Broad-Spectrum Inhibitor of Bacterial Polyphosphate Homeostasis Attenuates Virulence Factors and Helps Reveal Novel Physiology of Klebsiella pneumoniae and Acinetobacter baumannii

Acinetobacter baumannii and <i>Klebsiella pneumoniae</i> currently rank amongst the most antibiotic-resistant pathogens, responsible for millions of infections each year. In the wake of this crisis, anti-virulence therapeutics targeting bacterial polyphosphate (polyP) homeostasis have been lauded as an attractive alternative to traditional antibiotics. In this work, we show that the small molecule gallein, a known G-protein βγ subunit modulator, also recently proven to have dual-specificity polyphosphate kinase (PPK) inhibition in <i>Pseudomonas aeruginosa</i>, in turn exhibits broad-spectrum PPK inhibition in other priority pathogens. Gallein treatment successfully attenuated virulence factors of <i>K. pneumoniae</i> and <i>A. baumannii</i> including biofilm formation, surface associated motility, and offered protection against <i>A. baumannii</i> challenge in a <i>Caenorhabditis elegans</i> model of infection. This was highlighted most importantly in the critically understudied <i>A. baumannii</i>, where gallein treatment phenocopied a <i>ppk1</i> knockout strain of a previously uncharacterized PPK1. Subsequent analysis revealed a unique instance of two functionally and phenotypically distinct PPK1 isoforms encoded by a single bacterium. Finally, gallein was administered to a defined microbial community comprising over 30 commensal species of the human gut microbiome, demonstrating the non-disruptive properties characteristic of anti-virulence treatments as microbial biodiversity was not adversely influenced. Together, these results emphasize that gallein is a promising avenue for the development of broad-spectrum anti-virulence therapeutics.