It was 1917, the height of the First World War, and German soldiers stationed in the Balkans were extremely sick. While the rest of his comrades endured prolonged bouts of diarrhea, one soldier remained completely fine.

At the time, Alfred Nissle, then a medical assistant at the Albert-Ludwigs-University of Freiburg, studied how certain bacterial strains kill other bacteria. He wondered if this healthy soldier might have beneficial microbes in his gut protecting him from the infection plaguing his fellow soldiers. Nissle took a stool sample from the healthy soldier and isolated a strain of Escherichia coli from it (1). This new strain — which scientists later named E. coli Nissle 1917 (EcN) in Nissle’s honor — inhibited multiple bacterial pathogens and went on to become one of the best studied bacterial probiotics ever (2).

While EcN protected this soldier from what Nissle later discovered was likely a Shigella bacterial infection, they can’t defend against all kinds of diarrhea-causing pathogens. Neel Joshi, a microbial engineer at Northeastern University, however, reasoned that he and his team could use EcN as a starting point and engineer them with even better therapeutic abilities.

“It is E. coli, so the genetic tools that we have are compatible with engineering it,” said Joshi. “It also has the ability to survive in the gut and a long track record of use and safety in humans.”

Joining forces with his colleagues Charles Shoemaker and John Leong at Tufts University who are experts in infectious diseases, Joshi and the research team engineered EcN to target and sequester a variety of diarrhea-causing pathogens (3). Patients can take EcN orally. EcN are easy to manufacture. Because the engineered EcN are specific for diarrheal pathogens, so these engineered EcN may present an inexpensive and scalable solution for treating diarrheal disease worldwide.

Just like Nissle’s soldiers, diarrheal disease can affect people of all ages who ingest the bacteria, viruses, or parasites that cause it, but children have the worst outcomes. The World Health Organization estimates that every year, there are almost 1.7 billion cases of childhood diarrheal disease and that approximately 525,000 children under age five die from these infections (4). Frequent bouts of diarrhea also deprive children of the nutrients they need to grow and thrive, making it a leading cause of childhood malnutrition.

To endow EcN with diarrhea-fighting abilities, Joshi and his colleagues first needed to find a way for the bacteria to recognize and bind to the disease-causing pathogens. Antibodies are great at binding and neutralizing unwanted pathogens, but they are too big for bacteria like EcN to express. The researchers found their solution in the form of a furry South American mammal: the alpaca.

“For whatever quirk of evolution, camelids — camels, alpacas, llamas — have these antibody domains that are stripped-down, simplistic versions of the antibodies that exist in our bodies,” said Joshi. “It’s convenient for making in bacteria, which can't sometimes make the types of antibodies that we have in our bodies.”

Shoemaker, who is an expert in developing these tiny camelid nanobodies called single variable domain on a heavy chain (VHH), immunized alpacas with virulence factors from multiple strains of E. coli that cause diarrhea. He and his team screened the resulting VHH for the ones that bound the pathogens best.

This idea to have this probiotic having this net with the expression of the nanobody in a mucus, I think is wonderful. I am sending the paper to all my students to say, ‘Oh, let's do something like that!’ 
– Viviana Parreño, National Scientific and Technical Research Council

Joshi and his team then selected some of the top-performing E. coli-binding VHH along with previously validated VHH that bind to the bacteria Shigella flexneri and the parasite Cryptosporidium parvum, both of which cause diarrheal disease.

The researchers then expressed these VHH on EcN’s curli fibers, which are proteinaceous fibers that EcN use to form a mesh-like biofilm. They reasoned that when these engineered EcN reached the gut, they would create a biofilm containing these VHH antibodies which would provide a sticky surface to capture pathogens before they have a chance to cause disease. Then, through the normal passive process of digestion, the pathogens ensnared by the engineered EcN would move through the digestive tract and get excreted.

“This idea to have this probiotic having this net with the expression of the nanobody in a mucus, I think is wonderful,” said Viviana Parreño, a biochemist at the National Scientific and Technical Research Council who develops VHH to treat viral gastrointestinal diseases and who was not involved in this study. “I am sending the paper to all my students to say, ‘Oh, let's do something like that!’”

Joshi and his team found that the VHH-expressing EcN curli fibers bound and neutralized soluble toxins from both S. flexneri and multiple diarrhea-causing strains of E. coli in vitro. Most exciting, though, was how well the engineered EcN bound to the parasite C. parvum.

“That one in particular was a more ambitious thing than some of the other targets,” said Ilia Gelfat, the lead author of the study who is now a molecular biologist at GreenLight Biosciences. “It’s this larger, more complex eukaryotic parasite… I remember being very excited when seeing the data for the first time, that it actually binds to it.”

Parreño too found this result very exciting.

“For parasites, so far we don't have vaccines, and it's really a big problem. So, this is very promising in terms of parasites, not only for human but also for animal health,” she said. “Calves in the very early days of life, they die from diarrhea very, very easily. Cryptosporidum is the most important, so really, this work is nice.”

Moving forward, Parreño would like to see how well the engineered probiotics work in animals. Mehdi Arbabi-Ghahroudi, a molecular immunologist at the National Research Council Canada with expertise in developing VHH who was also not associated with the study, added that he would like to see how the authors determine the right dose of VHH-expressing EcN to give an animal.

“How many VHH will be expressed in this system?” he asked. “How stable [is] this net system or biofilm system?”

Joshi and his team are already working on these in vivo experiments, and they are excited to study and develop this technology further. Joshi hopes that because this engineered probiotic acts as both a drug factory and delivery vessel, it will make this treatment well-suited for resource scare areas.

“Bacteria make more of themselves easily and cheaply and maybe could also be deployed without any or with minimal refrigeration. It's a somewhat unique platform for deployment that makes it attractive,” he said.

Gelfat added, “It's a new way of looking at drugs and drug production and drug delivery, and it has all these benefits. In the end, it's relatively simple.”

References

1. Sonnenborn, U. Escherichia coli strain Nissle 1917—from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiol Lett  363, fnw212 (2016). 
2. Behnsen, J. et al. Probiotics: Properties, Examples, and Specific Applications. Cold Spring Harb Perspect Med  3, a010074 (2013).
3. Gelfat, I. et al. Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917. PLoS Pathog  18, e1010713 (2022).
4. World Health Organization. Diarrhoeal disease. Retrieved March 16, 2023, from https://www.who.int/news-room/fact-sheets/detail/diarrhoeal-disease.