One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg 2 + , a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella's resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella. Bacteria that can resist clinically used antibiotics pose an urgent threat to worldwide public health. One concerning antibioticresistant pathogen is the gram-negative bacterial species Salmonella enterica. [1-3] Non-typhoidal serotypes of Salmonella cause food-borne gastroenteritis and can be transmitted from animals to humans. [1] Typhoidal serotypes of Salmonella cause typhoid fever and include human-specific serotypes. [2] The World Health Organization has listed Salmonella as a "highpriority" pathogen for the development of new antibiotics, [4] and the Centers for Disease Control in the United States has deemed Salmonella as a "serious threat" to public health. [5] A strategy to combat antibiotic resistant bacteria is to target molecular processes that are required for bacterial virulence. [6-8] There has been to our knowledge no clinical development of compounds that target Salmonella virulence. [8] Here, we report [a] M.

Salmonella enterica, a major bacterial cause of food‐borne illness, can survive in harsh environments due to its ability to sense and respond to toxic conditions. Salmonella senses a threatening environment, such as antibacterial conditions produced by the body’s immune system, and then activates the expression of genes necessary to survive within that environment. These genes are activated through the use of two component systems. Two component systems consist of an inner membrane‐bound histidine kinase and a response regulator protein located in the cytoplasm of the cell. The PhoP/PhoQ two component system is critical for the virulence of Salmonella and for Salmonella to resist antibiotics such as polymyxin B. Therefore, we tested commercially available small molecules capable of inhibiting the sensory histidine kinase to see if they were capable of sensitizing Salmonella to polymyxin B. Indeed, a few small molecules were successful at sensitizing Salmonella to polymyxin B. To determine how these molecules decrease polymyxin B resistance within Salmonella, we carried out several mechanistic studies. First, we tested whether these small molecules influenced Salmonella’s outer membrane permeability. This was tested by combining small molecules with vancomycin. Vancomycin is an antibiotic used effectively on gram‐positive bacteria that only contain a single membrane, but gram‐negative bacteria are intrinsically resistant to vancomycin due to their outer membrane. We found that vancomycin did not impact Salmonella’s resistance to polymyxin B. These results suggest that the small molecules do not affect outer membrane permeability. We also tested whether these molecules affect expression of PhoP‐activated genes. These molecules have been seen to decrease expression of a PhoP activated gene when transcriptional reporters were used. Using reverse transcription quantitative polymerase chain reaction (RT‐qPCR), we found that the small molecules decreased expression of some, but not all, PhoP‐activated genes. Overall, our results demonstrate that small molecules can target Salmonella’s ability to regulate gene expression and resist toxic conditions. This discovery represents a promising new way to develop antibiotic drugs to treat bacterial infections. Support or Funding Information This research was supported by the following funding: UWL Undergraduate Research and Creativity Grants (MKT), UWL College of Science and Health Dean’s Distinguished Fellowship (MKT), Research Corporation for Science Advancement, Cottrell Teacher Scholar Ambassadors for PUI – R1 Partnerships Award (EEC and JFM)