Nestled beneath a layer of leaves in the damp mossy soil on the forest floor, a deadly hunt is underway. Amoebae, single celled eukaryotes, push their cytoplasm outward like feet as they creep toward their unsuspecting prey: bacteria. But the bacteria don’t make it easy for them.

“Bacteria, in general, don't really exist as single cells for very long in the environment because it's stressful. There're things hunting them, trying to kill them, so one of the ways that they protect themselves is by forming biofilms,” said Brad Borlee, a microbiologist at Colorado State University. 

When bacteria encounter a surface, they secrete a matrix of proteins, sugars, lipids, and bits of DNA to create the mesh-like fortress that is a biofilm (1). These biofilms help bacteria survive temperature and desiccation stresses as well as resist killing by the immune system and antibiotics. Recently, researchers have also found that antibiotic resistance genes can spread among bacteria within biofilms (2).

Biofilms are particularly dangerous in healthcare settings. They can grow on implanted medical devices and in human tissues, leading to difficult-to-treat chronic infections (1). Often, the doses of antibiotics that would be required to treat these infections are too high to safely give people.

To find better strategies to break down biofilms, researchers turned to amoebae — bacteria’s natural predators. While the field of active researchers probing amoebae biology for antibiofilm factors is small, recent advances have identified several amoebae-based molecules that may lead to new therapies for these treatment-resistant infections.

Move over MRSA and mycobacteria

Borlee and his colleagues have always wanted to better understand how microbes interact with one another to find ways to take advantage of that interaction for the benefit of human health. When they received a grant from the Department of Defense to investigate the interaction between amoebae and biofilms, they jumped at the chance.

“We wanted to see if we could use amoebae to break open biofilms,” Borlee said. “If they can get [bacterial cells] to leave that protection, then they can actually engulf them and eat them.”

On the bacteria side, he and his team started with two biofilm-forming species with important public health implications: methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis (3). MRSA is a common hospital acquired infection, and M. bovis causes bovine tuberculosis, which can lead to zoonotic tuberculosis in humans.

I was just shocked at how rapidly they were able to destroy the biofilms of Staph aureus. It looked like Pac-Man chewing through the biofilm. 
- Brad Borlee, Colorado State University

On the predatory amoebae team, they assembled five different species of soil amoebae: three Acanthamoeba species, Vermamoeba vermiformis, and Dictyostelium discoideum. They found that all five amoebae species disrupted the M. bovis biofilms, and all three Acanthamoeba species dispersed the MRSA biofilms. 

“I was just shocked at how rapidly they were able to destroy the biofilms of Staph aureus,” said Borlee. “It looked like Pac-Man chewing through the biofilm.” 

While the amoebae could block these bacteria from forming biofilms, Borlee was most excited to see that the amoebae could also destroy biofilms that had already formed, “which is the hardest stage of the biofilm to disrupt,” he added.

When Borlee and his team isolated the liquid in which the different Acanthamoeba species grew, they found that factors secreted by the amoeba into the culture medium also destroyed the MRSA biofilms.

Borlee’s next step is to identify the molecules the amoebae secrete to break down the biofilms. Not all of the molecules the amoebae secrete will likely directly harm the biofilm. Some of those molecules may instead act as signals to alter the behavior of the bacteria in the biofilm, potentially making the biofilm easier to disrupt. 

“Both partners are going to be secreting molecules that probably provide information to the other organism,” Borlee said. “There's quite a bit of research focused on trying to understand microbe-microbe interactions so we can mimic them to develop new therapeutics.”

A parasite protease

While Borlee looked to nonpathogenic amoebae for antibiofilm molecules, parasitologist Serge Ankri at Technion and biofilm researcher Ilana Kolodkin-Gal at Reichman University turned to the biofilm-eating ability of the parasitic amoeba Entamoeba histolytica.

E. histolytica is a gastrointestinal parasite that can cause stomach pain and bloody stool in severe infections resulting from consuming contaminated food or water. The amoeba causes these symptoms by feeding on gut bacteria and by damaging the mucus layer and epithelial cells that line the intestine (4). Ankri has studied this interaction between E. histolytica and the gut microbiome for many years. Although scientists knew that bacteria form biofilms in the inflamed human gut, many didn’t realize until recently that bacteria also aggregate into biofilms in the healthy gut, albeit weaker and smaller ones (5). 

“People were interested to understand what the microbiota is doing to the parasite and vice versa: shaping the virulence, regulating gene expression. But all of these studies have been done with bacteria in the planktonic form,” Ankri said.

Then, one day Ankri attended one of Kolodkin-Gal’s scientific talks about her biofilm research, and a new collaboration was born.

“If you want to understand how parasitic amoebae would interact in the GI, whether it's with probiotic bacteria or with pathogenic bacteria, you need to study them in the relevant form, which is biofilm form,” said Kolodkin-Gal.

To better understand this parasite-biofilm interaction, they decided to characterize the interactions between E. histolytica and the bacteria Bacillus subtilis (6). B. subtilis is commonly found in fermented foods and acts as a probiotic in the human gut (7). Analyzing the interaction between B. subtilis biofilms and E. histolytica was no easy feat, but Eva Zanditenas, a graduate student in Ankri’s group, found a way. She developed an entirely new system for studying both the amoebae and biofilms in compatible conditions and for quantifying outcomes such as E. histolytica’s penetration rate into the biofilm, how quickly it destroys the biofilm, and how that behavior affected the amoebae. 

The researchers then performed transcriptomics on E. histolytica under three conditions: alone, incubated with planktonic B. subtilis, or incubated with a B. subtilis biofilm. While the presence of planktonic B. subtilis led to the differential expression of some genes, the amoebae’s proximity to the B. subtilis biofilm resulted in five times as many genes changing expression patterns compared to the control.

“We found that the behavior of the parasite [was] really really different when a biofilm was present compared to planktonic bacteria,” said Ankri. “We were very excited because, first of all, it says that the amoeba is able to distinguish between planktonic versus biofilm.”

When they looked at the amoebae genes that showed increased expression in the presence of the biofilm, a familiar gene family called cysteine proteases caught Ankri’s eye. Cysteine proteases are important virulence factors for E. histolytica. They help the amoebae lyse intestinal epithelial cells by breaking down the extracellular matrix (8).

Ankri, Kolodkin-Gal, and their teams silenced one or a set of E. histolytica cysteine proteases, and they found that the amoebae’s biofilm degrading activity was significantly impaired. They then wondered if E. histolytica could also break down biofilms formed by pathogenic gut bacteria. E. histolytica easily broke down Escherichia coli biofilms, and factors released from the amoebae also significantly degraded Salmonella enterica serotype Typhimurium and Enterococcus faecalis biofilms.

Now, Ankri and Kolodkin-Gal are interested in identifying the kinds of molecules E. histolytica expresses when faced with other kinds of biofilms, including multispecies biofilms which are common in the human gut. By analyzing the genes expressed by the amoebae, they can see the strategies they evolved to destroy biofilms.

“The predator itself tells you what is the optimal antibiotic cocktail,” Kolodkin-Gal explained. “We as researchers can see what it produces and mimic it.”

Ankri agreed. “[There is] a fantastic reservoir of antibiofilm compounds from this amoeba, and now it's a goldmine that we need to explore.”

Three thousand amoebae

Somewhere in Madison, Wisconsin there are more the 3,000 samples of amoebae spores frozen in glass ampules. Hailing from the icy reaches of Alaska to the fertile soil of the Amazon, these social amoebae make up a collection of Dictyostelium species at the company AmebaGone.

“The goal here ultimately is to find a compound or series of compounds isolated either from one or multiple amoebae,” said Nathan Chesmore, a research and development scientist at AmebaGone. “We want to be able to treat a range of medically relevant pathogens with one approved compound.”

The company was originally founded in 2010 by Marcin Filutowicz, a microbiologist at the University of Wisconsin-Madison who has since passed away. Filutowicz inherited his collection of Dictyostelium from famed University of Wisconsin-Madison mycologist Kenneth Raper who was known as the “master of molds.”

One day, Filutowicz was watching the amoebae munching on both single bacterial cells and bacteria in a biofilm when he saw that the amoebae were eating the bacteria in both conditions at the same rate.

“Marcin immediately recognized that this [was] something special, and it might be that we can isolate or use the amoebae even themselves as a treatment for biofilm infections,” Chesmore said. “He immediately got to work with founding a company specifically to exploit that process.”

For regulatory reasons, using the amoebae is a bit of a nightmare. So, the compounds they produce are much more realistic in terms of getting it approved. 
– Nathan Chesmore, AmebaGone

Each of the 3,000 Dictyostelium isolates in their collection has a different preference in terms of the biofilms it likes to eat. So, when the team screens their collection against different biofilms, they can sometimes go through as many as 200 different amoebae isolates before they find one that devours a particular biofilm.

“For regulatory reasons, using the amoebae is a bit of a nightmare,” said Chesmore. “So, the compounds they produce are much more realistic in terms of getting it approved.”

So far, they have identified compounds released by amoebae that work synergistically with antibiotics to lower the concentration of antibiotics needed to kill a particular bacterial species. They also have some preliminary data in an ex vivo pig model of biofilm skin infections. The researchers found that when they treated the biofilm-infected pig skin wounds with amoeba-derived compounds, they didn’t see any adverse immune reactions.

“There is some work underway right now about looking at the efficacy of killing in those ex vivo tissues or skin explants, but it's TBD in terms of the CFU reduction and efficacy,” said Chesmore. “Ultimately, we're looking for funding. We know this works in a Petri dish, and we have visual scanning electron microscopy evidence that it works in these tissue explants that are ex vivo. So, we expect it to work in the animal model. It's just that we need the funding to do it properly.”

Difficulty in securing funding for amoebae research is not unique to AmebaGone. Borlee is working on finding additional funding to continue his amoebae-biofilm research as well. “The biggest challenge for studying amoebae is funding,” Borlee explained. “There's not a lot of funding available for this type of research. There's not a lot of people who work in this area.”

Kolodin-Gal echoed this same sentiment: “The community was not aware of the existence of the amoebae-biofilm interaction pathway as much as we hoped, and one of the consequences is we don't have a big mass of people who are working in this direction.” But she hopes that as scientists become aware of amoebae and their naturally potent antibiofilm activity, more researchers will join this small field of researchers on their quest to mine amoebae for new antibiotic compounds. “We'll show it in conferences,” she said, “and it's received really, really well.”

For now, the small but persistent field of amoebae-biofilm researchers pushes forward and looks to the amoebae themselves to guide the way to the best antibiofilm compounds.

“We really think that to take the solutions from nature, maybe tweak them a little bit, we generate many elegant solutions,” said Kolodkin-Gal.

References

1. Sharma, D., Misba, L., and Khan, A.U. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob Resist Infect Control  8, 76 (2019).
2. Ridenhour, B.J. et al. Persistence of antibiotic resistance plasmids in bacterial biofilms. Evol Appl  10, 640-647 (2017).
3. Martin, K.H. et al. Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms. Microbiology  166, 695-706 (2020).
4. Haque, R. et al. Amebiasis. N Engl J Med  348, 1565-1573 (2003).
5. Bollinger, R.R. et al. Biofilms in the normal human large bowel: fact rather than fiction. Gut  56, 1481-1482 (2007).
6. Zanditenas, E. et al. Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity. npj Biofilms Microbiomes  9, 77 (2023).
7. Lee, N.K., Kim, W.S., and Paik, H.D. Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier. Food Sci Biotechnol  28, 1297-1305 (2019).
8. Que, X. and Reed, S.L. Cysteine Proteinases and the Pathogenesis of Amebiasis. Clin Microbiol Rev  13, 196-206 (2000).