IDENTIFICATION OF AN HYDRAZONE CAPABLE OF INHIBITING THE PhoP/PhoQ VIRULENCE SYSTEM OF Salmonella

RICARDO FURLÁN; CARLOS A. LOBERTTI; ELEONORA GARCÍA VÉSCOVI; IGNACIO CABEZUDO

Evento virtual debido a la pandemia 2020

Congreso; Reunión Conjunta SAIB-SAMIGE; 2020

SAIB-SAMIGE

Salmonella is a versatile enterobacterial pathogen capable of infecting a wide range of animal hosts and cause a variety of diseases. PhoP/PhoQ is a two-component regulatory system (TCS) found in a variety of Gramnegative bacteria. This TCS consists of an inner membrane-bound histidine kinase PhoQ and its cognate transcriptional regulator PhoP. In Salmonella enterica serovar Typhimurium (S. Typhimurium), the PhoP/PhoQ system responds to input signals that bacteria encounter during their life cycle, such as the availability of Mg2+, the presence of long chain unsaturated fatty acids (LCUFAs) or cationic antimicrobial peptides (CAMPs) and acidic pH. In addition, the system controls key virulence phenotypes such as the invasion, survival and proliferation within host cells. As signal transduction in mammals does not involve TCS, the PhoP/PhoQ system is an attractive target to develop new antimicrobial agents. Through a TLC-overlay strategy previously reported for the search and identification of antimicrobial agents targeting the PhoP/PhoQ system, we screened a combinatorial library of hydrazones and thiocarbazones in the search for inhibitors and we identified a potential inhibitor, the hydrazone A16B4. This compound could be synthesized in its pure form, characterized, and it was confirmed that it does not affect the growth of Salmonella. By quantitative β-galactosidase assays we confirmed its inhibitory activity and it was found that the response was dose-dependent and selective as well. In order to define the A16B4 mechanistic action over the PhoP/PhoQ TCS we performed autophosphorylation activity assays which revealed that A16B4 is able to target the PhoQ autokinase activity. Taking into account that A16B4 negatively affects PhoQ activity, we analyzed the PhoQ domain targeted by the compound. Fluorescence-based thermal shift assay (FTS) results suggest that A16B4 could interact with the PhoQ periplasmic sensor domain. As the PhoP/PhoQ system is involved in the resistance to antimicrobial cationic peptides, we evaluated whether A16B4 was capable of affecting the ability of Salmonella to respond to them. We determined that A16B4 was able to increase the susceptibility of Salmonella to an antibiotic used in clinical settings. Taken together, these results position A16B4 as a promising compound for the development of an anti-Salmonella therapeutic agent.