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.

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence

“…Gallein also synergized with the antibiotics tetracycline and ciprofloxacin to improve C. elegans survival following P. aeruginosa infection [ 57 , 82 ]. In a follow-up study, we also demonstrated that gallein inhibits A. baumannii and K. pneumoniae PPK2s in vitro [ 42 ] ( Table 2 ).…”

“…ppk2 genes have been identified in many species of both Gram-positive and Gram-negative bacteria [ 35 ], with several species encoding multiple distinct isoforms [ 35 , 39 , 40 ] ( Table 1 ). For example, P. aeruginosa possesses three distinct PPK2s [ 39 ], whereas other pathogenic bacteria such as Mycobacterium tuberculosis [ 41 ] or Klebsiella pneumoniae [ 42 ] possess only a single PPK2 isoform. In a more extreme instance, the environmental bacterium Ralstonia eutropha contains five distinct PPK2 enzymes, further illustrating the genetic abundance and variability among PPK2s [ 40 ] ( Table 1 ).…”

“…In a more extreme instance, the environmental bacterium Ralstonia eutropha contains five distinct PPK2 enzymes, further illustrating the genetic abundance and variability among PPK2s [ 40 ] ( Table 1 ). By contrast, bacteria rarely encode more than one ppk1 allele [ 35 , 40 , 42 ].…”

“…As a result, our understanding of polyP dynamics in species other than E. coli has suffered. For example, in Acinetobacter baumannii —which is one of the few species known to encode multiple ppk1 genes in addition to ppk2 —knockout of individual ppk1 s had differing consequences on virulence [ 42 ]. P. aeruginosa possesses three PPK2s, only two of which, however, can synthesize polyP in vivo to compensate for loss of PPK1 [ 39 , 57 ].…”

“…These studies challenge the validity of generalizing certain aspects of E. coli polyP biology to organisms that possess PPK2s, since these enzymes may play species-specific niche roles. Furthermore, the presence of PPK2s in many of the current priority bacterial pathogens such as P. aeruginosa and A. baumannii highlights the importance of understanding PPK2 biology in the fight against bacterial infections ( Table 1 ) [ 42 , 57 ].…”