Polyphosphate Metabolism and Pyoverdine Production Modulate Zebrafish Survival Following Larval Immersion Exposure to Pseudomonas aeruginosa

Bacterial metabolism is increasingly recognized as a central determinant of bacterial virulence, linking the physiological state of the pathogen to its ability to survive and damage the host. Understanding how bacterial metabolism shapes virulence requires models that are both biologically informative and experimentally accessible. Here, we used zebrafish (Danio rerio) larvae for static immersion assays as a simple model to study Pseudomonas aeruginosa host–pathogen interactions and virulence regulation. Using this approach, we show that disruption of polyphosphate (polyP) synthesis by loss of ppk1 gene (Δppk1) leads to marked attenuation of virulence, whereas interrupting ppk2 gene (Δppk2) results in increased mortality in zebrafish larvae. Phenotypic assays and quantitative proteomics revealed that loss of polyP impairs siderophore production, with minimal effects on other canonical virulence factors. Consistently, a pyoverdine-deficient mutant (ΔpvdF) exhibited reduced virulence in zebrafish, validating the capacity of the zebrafish model to functionally resolve key virulence factors. Together, our results highlight polyphosphate metabolism as a central regulator of P. aeruginosa virulence and position zebrafish immersion assays as an efficient and ethically aligned alternative to mammalian models for studying bacterial pathogenesis.