Identification and characterization of type III secretion inhibitors in Erwinia amylovora, the causal agent of fire blight of apple and pear

Fire blight is a destructive bacterial disease of apples and pears as well as other rosaceous plants and causes millions of dollar losses around the world each year. Erwinia amylovora is the causative agent of fire blight. The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two major yet separate virulence factors in E. amylovora. Current fire blight management mainly relies on application of copper compounds and antibiotics. However, development of streptomycin resistant E. amylovora isolates in the US and other countries has made it ineffective. It is reasonable to believe that disabling the T3SS function may provide another way of controlling bacterial diseases. High-throughput screening of chemical libraries have identified small molecule inhibitors that attenuate T3SS of mammalian pathogens, but no study has been reported so far for plant pathogenic bacteria. In this study, three small molecules were identified to delay hypersensitive response (HR) development in tobacco. Using GFP as a reporter, six chemicals were identified to suppress T3SS gene expression of E. amylovora under hrp inducing conditions. Five of them belong to salicylidene acylhydrazides. Among them, chemical # 3 and # 9 were most potent in inhibiting T3SS gene expression in a dose-dependent manner without affecting bacterial growth. Furthermore, this group of chemicals also inhibited exopolysaccharide amylovoran production. Our results further demonstrated that chemical # 3 treatment resulted in growth reduction and symptom suppression on crab apple blossom. To better understand the mode of action of this class of compounds, we carried out a microarray analysis of E. amylovora treated with chemicals # 3 and # 9. A total of 534 and 183 genes were identified to be significantly differentially regulated by chemicals # 3 and # 9 treatment, respectively. The majority of genes in E. amylovora T3SS cluster including hrpL as well as effectors including avrRpt2 and hopC1 were down-regulated more than two folds by both chemicals # 3 and # 9. Chemical # 3 also suppressed the transcription of all amylovoran biosynthesis genes. Interestingly, the most significant upregulated genes were those involving iron acquisition and utilization. In addition, other compounds of this group (chemicals # 1, # 2, # 4) also exhibited a similar effect on gene expression, i. e suppressing T3SS and ams gene expression, while promoting iron uptake gene expression. Our results suggested that a common inhibition mechanism may be shared by these compounds. To test whether salicylidene acylhydrazides affect secretion of T3SS proteins, total secreted proteins from wild type bacteria grown in the presence or absence of chemicals # 3 or # 9 were quantified. Our results showed that chemical # 9 exhibited a dose-dependent inhibition of protein secretion in E. amylovora; while chemical # 3 was more effective, causing a complete blockage of secretion of T3SS proteins at as low as 5 μmol