High Throughput Screening For Type Three Secretion System Inhibitor In Acidovorax Citrulli

Acidovorax citrulli can infect melon and cause bacterial fruit blotch. Type three secretion system (TTSS) plays a critical role in bacterial infection. When TTSS function is suppressed, A. citrulli infection can be prevented or inhibited completely. In this study, a high throughput screening system was established by using TTSS as a chemical target. The reporter used for screening is based on a fusion of TTSS signal peptide and lactamase, which provides a yellow-red color read out. Totally ca.12000 compounds have been screened, and 463 of them showed TTSS inhibition activities. The compounds contianing malononitrile oxime ether group exhibited the highest activity and was further investigated for action models and disease suppression in pot experiments.The reporter vector contains a translational fusion of TTSS N-terminal signal peptide of a HopAO1 homolog and a β-lactamase gene. When expressed in TTSS-wild-type strain A. citrulli MH21, P-lactamase secreted into extracellular space and hydrolyzed the chromogenic substrate-nitrocefin to give a clear yellow-red color read out. TTSS induction conditions, such as induction medium, temperature, pH and ion concentration, were optimized and the best conditions determined is LB culture medium containing 10 mM mg2+ at pH5.8. The high throughput method for screening TTSS inhibitors was finally established in a 96 well culture plate format with a colorimetric readout (yellow-red) as the reporter.Totally about 12000 synthetic compounds have been screened through TTSS high throughput system, and 463 compounds were found to have TTSS-inhibitory acitvities at a certain level. Further test confirmed that the compounds belong tomalononitrile oxime ether group have significantly higher acitivities than others.Structure-activity analysis shows that compouds with para-malononitrile oxime ether have highly active. Replacing one of the malononitrile oxime ether with halogen or nitro could further increase inhibitory activity of the compounds. In vivo, malononitrile oxime ether compound 33962 significantly suppressed the A. citrulli MH21 infection of melon seedlings with a control efficiency of 82%. qRT-PCR analysis of malononitrile oxime ether-treated stain MH21 revealed an obvious impact on the expression of TTSS encoding genes, and the expression of some genes decreased more than 80%. Based on the above data, malononitrile oxime ether was confirmed as a novel inhibitor of bacterial TTSS.Malononitrile oxime ether derivatives found in our work are novel TTSS inhibitors that can prevent plant disease by suppressing the key pathogenic factor of bacterial infection. These compounds, which are different from traditional bactericides, do not inhibit bacterial growth at the test concentrations. This provides them a great potencial to incorporate with low dosages of chemical pesticides for plant disease management.