Antimicrobial activity of iron-depriving pyoverdines against human opportunistic pathogens

Reviewer #1 (Public Review):

Summary:
In an era of increasing antibiotic resistance, there is a pressing need for the development of novel sustainable therapies to tackle problematic pathogens. In this study, the authors hypothesize that pyoverdines - metal-chelating compounds produced by fluorescent pseudomonads - can act as antibacterials by locking away iron, thereby arresting pathogen growth. Using biochemical, growth, and virulence assays on 12 opportunistic pathogens strains, the authors demonstrate that pyoverdines induce iron starvation, but this effect was highly context-dependent. This same effect has been demonstrated for plant pathogens, but not for human opportunistic pathogens exposed to natural siderophores. Only those pathogens lacking (1) a matching receptor to take up pyoverdine-bound iron and/or (2) the ability to produce strong iron chelators themselves experienced strong growth arrest. This would suggest that pyoverdines might not be effective against all pathogens, thereby potentially limiting the utility of pyoverdines as global antibacterials.

Strengths:
The work addresses an important and timely question - can pyoverdines be used as an alternative strategy to deal with opportunistic pathogens? In general, the work is well conducted with rigorous biochemical, growth, and virulence assays. The work is clearly written and the findings are supported by high-quality figures.

Weaknesses:
I do not think there are any 'weaknesses' as such. However, it is well known that siderophore production is highly plastic, typically being upregulated in response to metal limitation (as well as toxic metal stress). Did the authors quantify whether pyoverdine supplementation altered siderophore production in the focal pathogens (either through phenotypic assays / transcriptomics)? Could such a phenotypic plastic response result in an increased capacity to scavenge iron from the environment? Importantly, increased expression of siderophores has been shown to enhance pathogen virulence (e.g. Lear et al 2023: increased pyoverdine production is linked with increased virulence in Pseudomonas aeruginosa). I really appreciate the amount of work the authors have put into this study, but I would suggest expanding the discussion a bit to include a few sentences on (1) unintentional consequences of pyoverdine treatment (e.g. changes in gene expression and non-siderophore-related mutations (e.g. biofilm formation)) on disease dynamics/pathogen virulence , and (2) the efficacy of siderophore treatment under more natural conditions, i.e. when the pathogens have to compete with other species in the resident community (i.e. any other effects than resistance evolution through HGT of pyoverdine receptors as mentioned).

Reviewer #2 (Public Review):

In this work, Vollenweider et al. examine the effectiveness of using natural products, specifically molecules that chelate iron, to treat infectious agents. Through the purification of 320 environmental isolates, 25 potential candidates were identified from natural products based on inhibition assays and were further screened. The structural information and chemical composition were determined.

The paper is well-structured and thorough; targeting virulence factors in this manner is a great idea. My enthusiasm is dampened by the mediocre effects of the compounds. The lack of a dose-response curve in the survivability assays suggests a limited scope for these molecules. While it is encouraging that the best survivability occurred at the lowest toxicity level, it opens questions as to how effective such molecules can be. Either the reduction in mortality was offset by using higher concentrations, which was not observed in the compound-alone test, or there is no dose-response curve. The latter would suggest to me that the variation in survivability is not due to the addition of siderophores.

I would also like to see how these molecules compare to other iron-chelating molecules. Desferoxamine is a bacteria-derived siderophore that is FDA-approved. However, it is not used to treat infections. Would the author consider comparing their candidate molecules to well-studied molecules? This also raises questions about the novelty of this work; I think the authors could rephrase the discussion to better reflect that bioprospecting for iron-chelating molecules has previously occurred and been successful.

Finally, I am concerned about the few mutations reported in the resistance study. Looking at the SI, it appears that very few mutations were seen. It is unclear what filtering the authors used to arrive at such a low number of mutations. Even filtering against mutations that were selected by adaptation to the media, it seems low that only a handful of clones had distinct mutations.

This paper has a lot of strengths. The workflow is logical and well-executed; the only significant weakness is the effect of the molecules and the lack of an explanation for a dose-response curve in the survivability assay, especially when compared to the data reported in Figure 3. As the authors describe in lines 214-217.