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Discovering the dynamics of prophage induction in the gut ecosystem

The human gut is a complex ecosystem dominated by bacteria and their viruses, i.e. phages. Approximately half of the viruses that reside in our intestine are derived from lysogens, bacteria that contain normally dormant viruses – prophages — in their genome.

When lysogenic bacteria are exposed to stressful conditions, the prophages become activated and produce viruses that are released in the gut. While it is well established that bacteria in our gut play a key role in maintaining health, the evidence is accumulating that phages can also modulate our immune system.

Therefore, it is important that we understand the dynamics and triggers of prophage induction in the gut ecosystem, as this may create novel avenues to promote human health.

Developing Gene Editing tools for use in Lactobacillus

My research team aimed to understand the mechanism that leads to phage production in the gut. Critical to understanding the mechanism is the ability to inactivate genes, which requires genome editing tools.

Over the course of the last decade, we invested heavily in developing genome editing tools for use in Lactobacillus. One of the tools developed is single-stranded DNA recombineering, which allows the user to make subtle changes to the bacterial genome without the need for antibiotic selection. Changes as subtle as a single base can be generated, which would make the resultant strain genetically indistinguishable from a strain that has naturally mutated.

Our laboratory combined single-stranded DNA recombineering with CRISPR-Cas genome editing. Implementation of CRISPR greatly increased the efficiency by which mutations could be identified, opening up exciting applications.

In a matter of days, small deletions can be generated, which normally would take up to two weeks. Lastly, we developed a tool to delete large DNA fragments. Clearly, our laboratory has a carte-blanche to engineer a number of Lactobacillus species, including Lactobacillus reuteri, a gut symbiont species which is their model organism of choice.

Understanding the impact of fructose on L. reuteri phage production

Lactobacillus reuteri ATCC PTA 6475, a strain of human origin, contains two prophages in its genome. A simple assay was performed to determine if these prophages were active: a chemical was added that induces a stress response in L. reuteri. If the L. reuteri prophages can be activated upon exposure to this stress-inducing chemical, viruses start to replicate inside the L. reuteri cells which leads to the destruction of the L. reuteri cell wall — lysis — and subsequent release of the virus particles.

This experiment clearly demonstrated L. reuteri can produce viruses. Now experiments could be initiated to delve into the mechanisms of virus production in response to sugar metabolism.  First, L. reuteri was engineered to yield a strain that is susceptible to its own phages. This platform is critical to study phage production and can be used to quantify L. reuteri viruses in biological samples.

Next, we aimed to understand to what extent fructose impacted L. reuteri phage production. Fructose is a sugar that is present in a wide range of sweetened foods and beverages, and consumption has increased nearly 5-fold since the introduction of high-fructose corn syrup in our food chain.

First, we demonstrated in the laboratory the growth of L. reuteri in the presence of fructose by increased virus production. To understand which genes are linked to fructose consumption and virus production, we mapped the metabolic pathway of Lactobacillus reuteri and systemically inactivated genes in this pathway. One of the mutants generated abolished the ability of L. reuteri to produce acetic acid, an end-product of fructose metabolism, and also reduced virus production nearly 10,000-fold.

Further research revealed that the key trigger of virus production was acetic acid itself. This was an interesting finding as acetic acid is also the dominant short-chain fatty acid in the colon of mammals. My team found that, in addition to acetic acid production, that exposure to acetic acid or other short-chain fatty acids also promoted virus production by L. reuteri. Intriguingly, these activation pathways were dependent on the global stress response system in the bacterial cell.

Finally, experiments in mice confirmed the laboratory findings, showing that increased intake of fructose also increased the L. reuteri virus production.

Conclusion

This work revealed mechanistic insights by which the dietary sugar fructose promotes phage production in the gut. Future work will focus on unraveling how fructose metabolism, or exposure to short-chain fatty acids, activate the global stress response mechanism in the bacterial cell. In addition, experiments are ongoing to unravel the biological role of these viruses in Lactobacillus reuteri.

Jan Peter van Pijkeren

 

Jan Peter Van-Pijkeren is Assistant Professor at the University of Wisconsin-Madison, Department of Food Science. His lab’s work on bacteriophage production in the gut was the subject of his presentation at the 3rd Probiotics Forum: Europe.

 

There is still time to register for the 4th Probiotics & Prebiotics Congress: USA. Download the agenda today.

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