An allosteric inhibitor of the PhoQ histidine kinase with therapeutic potential against Salmonella infection

Abstract

Background: The upsurge of antimicrobial resistance demands innovative strategies to fight bacterial infections. With traditional antibiotics becoming less effective, anti-virulence agents or pathoblockers, arise as an alternative approach that seeks to disarm pathogens without affecting their viability, thereby reducing selective pressure for the emergence of resistance mechanisms. Objectives: To elucidate the mechanism of action of compound N′-(thiophen-2-ylmethylene)benzohydrazide (A16B1), a potent synthetic hydrazone inhibitor against the Salmonella PhoP/PhoQ system, essential for virulence. Materials and methods: The measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes was used to evaluate the specificity of A16B1 to the PhoP regulon. Autokinase activity assays with either the native or truncated versions of PhoQ were used to dissect the A16B1 mechanism of action. The effect of A16B1 on Salmonella intramacrophage replication was assessed using the gentamicin protection assay. The checkerboard assay approach was used to analyse potentiation effects of colistin with the hydrazone. The Galleria mellonella infection model was chosen to evaluate A16B1 as an in vivo therapy against Salmonella. Results: A16B1 repressed the Salmonella PhoP/PhoQ system activity, specifically targeting PhoQ within the second transmembrane region. A16B1 demonstrates synergy with the antimicrobial peptide colistin, reduces the intramacrophage proliferation of Salmonella without being cytotoxic and enhances the survival of G. mellonella larvae systemically infected with Salmonella. Conclusions: A16B1 selectively inhibits the activity of the Salmonella PhoP/PhoQ system through a novel inhibitory mechanism, representing a promising synthetic hydrazone compound with the potential to function as a Salmonella pathoblocker. This offers innovative prospects for combating Salmonella infections while mitigating the risk of antimicrobial resistance emergence.