Lysine polyphosphate modifications contribute to virulence factors in Pseudomonas aeruginosa

Abstract

Inorganic polyphosphate (polyP) is a universally conserved polymer involved in various biological processes, but its role as a direct protein regulator remains largely unexplored. Lysine polyphosphate modification (KPM), a strong but non-covalent interaction between polyP and lysine-rich protein sequences, has not been functionally characterized until now. In this study, we present the first investigation into KPM's biological significance using Pseudomonas aeruginosa, a critical priority pathogen known for its antibiotic resistance and virulence. We identified two essential bacterial proteins, EngA and SrmB, as novel KPM targets. Through site-specific lysine deletions, we demonstrated that disrupting lysine-polyP interactions severely impairs biofilm formation and significantly reduces the production of key virulence factors, including pyoverdine and pyocyanin. These findings establish a direct functional link between polyP and bacterial pathogenicity mediated by KPM. Our results highlight KPM as a previously unrecognized regulatory mechanism critical for controlling bacterial virulence factors. This work uncovers the first functional role of KPM and its importance in regulating virulence phenotypes in a major human pathogen.IMPORTANCEPolyphosphate is commonly known for its roles in metabolism and stress response. How inorganic polyphosphate (polyP) facilitates bacterial virulence has remained largely elusive. This study reveals that lysine polyphosphate modification (KPM), a chemical interaction between polyP and lysine-rich proteins, is essential for bacterial survival and pathogenicity in P. aeruginosa, a harmful microbe responsible for difficult-to-treat infections. We discovered that disrupting KPM in key proteins impairs the bacteria's ability to form protective biofilms and produce harmful toxins. This previously unknown biological process links polyP to protein function in controlling bacterial virulence factors. Our findings open new possibilities for developing anti-virulence therapies aimed at reducing bacterial infections without promoting antibiotic resistance.

Keywords: EngA and SrmB; Pseudomonas aeruginosa virulence; biofilm formation; lysine polyphosphate modification; lysine-rich motif.

Fig 1

Screening of selected KPM targets in P. aeruginosa. (A) NuPAGE analysis of poly-lysine P. aeruginosa targets recombinantly expressed in BL21 RIPL E. coli cells in a custom MBP-fusion tag vector, HT25 (N-MBP-Protein). (B) NuPAGE analysis of C-terminal truncations of SrmB and EngA MBP fusions lacking their respective lysine-rich regions following in vitro polyP treatment as in A. SrmB ∆K consists of residues 1-386 (residues 387-446 deleted), and EngA ∆K consists of residues 1-472 (residues 473-493 deleted). (C) List of top 12 protein candidates that were identified and ranked based on total number of lysine residues in a sliding 20-residue window; red indicates the poly-lysine bacterial proteins that were screened for polyP binding in this study. For a full list, see Table S4.

Fig 2

KPM modulates P. aeruginosa virulence factors. (A) Effect of recombinant overexpression of WT EngA and SrmB compared to ∆K mutants on biofilm formation. Biofilm formation was observed in WT PA14 and ∆polyP PA14 strains and quantified via crystal violet staining. (B) Representative image of biofilm ring formation. (C) Effect of recombinant overexpression of WT EngA and SrmB compared to ∆K mutants on pyoverdine production. Pyoverdine production was observed in WT PA14 and ∆All PA14 strains and quantified via absorbance at 403 nm. (D) Representative image of pyoverdine production. (E) Effect of recombinant overexpression of WT EngA and SrmB compared to ∆K mutants on pyocyanin production. Pyocyanin production was observed in WT PA14 and ∆polyP PA14 strains and quantified via absorbance at 520 nm following chloroform extraction and HCl treatment. Proteins were expressed using the pHERD20T arabinose-inducible expression vector (1% L-arabinose wt/vol). ns, P >0.05; ^*, P <0.05; ^**, P <0.01; ^***, P <0.001 (unpaired t-test, n = 3 biological replicates). Error bars are ±standard deviation.