The emergence and spread of themcr-1gene and its mutants has immensely compromised the efficient usage of colistin for the treatment of drug-resistant Gram-negative bacterial infection in clinical settings. However, there are currently no clinically available colistin synergis. Here we identify artemisinin derivatives, such as dihydroartemisinin (DHA), that produces a synergistic antibacterial effect with colistin against the majority of Gram-negative bacteria (FIC < 0.5) without induced resistance, particularly those carrying themcr-1gene. Mechanism analysis reveals the direct engagement of DHA with the active center of MCR-1 to inhibit the activity of MCR-1. Meanwhile, the results from transcriptome and electron microscope analysis show that DHA could also simultaneously affect the flagellar assembly and the energy metabolism of bacteria. Moreover, in the mouse infection models of Gram-negative bacteria, combination therapy shows remarkable treatment benefits, as shown by an improved survival rate, reduced morbidity, alleviated pathological injury and decreased bacterial loading. Due to the generally safe profile of specialized malaria medication administration in humans, artemisinin derivatives are a promising class of multi-target inhibitors on bacterial resistance and virulence that can be used to extend the usage life of colistin and to tackle the inevitability of serious bacterial infection with colistin.

The invasiveness of Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely associated with the Salmonella pathogenicity island (SPI)‐encoded type Ⅲ secretion system (T3SS), which can directly inject a series of effector proteins into eukaryotic cells to enable bacterial infection. In this study, syringaldehyde was identified as an effective inhibitor of the S. Typhimurium T3SS using an effector protein‐lactamase fusion reporter system. Syringaldehyde treatment could inhibit the expression of important effector proteins (SipA, SipB and SipC) at a concentration of 0.18 mM without affecting bacterial growth. Additionally, significant inhibition of bacterial invasion and cellular injury was observed following the syringaldehyde treatment in the co‐infection system of HeLa cells and S. Typhimurium. Furthermore, treatment with syringaldehyde provided systemic protection to mice infected with S. Typhimurium, reducing mortality (40.00%) and bacterial loads and relieving caecal damage and systemic inflammation. The results presented in this study indicate that syringaldehyde significantly affects T3SS activity and is a potential leading compound for treating S. Typhimurium infections.

The increasingly serious problem of bacterial antibiotic resistance limits the clinical application of antibiotics, which increases the need for the development of antivirulence agents. The type III secretion system (T3SS) plays a critical role in host cell invasion and pathogenesis of Salmonella and becomes a popular target for antivirulence agents screening.

Listeria monocytogenes is a ubiquitous Gram-positive foodborne pathogen that is responsible for listeriosis in both humans and several animal species. The bacterium secretes a pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), a major virulence factor involved in the activation of cellular processes. The ability of LLO to lyse erythrocytes is a measure of LLO activity. We used hemolytic activity assay to screen the LLO inhibitors. Acacetin was found to be an LLO inhibitor, which is a di-hydroxy and mono-methoxy flavone present in various plants, including Black locust, Damiana, and Silver birch. As the features of acacetin are of low toxicity and have less acquired resistance, it comes to a hotspot in drug development. In our study, we report that acacetin antagonized the hemolytic activity of L. monocytogenes culture supernatants and purified LLO by directly interfering with the formation of oligomers without inhibiting the bacterial growth and the expression of LLO. Acacetin also relieved the injury of alveolar epithelial cells by inhibiting LLO activity. Further, acacetin significantly promoted the clearance of L. monocytogenes and alleviated the histopathological damage, thereby raising survival rate, which conferred mice with effective protection against L. monocytogenes infection. Using molecular docking and dynamics simulation, we further proved the mechanism of acacetin antagonizing LLO pore-forming activity by direct binding to the second membrane-inserting helix bundle (HB2) of LLO domain 3. These data suggested that acacetin recedes the virulence of L. monocytogenes both in vivo and in vitro, and this study provided a promising candidate and potential alternative for the prevention and treatment of L. monocytogenes infections.

AimsThis study was conducted to screen the type Ⅲ secretion system (T3SS) inhibitors of Salmonella enterica serovar Typhimurium (S. Typhimurium) from natural compounds. Through systemic analysis the pharmacological activity and action mechanism of candidate compounds in vivo and in vitro. Methods and resultsUsing an effector-β-lactamase fusion reporter system in S. Typhimurium, we discovered that quercitrin could block effector SipA translocation into eukaryotic host cell without affecting bacterial growth, and inhibit invasion or epithelial cells damage. Using β-galactosidase activity and Western blot assay, it was found that quercitrin significantly inhibits the expression of SPI-1 genes (hilA and sopA) and effectors (SipA and SipC). The animal experiment results indicated that quercitrin reduces mortality, pathological damages and colony colonization of infected mice. ConclusionsSmall-molecule inhibitor quercitrin directly inhibits the founction of T3SS in S. Typhimurium, and provids a potential alternative antimicrobial against Salmonella infection.Significance and impact of the studyNatural compounds have become valuable resources for antibacterials discovery due to their widely structures and biological activities. However, the potential targets and molecular action mechanisms of candidate compounds responsible for anti-infections remain elusive. The T3SS plays a crucial role in bacterial invasion and pathogenesis process in S. Typhimurium. Compared with traditional antibiotics, small molecular compounds can inhibit the T3SS of Salmonella and achieve the effect of anti-infection. They have less pressure on bacterial survival and are not easy to produce drug resistance. This provides strong evidence for development novel anti-virulence drugs against Salmonella infection.