Ortiz-Severin, J. P.; Lecaros, C.; Perez, I.; Varas, M.; Allende, M. L.; Chavez, F.

Understanding how bacterial metabolism shapes virulence requires infection models that are both biologically informative and experimentally accessible. Here, we used zebrafish (Danio rerio) larvae infection by static immersion as a simple and robust vertebrate model to study Pseudomonas aeruginosa host-pathogen interactions and virulence regulation. Using this approach, we show that disruption of polyphosphate synthesis by loss of ppk1 gene ({Delta}ppk1) leads to marked attenuation of virulence, whereas interrupting ppk2 gene ({Delta}ppk2) results in a hypervirulent phenotype. Phenotypic assays and quantitative proteomics revealed that loss of polyP selectively impairs pyoverdine production, with minimal effects on other canonical virulence factors. Consistently, a pyoverdine-deficient mutant ({Delta}pvdF) exhibited reduced virulence in zebrafish, validating the model's capacity to functionally resolve key pathogenic determinants. 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.