Pilar and the Petri dishes

Published on 03/10/2022

It is the fungal models that first catch our eye as we walk with Pilar Junier in the research wing of the Microbiology Lab at the University of Neuchâtel. On this sunny spring morning, about a dozen of these wondrous specimens stand in the window niches of the modern building high above Lake Neuchâtel, which offer a spectacular view of the Alps.

Poisonous and edible species are clearly labeled, and we quickly realize the danger behind the seemingly harmless common potato puffball and the fiery toadstool.

Even though the diverse colors and shapes of the mushrooms appear quite exotic on the bare concrete of the window niches, they are ubiquitous in nature and thus an obvious and central object for Junier and her 30-member laboratory and research team.

In the labs, the professor also shows us the numerous Petri dishes and analytical equipment used to study the interaction of fungi and other microbial partners within the environment. The doors are usually open, and the hustle and bustle of the graduate and Ph.D. students in the corridors and laboratories seems like a microcosm of its own within the university, intensely and very intently dedicated to a mysterious task.

This microcosm clearly revolves around 44-year-old Pilar Junier: A quick glance into one of her 12 research labs is enough for her to give the people working in there the necessary instructions in exactly the language they understand best – French, English, German or even in Spanish, her mother tongue.

Professor and more

Pilar Junier was born in Colombia, where she also studied. The country lies on the Pacific Ring of Fire, and six of the country’s ten volcanoes are still considered active. Junier obviously brought some of this energy from her homeland, because it would otherwise be hard to explain how she is able to reconcile her diverse tasks.

As a professor, she heads the laboratory of microbiology at the University of Neuchâtel, where she also lectures, mentors Ph.D. and Master’s students and trains biology lab technicians. In addition, she develops new projects and ideas and raises funds to keep the labs running. Then, if there is still time, she works on turning the dream of owning her own company into a reality.

With the Novartis Foundation’s FreeNovation project, launched in 2016 to promote innovative ideas in medicine, Junier was able to complete several items on her to-do list. But how does an environmental microbiologist who has never been involved in medicine in her career get a grant from a medical foundation?

A crazy idea

To apply for FreeNovation, Junier did not have to submit any patient- related data that supported her idea. Nor would that have been possible at the time, she tells us candidly. Another positive aspect was that the decision in the selection process was made by a jury who quite deliberately was not given any insight into Junier’s CV, but only had to judge the quality of the submitted idea. “That is why they also allowed an environmental microbiologist to do a medical project. It’s not like I had a background in pulmonology or anything. But I just had this crazy idea to work with people,” Junier recalls.

The “crazy idea” did not come to Pilar Junier and her team in a dream, however, but because of their many years of research in environmental microbiology. Junier, along with a postdoctoral researcher and a doctoral student, Fabio Palmieri, 30, and Aislinn Estoppey, 29, had already conducted numerous projects with soil samples. Their main interest was the interaction between oxalic acid, fungi and bacteria – a traditional approach in the field of environmental microbiology that is also being studied, for example, to improve soil fertility or to store CO2 in the soil.

“The basic question in our FreeNovation application was: Why do we not look at people as if we were looking at our soil samples, as something that is just a bit more complex than human cells?” says Junier, and immediately provides a concrete task to go with it: “Every year, around 1.5 million people die from fungal diseases, and current treatment methods are very limited. That is why we wanted to use the knowledge we have gained about fungi over the years and investigate whether and how we could use it as a therapeutic alternative to control pathogenic fungi in human medicine.”

Two sides of the same substance

In their previous experiments, Junier and her team had found that oxalic acid is a critical environmental factor in the interaction of two fundamental elements of the human microbiome: bacteria and fungi. In environmental microbiology, for example, the secretion of oxalic acid by fungi is not only common, but even essential for their survival.

In humans, however, the salts of oxalic acid in particular, such as calcium oxalate, can cause health problems. Crystals of calcium oxalate have also been found in the lungs of immunocompromised patients suffering from mold infections. However, inhaling fungal spores alone is not a cause for concern, as Junier explains. “Everyone breathes in thousands of spores every day, but in healthy individuals, this does not trigger any problems. In immunocompromised patients, on the other hand, this can lead to colonization of the lung tissue with spores and eventually to fungal infections.”

On this basis, Junier and her team formulated their hypothesis for the FreeNovation project, which states that fungi also produce oxalic acid in human lungs to create an optimal environment for their further development.

Another finding of the research group even had the potential to contribute significantly to the successful development of an alternative treatment method for fungal diseases. In previous studies of oxalates in soil samples, Junier and her team were able to identify a bacterial metabolism that explains the consumption of oxalates.

The crucial question now was whether oxalate-degrading bacteria could limit the growth potential of pathogenic fungi, which use these salts as food. To test their assumptions, Junier and her team chose the widespread black mold (Aspergillus niger) as the pathogenic fungus and the garden soil isolate Cupriavidus oxalaticus as the oxalate-consuming bacterium.

Thus, the innovative treatment approach, which they developed as part of their FreeNovation project, was to modify the patient “environment” in which the pathogen thrives, rather than directly targeting the pathogen.

Petri dishes and numbers

To test this hypothesis, the mold was first cultured in a medium used to grow human lung cells. The results were consistently in line with the research team’s expectations. In those Petri dishes that contained the oxalate-consuming bacterium as well as the fungus, the pH remained at the physiological level of 7.5, the concentration of oxalic acid was reduced by about 90 percent, and the growth of the fungus was significantly restricted by the bacterium.

“All other samples containing only the mold and the medium showed a yellow discoloration and a change in pH to 4.5, so the fungus was producing oxalic acid to support its own growth,” Junier said, describing the observations and thus confirming her hypothesis.

But what succeeds in the Petri dish can often enough fail in the far more complex “real world.” Therefore, the research team decided to verify these results with real human lung cells, which was absolutely necessary for another important step towards medical application in patients. However, there was no suitable partner for this research in Switzerland.

International collaboration

For this next stage, Ph.D. student Fabio Palmieri had to travel to the USA in 2019, where he conducted experiments on bronchial lung tissue together with a team from the Los Alamos National Laboratory in New Mexico. Again, the experiments were conducted both with and without the oxalate-consuming bacterium, and the results were measured and assessed using three parameters: pH, concentration of calcium, and concentration of oxalic acid.

“What we saw in these experiments actually exceeded our hopes and expectations,” reports Palmieri. “Where we used only the mold on the lung tissue, both the pH and calcium levels, as well as the free oxalic acid content, were lower than in the samples with the bacterium. We were even able to detect crystals of calcium oxalate on the bronchial cells with the mold, while this was not the case in the presence of the bacterium.” Moreover, these experiments also showed the aggressiveness of the mold, which completely destroyed the bronchial tissue.

The liberated look

Pilar Junier and her team are very pleased with what they have achieved in the FreeNovation project. “We started with a crazy idea and an unusual approach, and now we have tangible evidence that we are not quite as crazy as it might have seemed when we started,” Pilar Junier says in retrospect, “because our results clearly show that the use of bacteria has a measurable impact on the course of a fungal infection, even in human lungs.”

But there is still a long way to go before a new, innovative therapy based on their basic research can be introduced, and the three researchers on the core team, Junier, Palmieri and Estoppey, are absolutely aware of that. For example, the results obtained so far still need to be validated in an in vivo mouse model. But their work still provides the foundations and exciting prospects for further projects focusing on the isolation and characterization of novel oxalotrophic bacteria from the lung microbiome.

As we say goodbye to Pilar Junier and her team, we step out of the former prison building that now forms the middle section between the two modern additions to the university building high above the old town of Neuchâtel. It is a bit like being in a movie, because the view of Lake Neuchâtel and the still snow-covered peaks of the Alps is breathtakingly beautiful. And it is like so often with FreeNovation projects: The step out of the prison of traditional approaches to research opens up unique perspectives!