Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA, United States.
Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, CA, United States.
Front Cell Infect Microbiol. 2018 Nov 22;8:404. doi: 10.3389/fcimb.2018.00404. eCollection 2018.
Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic as the workhorse T3SS-expressing model organisms for this experimental pipeline, as is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against .
数十种革兰氏阴性病原体使用一种或多种 III 型分泌系统（T3SS）在感染宿主生物时解除宿主防御或占据有利生态位。虽然 T3SS 是一个有吸引力的药物靶点，并且已经鉴定出数十种具有 T3SS 抑制活性的化合物，但很少有 T3SS 抑制剂得到验证并确定其作用模式。一个问题是缺乏高通量筛选后的标准化正交测定。使用先前显示具有 T3SS 抑制活性的商业上可获得的化合物的训练集，我们展示了由六个不同测定组成的实验管道的实用性，以评估受影响的 III 型分泌阶段：T3SS 基因拷贝数、T3SS 基因表达、T3SS 基底体和针组装、货物通过 T3SS 的分泌以及 T3SS 效应蛋白向宿主细胞的易位。我们使用肠致病性 作为该实验管道的工作型 T3SS 表达模型生物，因为 对我们测试的所有 T3SS 抑制剂都敏感，包括对其他 T3SS 表达病原体有效的抑制剂。我们发现，该实验管道能够快速区分在 T3SS 组装和功能的不同点中断 III 型分泌过程的 T3SS 抑制剂。例如，我们的数据表明，二羟丙氨酸二酰胺化合物 3 阻断组装的 T3SS 的活性或改变 T3SS 的结构，以阻止 T3SS 货物分泌，但不阻止 T3SS 针的抗体识别。相比之下，我们的数据预测，卤代磺酰胺苯甲酰胺化合物 4 破坏 T3SS 针亚基的分泌或组装。此外，我们建议，pYV 毒力质粒（编码 T3SS）的拷贝数控制的失调应被视为具有针对 的 T3SS 抑制活性的化合物的可能作用模式。
