Chemical Inhibitors of DksA1, a Conserved Bacterial Transcriptional Regulator, Suppressed Quorum Sensing-Mediated Virulence in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium of clinical importance. As an opportunistic pathogen, its virulence is dependent on quorum sensing (QS). Stringent response (SR), a conserved mechanism in the bacterial kingdom, is activated under diverse stress conditions including nutrient starvation. DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in P. aeruginosa SR. Our recent study clearly demonstrated that, apart from SR, DksA1 regulates a wide range of phenotypes including QS-mediated virulence. This suggests that DksA1 is a potential target to be inhibited to control P. aeruginosa infections. Here, we screened a library of 6,970 chemical compounds and identified two compounds (termed Dkstatin-1 and Dkstatin-2) that specifically suppress DksA1 activity. Two Dkstatins substantially suppressed production of elastase and pyocyanin and protected a murine host against lethal infection with PAO1, a prototype strain of P. aeruginosa. Dkstatins also suppressed the production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced in Dkstatin-treated cells was almost identical to that in the {Delta}dksAI mutant of PAO1. Our RNASeq analysis showed that transcript levels of genes involved in QS and virulence were markedly reduced in Dkstatin-treated PAO1 cells, further demonstrating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased antibiotic susceptibilities of PAO1, particularly to the antibiotics that inhibit protein synthesis. Our co-immunoprecipitation assay demonstrated that Dkstatins interfere with the binding of DksA1 to the {beta} subunit of RNA polymerase, which potentially explains the mode of Dkstatin action. Collectively, our results suggest that the inhibition of P. aeruginosa QS, which has often been attempted, can be achieved by DksA1 inhibitors. Dkstatins will prove to be useful when establishing infection control strategies. Author summaryBacterial cells developed numerous systems to handle environmental stresses. Stringent response (SR) is one of those systems, and it is activated under the stress of nutrient starvation. Active SR requires DksA1, a protein conserved across bacterial species. Besides this well-recognized function, DksA1 in P. aeruginosa positively regulates its virulence by activating quorum sensing (QS). This finding led us to hypothesize that inhibiting DksA1 would result in virulence attenuation of the pathogenic P. aeruginosa. Herein, we identified two molecules that effectively suppressed DksA1 function through a large-scale compound library screen. These two compounds suppressed P. aeruginosa virulence and therefore protected a murine host from a deadly infection of P. aeruginosa. Moreover, the compounds also rendered P. aeruginosa more susceptible to antibiotics. Our results demonstrate that dangerous P. aeruginosa infections can be controlled by inhibiting DksA1.