A natural product from Streptomyces targets PhoP and exerts antivirulence action against Salmonella enterica

Background The overprescription and misuse of classical antimicrobial compounds to treat gastrointestinal or systemic salmonellosis have been accelerating the surge of antibiotic-recalcitrant bacterial populations, posing a major public health challenge. Therefore, alternative therapeutic approaches to treat Salmonella infections are urgently required. Objectives To identify and characterize actinobacterial secreted compounds with inhibitory properties against the Salmonella enterica PhoP/PhoQ signal transduction system, crucial for virulence regulation. Methods The methodology was based on a combination of the measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes and bioguided assays to screen for bioactive inhibitory metabolites present in culture supernatants obtained from a collection of actinobacterial isolates. Analogues of azomycin were used to analyse the functional groups required for the detected bioactivity and Salmonella mutants and complemented strains helped to dissect the azomycin mechanism of action. The tetrazolium dye colorimetric assay was used to investigate azomycin potential cytotoxicity on cultured macrophages. Salmonella intramacrophage replication capacity upon azomycin treatment was assessed using the gentamicin protection assay. Results Sublethal concentrations of azomycin, a nitroheterocyclic compound naturally produced by Streptomyces eurocidicus, repressed the Salmonella PhoP/PhoQ system activity by targeting PhoP and inhibiting its transcriptional activity in a PhoQ- and aspartate phosphorylation-independent manner. Sublethal, non-cytotoxic concentrations of azomycin prevented Salmonella intramacrophage replication. Conclusions Azomycin selectively inhibits the activity of the Salmonella virulence regulator PhoP, a new activity described for this nitroheterocyclic compound that can be repurposed to develop novel anti-Salmonella therapeutic approaches.