In this interview, we speak with Echo Pan, Ph.D. student at North Carolina State University about his latest research that used CRISPR to discover more about probiotics.
Could you please introduce yourself and tell us what inspired your latest probiotic research?
My name is Echo Pan. I am a Ph.D. student at NC State University, majoring in Functional Genomics. I joined Rodolphe’s lab in 2017 after receiving my bachelor’s degree in food science from the University of Wisconsin – Madison.
I have always wanted to learn more about probiotics and the human microbiome. It fascinated me that these tiny bacteria can have such drastic impacts on our health. I had only heard of CRISPR once in my microbial genetics class when I joined the lab and had no idea that I would be working with it for my graduate research.
Our laboratory has always been at the frontier of probiotics research, especially Lactobacillus, the no. 1 bacterial genus used in commercial probiotics. We applied our CRISPR expertise to manipulate Lactobacillus genomes to better study their probiotic efficacy. On the other hand, limited molecular tools have been developed for Bifidobacterium, which leaves a gap in understanding the underlying mechanisms of Bifidobacterium probiotic efficacy. We wanted to leverage our expertise in both CRISPR and microbiology to help fill this technical gap.
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What are probiotics and why are they commonly used?
The FAO/WHO definition of a probiotic is “live micro-organisms which when administered in adequate amounts confer a health benefit on the host”.
Probiotics are often called the “good bacteria”. You can find them in supplements and fermented foods like yogurt and cheese. Doctors will sometimes recommend that patients take probiotics when prescribed antibiotics. The idea is that antibiotics tend to wipe out your native microbiome (both good and bad bacteria) and probiotics can help restore the normal microbiome. Probiotics have been associated with a number of health benefits such as digestive health, immune health, child health, brain health, etc.
Scientists have discovered some underlying mechanisms of how these beneficial microorganisms confer health benefits on humans. For the most part, however, researchers are still trying to understand exactly how probiotics work.
Bifidobacterium, a gut bacterium commonly used to help maintain healthy microbiomes, was the subject of your latest research. Although found in many probiotics, it is difficult to characterize. Why is this?
Unlike other genetically tractable bacteria, genome engineering in Bifidobacterium is hampered by different factors, including a limited molecular biology toolbox, a refractory cell wall structure, a large number of restriction systems and modification (RM) and the lack of a universal replicon that replicates in a wide range of Bifidobacterium species.
Together, these limiting factors hinder our ability to investigate and manipulate this important genus (the second most commonly formulated genus in commercial probiotics after Lactobacillus), and new molecular tools will allow the engineering of Bifidobacterium strains with improved probiotic efficacy and the development of biotherapeutic applications.
In your last research, you investigated the genetics behind Bifidobacterium. Can you tell us how you did your research and the genetic tools used?
In this paper, we reused the endogenous type IG CRISPR-Cas system and adopted an exogenous CRISPR base editor for genome engineering in B. animalis subsp. dairy We used a combination of different approaches, including genomics, transcriptomics, computational analysis and in vitro TXTL tests, to characterize endogenous CRISPR-Cas systems in Bifidobacterium, which was crucial for aquatic genome engineering applications down We also characterized the epigenomes of these bacteria, which helped us understand how these closely related strains differ from each other.
Image credit: Marc Hall, NC State University
What did you discover about the bacterium?
The results of this paper highlighted the importance of combining genomics and epigenetics to democratize genetic engineering in recalcitrant bacteria such as Bifidobacterium. Despite sharing very homogeneous genomes, these strains express different epigenetic patterns, which contribute to the different levels of genetic accessibility between the strains.
We predict that heterogeneous epigenetic patterns could also contribute to differential probiotic performance beyond genetic accessibility. A better understanding of the effect of the epigenome on the efficacy of probiotics is something we are interested in exploring. As we showed in the paper, establishing a genome editing platform would be crucial for discovery.
What implications will your findings have for the field of microbiology and, in particular, probiotics?
The strategies established in this paper can be exploited and widely applied to other important bacteria, including the human gut microbiome and microbiomes in environmental niches such as water (oceans, lakes) and soil (for Ag and beyond). Because Bifidobacterium is a relatively understudied genus with limited tools for DNA delivery and genome manipulation, the approaches we used may facilitate and accelerate the deployment of molecular microbiology and genome editing in numerous important genera but little researched
In addition to antibiotic resensitization, as demonstrated in the paper, the strategies we set out in the paper open up new opportunities for probiotic improvement. For example, we can fine-tune carbohydrate metabolism to improve the utilization of HMOs in breast milk by Bifidobacterium, which is strongly associated with a healthy infant microbiome. To control inflammation, we can also engineer cell surfaces to modulate the molecular interaction with host epithelial and immune cells. We can also develop living biotherapeutics (LBPs), designing probiotics as a scaffold to deliver enzymes or small molecule therapeutics. There is a wide range of applications for deciphering and improving the genetic attributes that make bifidobacteria highly relevant to human health.
For your latest research, you used CRISPR-Cas. Since this is still a relatively new gene editing technology, how has this influenced microbiology research? Are you hopeful that with continued advances in technology, discoveries within microbiology will continue to emerge?
In nature, CRISPR-Cas systems serve as adaptive immune systems in bacteria again in bacteriophage attacks (first reported by my PI Dr. Rodolphe Barrangou in 2007). CRISPR has been used in the fermentation industry to immunize the starter culture against phages long before it was repurposed as a genome editing tool. Microbiology scientists also took advantage of the hypervariable CRISPR array sequences to differentiate strains of bacteria that are more than 99% identical in genomes. In this sense, CRISPR has influenced microbiological research long before it was ever used to edit human cells or plant cells.
Since the introduction of the CRISPR-Cas9 system, scientists have used CRISPR-based genome editing tools to manipulate genomes across the tree of life, revolutionizing the field of genetics. Although CRISPR has been rapidly adopted for genome engineering in eukaryotes, its deployment in prokaryotes has been delayed due to many intrinsic factors such as nuclease lethality. This is where our research comes in, and hopefully we take a small step to help fill this huge gap. There is much unknown about our microbiome. I am sure that more discoveries will continue to emerge within microbiology.
Image credit: elenabsl/Shutterstock.com
One sector that has become interested in probiotics is the wellness industry. How has this industry influenced probiotics research?
Recent advances in microbiome research have led to a new wave of enthusiasm for probiotic bacteria for human health applications. Our industry partner, IFF, one of the largest suppliers of probiotics in the world, has also seen a growing demand from its customers for products supplemented with probiotics such as protein bars, drinks, snacks, etc.
On the one hand, it’s great that the public is more health conscious and educated to make informed choices. Especially after the onset of COVID-19 in 2020, there has been an increase in public interest in health and wellness related products such as probiotics. On the other hand, not all probiotics are created equal.
Unfortunately, many commercial probiotic products do not match the correct taxonomic group listed in the ingredients list or are short on viable cell counts before their expiration dates. All of these shortcomings will negatively influence the probiotics industry and public confidence in this market. Structured regulations and effective and affordable methods for formulating and testing commercial probiotic products would be crucial for the probiotic industry in the long term.
Your research was supported by NC State and the North Carolina Agricultural Foundation, and was a collaborative effort among many researchers. How important are funding, support and collaboration to make new discoveries?
I am beyond grateful for the opportunity to work with so many excellent colleagues and collaborators; they have been a great support throughout this journey. The NC Ag Foundation has supported funding for translational agricultural research like the ones we have in the lab. The success of any modern scientific research these days largely depends on receiving sufficient funding.
I am very grateful to my PI, Dr. Rodolphe Barrangou, who always ensures that his students have secure funding. In today’s climate of tight funding, your academic career is often closely linked to your…