Table_1_Identification of Benzyloxy Carbonimidoyl Dicyanide Derivatives as Novel Type III Secretion System Inhibitors via High-Throughput Screening.pdf

The type III secretion system (T3SS) in many Gram-negative bacterial pathogens is regarded as the most critical virulence determinant and an attractive target for novel anti-virulence drugs. In this study, we constructed a T3SS secretion reporter containing the β-lactamase gene fused with a signal peptide sequence of the T3SS effector gene, and established a high-throughput screening system for T3SS inhibitors in the plant pathogenic bacterium Acidovorax citrulli. From a library of 12,000 chemical compounds, we identified a series of benzyloxy carbonimidoyl dicyanide (BCD) derivatives that effectively blocked T3SS-dependent β-lactamase secretion. Substitution of halogens or nitro groups at the para-position on the benzene ring contributed to an increased inhibitory activity. One representative compound, BCD03 (3,4-dichloro-benzyloxy carbonimidoyl dicyanide), dramatically reduced pathogenicity of A. citrulli on melon seedlings, and attenuated hypersensitive responses in the non-host Nicotiana tabacum caused by pathogenic bacteria A. citrulli, Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. tomato at sub-MIC concentrations. Western blotting assay further confirmed that BCD03 inhibited effector secretion from the above bacteria via T3SS in the liquid medium. Taken together, our data suggest that BCD derivatives act as novel inhibitors of T3SS in multiple plant bacterial pathogens.

诸多革兰氏阴性致病菌所具备的III型分泌系统（type III secretion system, T3SS），被视为最为关键的毒力决定因子，同时也是新型抗毒力药物极具吸引力的作用靶点。本研究中，我们构建了一款融合了T3SS效应基因信号肽序列的β-内酰胺酶（β-lactamase）报告基因，以此搭建了针对植物病原细菌瓜类噬酸菌（Acidovorax citrulli）T3SS抑制剂的高通量筛选体系。我们从包含12000种化合物的化学库中，筛选得到一系列可有效阻断T3SS依赖性β-内酰胺酶分泌的苄氧基碳亚胺基二氰化物（benzyloxy carbonimidoyl dicyanide, BCD）衍生物。在苯环对位引入卤素或硝基取代基，可有效提升该类衍生物的抑制活性。代表性化合物BCD03（3,4-二氯苄氧基碳亚胺基二氰化物）可在亚最低抑菌浓度（sub-MIC）下，显著降低瓜类噬酸菌对甜瓜幼苗的致病力，同时削弱病原细菌瓜类噬酸菌、稻黄单胞菌水稻致病变种（Xanthomonas oryzae pv. oryzae）及丁香假单胞菌番茄致病变种（Pseudomonas syringae pv. tomato）在非宿主植物本氏烟（Nicotiana tabacum）中引发的超敏反应。蛋白质免疫印迹试验（Western blotting assay）进一步证实，BCD03可在液体培养基中通过阻断T3SS，抑制上述病原菌的效应蛋白分泌。综上，本研究数据表明，BCD衍生物可作为多种植物病原细菌的T3SS新型抑制剂。