Izabela Ciastoń

Prototheca , a genus of unpigmented yeast-like microscopic algae, is an emerging pathogen of humans and animals. Treatment of Prototheca infections is often unsuccessful because the unusual algal biology of these pathogens limits the availability of effective therapeutics. Here, we screened the Pandemic Response Box, a library of 400 compounds with potential anti-infective activity that are either clinically approved or in preclinical development, and identified five promising compounds with anti- Prototheca activity: tafenoquine, OSU-03012, alexidine, ciclopirox, and eberconazole. Characterized alongside amphotericin B, which is currently often used for treatment, these identified compounds displayed potent activity against multiple Prototheca strains and species by inhibiting growth, killing the algae, and disrupting biofilms. Their diverse mechanisms of action included effects on cell walls and membranes, mitochondrial dysfunction, ROS overproduction, metabolic alterations, and induction of apoptosis. Most of the identified compounds were also effective against in vivo Prototheca infection in larval zebrafish, highlighting their translational potential and the possibility of future introduction into clinical practice. IMPORTANCE: This study addresses the emerging threat of infections caused by Prototheca , a group of unusual algae that can cause difficult-to-treat infections in both humans and animals. Current treatment options are limited and often ineffective, highlighting the need to develop new therapeutic strategies, especially as reported cases of Prototheca infections quickly increase. Here, we identified several compounds with strong activity against Prototheca . These agents not only blocked growth but also killed the microorganisms, damaged their biofilms, and reduced infection in an animal model. Importantly, many of the identified compounds are already used clinically for other indications, which raises the possibility of rapid repurposing against Prototheca . Moreover, we identified multiple mechanisms responsible for these compounds’ activity, providing a framework for the future development of even more potent anti- Prototheca therapies.

Unpigmented, yeast-like Prototheca algae are emerging pathogens responsible for unusual infections in humans and animals. Their treatment is often unsuccessful, as traditional antifungals and antibacterials show unsatisfactory activity against Prototheca . Therefore, the development of improved drugs that consider the peculiarities of algal biology is needed. Here, we describe a novel anti- Prototheca compound, the 2-methoxy-1,4-naphthoquinone, identified among molecules known to mediate plant and algal allelopathy. The 2-methoxy-1,4-naphthoquinone was highly active in vitro against a range of Prototheca isolates from different species, both planktonically and in biofilms. Its mode of action included the induction of toxic reactive oxygen species, and it targeted energy generation in the pathogen’s mitochondria. It effectively treated Prototheca bovis skin infection in a mouse model, demonstrating translational potential. Altogether, this study not only identified a new effective anti- Prototheca compound, but also identified 1,4-naphthoquinone backbone as a lead structure for further therapeutic development and demonstrated the feasibility of exploring known anti-plant compounds as a source of anti- Prototheca treatments.

Neutrophil extracellular traps (NETs) are associated with diseases linked to aberrant coagulation. The blood clotting cascade involves a series of proteases, some of which induce NET formation via a yet unknown mechanism. We hypothesized that this formation involves signaling via a factor Xa (FXa) activation of the protease-activated receptor 2 (PAR2). Our findings revealed that NETs can be triggered in vitro by enzymatically active proteases and PAR2 agonists. Intravital microscopy of the liver vasculature revealed that both FXa infusion and activation of endogenous FX promoted NET formation, effects that were prevented by the FXa inhibitor, apixaban. Unlike classical NETs, these protease-induced NETs lacked bactericidal activity and their proteomic signature indicates their role in inflammatory disorders, including autoimmune diseases and carcinogenesis. Our findings suggest a novel mechanism of NET formation under aseptic conditions, potentially contributing to a self-amplifying clotting and NET formation cycle. This mechanism may underlie the pathogenesis of disseminated intravascular coagulation and other aseptic conditions.