While looking at a fungus is not exactly like looking in a mirror, fungi and humans are more similar than meets the eye. Unlike their microbial cousins, bacteria, which are prokaryotes, fungi are eukaryotes like humans. The evolutionary similarity between fungi and humans can make it difficult to find drugs to target a fungal infection that do not harm the infected human.

Humans with healthy immune systems can usually fend off fungal infections, but fungi become a problem for immunocompromised people. In fact, around 1.7 million people globally die from fungal infections every year (1).

“We have increasing numbers of patients that fall into that [immunocompromised] category because of underlying diseases and treatments for other diseases like transplantation and cancer,” said David Andes, a medical microbiologist at the University of Wisconsin-Madison who studies antifungal drug resistance. Fungal infections are becoming more common.

Multidrug-resistant fungal infections frequently arise in hospital settings, where they often infect patients via breathing machines or catheters. For example, a COVID-19 outbreak in India earlier this year resulted in an associated outbreak of fungal infections in those patients.

The world desperately needs new antifungals to combat these infections, but their development is slow. Before the first drug in a new class of antifungals was approved in 2021 — ibrexafungerp for vaginal yeast infections — there had not been a new class of antifungals developed since 1970 (1).

“It's that combination of increasing numbers of patients with these infections, the development of anti-infective resistance to the agents we do have, and the fact that we have very few [antifungals] that make this a highly important area,” Andes said.

To find new antifungals, some scientists take a unique approach. Animals such as sea squirts in the crystal blue waters of the Florida Keys, ants and beetles from South America, and armadillos and opossums on the side of the road in Oklahoma — or the bacteria that live inside of these animals — are a bountiful source of never-before-seen molecules that may just supply the new antifungals we’ve been searching for.

The quest for new antifungals: From soil to animals

“Historically, bacteria have been an important source for drug discovery, especially for infectious disease,” said Tim Bugni, a natural products chemist at the University of Wisconsin-Madison. 

Whether sequestered inside the gut of a host or out in nature, bacteria live in close contact with other bacterial species, where they encounter diverse environmental pressures. Because of this, bacteria evolved to produce specific molecules that help them communicate with other microbes, wage war against encroaching microbial species, or adapt to new environments. These molecular signals, also called natural products, can sometimes have antimicrobial effects, making them useful for human health.

For example, the soil bacteria, Streptomyces nodosus produces the broad-spectrum antifungal amphotericin B (2), and a related species, Streptomyces noursei synthesizes nystatin, which is used to treat diaper rash and thrush (3).

In fact, Streptomyces species from the soil have been an important source of both antifungals and antibiotics that have made their way to the clinic. Streptomycin and tetracycline, for example, are two of the most well-known antibiotics produced by these species.

When scientists at pharmaceutical companies first realized the potential for soil bacteria to produce antimicrobial compounds, “they just went out and sampled soil or water and sampled diverse collections of microbes,” said Cameron Currie, a microbiologist at the University of Wisconsin-Madison. “[They] screened them for their potential to produce antimicrobials, and that was hugely successful.”

For many years, that worked. Recently, however, scientists noticed that they kept rediscovering the same molecules time and time again. Frustrated, many came to view species of soil Streptomyces as an exhausted source of new antimicrobial molecules.

So, some scientists thought, why not look in other environments where microbes interact with each other and evolve useful antimicrobial molecules? 

Bradley Stevenson (pictured) and Robert Cichewicz sampled bacteria from roadkill in the search for novel antimicrobials.

Credit: Bradley Stevenson

“The gut microbiome is a place where we would imagine these interactions having evolved, and that might be because there are so many microorganisms in the environment,” said Bradley Stevenson, a microbial ecologist at Northwestern University.

In addition to selecting for the production of new compounds under diverse selective pressures, some animals, like fungus-eating ants, associate with antimicrobial-producing superstars like Streptomyces species.

“Discovering that some insects have evolved to use antimicrobial producing bacteria led to the idea [that] maybe some of these would be useful as drug leads,” said Currie. “If you go and bio-prospect randomly for microbes, you're doing all the work. Whereas if you're looking in insect hosts, through evolution, they appear to be selecting the microbes for antimicrobial production. So, in some ways, they're doing some of the screening for us.”

Because these microbes have evolved with their animal hosts over time, if these animal-associated microbes produce antifungals, “there should be selection on those antifungals to have reduced animal toxicity because of the association with their hosts,” said Currie.

With the benefit of safe, potential antimicrobials tucked away in the guts of animals, scientists trained their gaze into the belly of the beasts.

Antifungals under the sea

Clinging to rocks and sprinkled across the ocean floor, sea squirts may seem like an unlikely source of novel antifungals for humans, but when Bugni and Andes started their collaborative project with these marine invertebrates, they saw potential.

“Starting in about 2000, there was a lot more documentation about the immense bacterial diversity in the oceans and how it differed from that found from terrestrial sources,” said Bugni. Sea squirts have a lot of interesting chemistry. “What people have found is that the chemistry there is actually produced by symbiotic microorganisms within the whole animal.”

Bacteria associated with sea squirts produce turbinmicin, a new antifungal that may help treat multidrug resistant fungal infections in humans.

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The team collected samples of brightly colored sea squirts off the coast of the Florida Keys, from which they profiled more than 1,400 bacterial isolates (4). One bright orange bacterial sample, a Micromonospora species, produced a molecule that looked like it had some unique chemistry. The team named it turbinmicin.

When Bugni and Andes’ team assessed turbinmicin’s activity against a barrage of 39 multidrug-resistant human fungal pathogens isolated from patients, even low concentrations of turbinmicin inhibited the virulent fungi. For example, a 0.25 ug/mL dose of turbinmicin inhibited visible growth of multidrug-resistant Candida auris, while inhibiting the same amount of growth with the established antifungal fluconazole required more than 256 mg/mL.

The researchers found no toxicity when they delivered turbinmicin every six hours to a mouse model of invasive C. auris infection, and the treatment reduced the fungal burden by 3.6 log₁₀, a significant improvement over untreated control mice and mice treated with the standard of care, micafungin. Turbinmicin also inhibited a drug resistant strain of Aspergillus fumigatus, which has a greater than 50% mortality rate in infected humans.

To investigate how exactly turbinmicin defeated these drug resistant fungi, the researchers screened yeast knockout and knockdown libraries treated with turbinmicin and identified the gene SEC14, which encodes a protein involved in vesicle trafficking, presenting a novel antifungal target.

In a prior study, Andes and Bugni’s team demonstrated that vesicle trafficking delivers components of the extracellular matrix to help the fungus maintain itself in a drug resistant biofilm (5). 

“Biofilms are just communities of organisms that grow attached to a surface,” Andes explained, but “when these pathogens grow in biofilms, they're typically much more resistant to the therapies we have available.”

By disrupting vesicle trafficking, turbinmicin inhibited the formation of Candida biofilms and sensitized the previously drug resistant biofilms to available antifungal drugs (6).

“We had hypothesized that it would have some effect because it was inhibiting the vesicle pathway, but given the kind of poor track record of other anti-infectives against biofilms, we were pleasantly surprised to see it had that profound effect,” Andes said.

With turbinmicin’s apparent safety and effectiveness against multidrug resistant fungi, Bugni and Andes are excited to see where turbinmicin goes from here. They are planning additional studies to refine turbinmicin’s exact fungal target and to test its safety in mammals.

“The further we push the molecule, it keeps getting more exciting,” said Bugni.

Buzz on by: Insect-derived antifungals

Currie has collected everything from butterflies to bees to fungus-growing ants around the world over the past ten years. These specimens aid his studies of the molecular ecology between microbes and their insect hosts, and their microbiomes produce molecules with potential as antimicrobials.

“We'll use an approach of saying, ‘Okay, what insects might we expect to be under more disease pressure?’ And we’ll look at those,” Currie said. In addition to probing the guts of these insects for interesting microbes, Currie’s team also takes “isolations from the surface of the insect, especially if they're from an interesting niche, like termites that live inside a nest or paper wasps.” 

Researchers in Currie’s lab collect insects in interesting ecological niches to find associated microbes that produce new molecules with antifungal potential.

Credit: Caitlin Carlson

In a recent study, Currie and his team screened Streptomyces bacteria isolated from the microbiomes of 1445 insects for their antifungal potential (7). They found that many of the Streptomyces species seemed to have evolved to uniquely associate with insects.

“It's really viewed as a soil organism, and finding a whole bunch of novel evolutionary space associated with insect hosts is pretty cool and exciting. And [it] suggests that some of these associations are really ancient,” Currie said.

This unique evolutionary association suggests that these bacterial species evolved to produce new compounds with antimicrobial potential. In fact, the researchers found that the majority of the insect-associated Streptomyces species had at least one unique biosynthetic gene cluster, which is a region of the microbial genome that contains genes that work together to synthesize a particular metabolite. Many of these metabolites have antimicrobial properties.

Cameron Currie investigates insect microbiomes in search of new antifungal molecules.

Credit: Cameron Currie

With further investigation, the team found that compounds isolated from the insect-associated Streptomyces species inhibited fungal pathogens in vitro and in an in vivo C. albicans-infected mouse model. One molecule isolated from Streptomyces in the gut of a fungus-eating ant stood out. Currie’s team named this molecule cyphomycin.

Cyphomycin inhibited drug-resistant fungal pathogens such as A. fumigatus and C. auris in vitro, and in an in vivo clinical mouse model of C. auris infection, cyphomycin treatment reduced the fungal burden in mouse kidneys.

Currie plans to further characterize cyphomycin in a mouse model, and he hopes to work with industry partners to develop cyphomycin into a potential antifungal drug.

The potential of insect-associated microbiomes for new antifungals is plentiful, and Currie is sure that these bacteria produce additional exciting molecules.

“One of the estimates of the number of strains you have to screen of soil streptomyces to get a new antimicrobial is a million strains,” he said, “and we’re getting new molecules for screening with several dozen to a couple dozen strains.”

It’s an armadillo! It’s an opossum! It’s roadkill?

Neither Robert Cichewicz, a natural product chemist at the University of Oklahoma, nor Bradley Stevenson, a microbial ecologist previously at the University of Oklahoma, who is now at Northwestern University, can remember who exactly came up with the idea to sample the gut microbiomes of roadkill  in search of novel antifungals (8). But it was absolutely their favorite research proposal to write.

Because mammalian microbiomes are home to many species of bacteria, Robert Cichewicz (pictured) and Brad Stevenson reasoned that they likely produce molecules with unique chemistry that may work as effective antifungals for humans.

Credit: Robert Cichewicz

“I wish I could have been in the review session to hear the discussion,” laughed Stevenson.

Cichewicz and Stevenson reasoned that the gut microbiome would be ripe with unique antimicrobial molecules that might be useful in the clinic. “The premise that we are operating on is that you need to have a diverse community that is actively competing for something,” Stevenson said.

They first considered looking in the human microbiome for antimicrobials, but with the many administrative hurdles involved in using human samples for research, they shifted their focus to mammals more broadly.

They considered farm animals, animals from the zoo, and then, Stevenson said, “We thought, well, there’s roadkill on highway nine, just south of campus every day, and I bet you that there’s not much paperwork involved in sampling.”

Collecting roadkill is not a new concept in scientific research. Normally, it goes by the more academic sounding phrase, “opportunistic sampling,” and it mainly comes up in wildlife surveys to determine how many animals live in a particular area.

For Stevenson and Cichewicz, the 48 km section of Oklahoma State Highway 9 just south of the University of Oklahoma was perfect. With a speed limit of 50 miles per hour and transitioning from residential suburbs, ranches, unmanaged land, and finally to the university campus, it was “the perfect recipe for disaster for the animals,” Stevenson added.

In the evening after work and in the morning as they came into the lab, Stevenson and Cichewicz would go out to the highway with their students to sample from whatever unlucky animals had met their fate in the past ten hours.

“Anything that wasn't there the day before was considered fresh, and we would stop and sample it, providing that at least one end of it was largely intact because we were taking mouth and gut samples,” said Stevenson.

Sampling roadkill from a busy highway is not for the faint of heart. “We were preoccupied with traffic, getting the right sample, [and] the smell if it was a skunk. Robert was covered in deer ticks at one point,” said Stevenson.

Bacteria isolated from the ear of an opossum inhibited the formation of a drug-resistant C. albicans  fungal biofilm.

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“Oh yeah, that was unpleasant. I didn’t know they moved that fast,” Cichewicz laughed. Another challenge, he added, was that “one of our main sources of competition was people basically grabbing roadkill before we could get to it. By the time you would get to the end of your route and circle back, it's like seriously, someone took that too?”

Despite the smell, traffic, and ticks, the team identified multiple molecules with antifungal effects. They found that Pseudomonas and Serratia bacterial species isolated from an opossum’s ear inhibited drug-resistant C. albicans biofilm formation.

While the team has not yet pursued further studies of these molecules, they are excited by the potential that animal microbiomes hold. For example, Cichewicz suggested that sampling roadkill at different times during the year might lead to an entirely different swath of antimicrobial molecules because of the differences in food availability as the seasons change.

“I mean, it is sad,” Stevenson said about sampling from roadkill. “We hope their memories live on by the natural products that we've discovered and the science they inspired.”

Whether it’s sea squirts in Florida, ants in South America, or an opossum in Oklahoma, animal microbiomes may house the antifungal-producing bacteria needed to treat multidrug-resistant fungal infections in humans. 

“It's an exciting area that's full of potential,” said Cichewicz. “I don't think it's even close to being understood or tapped as a resource.”

References

1. Fernandes, C.M. et al. The Future of Antifungal Drug Therapy: Novel Compounds and Targets. Antimicrob Agents Chemother 65, e01719-20 (2021).
2. Carolus, H. et al. Amphotericin B and Other Polyenes—Discovery, Clinical Use, Mode of Action and Drug Resistance. J. Fungi 6, 321 (2020).
3. Fjærvik, E., Zotchev, S.B. Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei. Appl Microbiol Biotechnol 67, 436-443 (2005).
4. Zhang, F. et al. A marine microbiome antifungal targets urgent-threat drug-resistant fungi. Science 370, 974-978 (2020).
5. Zarnowski, R. et al. Candida albicans biofilm–induced vesicles confer drug resistance through matrix biogenesis. PLoS Biol 16, e2006872 (2018).
6. Zhao, M. et al. Turbinmicin inhibits Candida biofilm growth by disrupting fungal vesicle–mediated trafficking. J Clin Invest. 131, e145123 (2021).
7. Chevrette, M.G. et al. The antimicrobial potential of Streptomyces from insect microbiomes. Nat Commun 10, 516 (2019).
8. Motley, J.L. et al. Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Nonanthropoidal Mammalian Microbiomes. J. Nat. Prod. 80, 598-608 (2017).