Whether on a plane ride back from a conference or within an overlooked fungal species, sometimes the answer just presents itself. This kind of serendipity led University of Oklahoma chemist Robert Cichewicz to devise a method for procuring leaf samples at an unprecedented rate. The novel system, in turn, guided the discovery of an antifungal compound. His findings may pave the way for a new treatment to fight fungal infections and speed up the discovery of potential drug candidates (1).

The study stemmed from a broader exploration of endophytes, a type of fungi that engages in symbiotic relationships with plants. Natural products are often a core driving force for the development of such relationships. Cichewicz’s team suspected that these products might also protect both endophytes and their host plants from various infections. However, studies of endophytes as a source for potential drug compounds are scarce (2). “Every plant that's ever been looked at has at least dozens of endophytes,” said Cichewicz, who therefore thought that these fungi were ripe for exploration. 

However, the time-intensive task of preparing a single sample proved a major bottleneck to his team’s research. “You’ve got to cut, sterilize, cut, sterilize, because you don't want to cross contaminate,” said Cichewicz. He was commiserating over this problem with a colleague at an American Chemical Society meeting when he decided to look for solutions. “On the plane ride back, believe it or not — I don’t know how many thousands of feet we were in the air — it just dawned on me,” he recounted. “Lasers! . . . How could we adapt a laser in order to carry out this stuff?”

The team soon created the Fast Laser-Enabled Endophyte Trapper (FLEET) system, in which they fit a commercially-available laser into a clean box with a HEPA filter and germicidal UV light. Researchers place the leaves on top of microplates, and the laser cuts them into small squares that fall directly into individual wells where they are ready for immediate culturing. The system fit up to six plates, enabling the preparation of 600 samples within ten minutes in a process that is ten times faster than doing it by hand. “It's a game-changer,” said Cichewicz.

Among all of the samples they obtained with FLEET, one pomegranate-colored fungus stood out. “As fungi grow on the plate, their hyphae start to interweave and eventually you just get this mat [that] looks like a tapestry,” said Cichewicz. Yet one leaf sample held a dark red dot within a clear zone that no other fungi grew into. “It was really cool looking,” he said. 

While the clear zone suggested some type of antifungal activity, sequencing results revealed that the fungus was a species of Elsinöe. Elsinöe is known to produce highly pigmented molecules, but there was no record of it containing antifungal metabolites.

Cichewicz and his colleagues isolated the red fungus and traced its antifungal activity back to a single metabolite. They probed the metabolite’s structure and discovered that it was a member of the aureobasidin family, which contain cyclic peptides with antifungal properties (3). The new peptide compound also contained a previously unknown amino acid structure. “That was pretty exciting,” said Cichewicz. “Where else do you get to discover new stuff on Earth these days?”  

The team named the metabolite persephacin and the amino acid persephanine after the Greek goddess Persephone, who is often symbolized by a pomegranate.

Besides its novelty as an amino acid, the persephanine residue’s location within the larger persephacin molecule holds potential clinical significance (4). “There was some literature from Japan, specifically some patents where they had suggested that if you start modifying this portion of the aureobasidin, you could expand the scope of fungi that this molecule worked against,” said Cichewicz (5). “We have found this new part here that seems to mimic what these medicinal chemists obviously worked on for hundreds and hundreds of hours to prepare. Here’s this little humble fungus making it for us in the lab.”

Finally, the team tested persephacin in in vitro models for fungal infections. The new molecule was effective against a broad range of pathogenic fungi, performing as well as or better than existing antifungal treatments. “That’s a wonderful sign,” said Cichewicz. “[Persephacin] has got all the hallmarks of the kind of stuff you want to see for a potential drug lead.” 

Martin Hoenigl, a clinical mycologist at the Medical University of Graz who was not involved in the study, cautioned against excessive optimism. “This looks great, in terms of [effective] concentrations and so on, but it’s only one small step,” he said. Hoenigl has seen plenty of antifungal drug candidates fail to make it through clinical trials; no new antifungal compounds have gained approval for use in more than 20 years. “The bar is just very high,” he said.  

Both Hoenigl and Cichewicz noted that fungal infections remain a major global health concern. “With climate change, disasters, et cetera, there’s just a growing population at risk,” said Hoenigl. “We need to be getting ready,” Cichewicz said.

While Cichewicz’s team collects more potential antifungal samples with their FLEET system, they also plan to investigate percephacin metabolism and toxicity in animal models. “My biggest hope is that it’s going to bring a big spotlight back to the aureobasidin family of molecules,” said Cichewicz. “Persephacin and its analogues have a lot of promise that needs to be explored.”

References

1. Du, L. et al. Persephacin Is a Broad-Spectrum Antifungal Aureobasidin Metabolite That Overcomes Intrinsic Resistance in Aspergillus fumigatus. J Nat Prod  86, 1980-1993. (2023)
2. Strobel, G. The Emergence of Endophytic Microbes and Their Biological Promise. J Fungi  4, 57 (2018).
3. Takesako, K. et al. Biological properties of aureobasidin A, a cyclic depsipeptide antifungal antibiotic. J Antibiot  46, 1414-20 (1993).
4. Wuts, P. G. M. et al. Generation of Broad-Spectrum Antifungal Drug Candidates from the Natural Product Compound Aureobasidin A. ACS Med Chem Lett  6, 645-9 (2015).
5. Hashida-Okado, T. et al. AUR1 novel gene conferring aureobasidin resistance on Saccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells. Mol Gen Genet  251, 236-244 (1996).