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

ABSTRACT: Acinetobacter baumannii and Klebsiella pneumoniae 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 Pseudomonas aeruginosa, in turn exhibits broad-spectrum PPK inhibition in other priority pathogens. Gallein treatment successfully attenuated virulence factors of K. pneumoniae and A. baumannii including biofilm formation, surface associated motility, and offered protection against A. baumannii challenge in a Caenorhabditis elegans model of infection. This was highlighted most importantly in the critically understudied A. baumannii, where gallein treatment phenocopied a ppk1 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.

SUBMITTER: Roberge N

PROVIDER: S-EPMC8576328 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5-15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ?galU, ?galE, ?rfaI, or ?rfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.

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

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