Tizoxanide associated with anti-virulent activity controls fire blight disease caused by Erwinia amylovora in Malus asiatica

Erwinia amylovora is a causative pathogen of fire blight disease, affecting apple, pear, and other rosaceous plants. This study aimed to discover new anti-virulence agents to control this plant bacterial disease by preventing its pathogenesis. To this end, 2485 small molecules were screened for targeting the type III secretion system (T3SS) which is a critical virulence factor in E. amylovora. Our screening identified tizoxanide as a potent inhibitor, significantly reducing the promoter activity of hrpA, which encodes the Hrp pilus protein essential for T3SS function. Beyond T3SS inhibition, tizoxanide also exhibited inhibitory effects on other virulence factors by down-regulating genes associated with these functions. Given its broad effects on various virulence factors, we conducted a proteomic analysis of E. amylovora treated with tizoxanide, and the results revealed differentially expressed proteins related to cell metabolism and pathogenesis. In a plant infection model, spray treatment with tizoxanide effectively protected Chinese pearleaf crabapple seedlings against E. amylovora. Furthermore, a synthesized tizoxanide derivative containing an isoxazole moiety further enhanced hrpA promoter inhibition and disease control efficacy compared to the tizoxanide. Taken together, our results highlight the potential of tizoxanide as an anti-virulence agent to prevent plant disease caused by E. amylovora.