Small Molecules as Tools in Biological Chemistry : Effects of Synthetic and Natural Products on the Type III Secretion System

Abstract: The increasing use of antibiotics has led to a huge problem for society, as some bacteria have developed resistance towards many of the antibiotics currently available. To help find solutions to this problem we studied small molecules that inhibit bacterial virulence, the ability to cause disease. The type III secretion system (T3SS) is a conserved virulence system found in several gram-negative bacteria, including human and plants pathogens, such as Yersinia spp., Pseudomonas aeruginosa, Chlamydia spp., Salmonella spp., Shigella spp, enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), and Erwinia spp. One class of virulence-blocking compounds is the salicylidene acylhydrazides. They were first identified in a screen towards the T3SS in Yersinia pseudotuberculosis and have since been shown to block the T3SS in a panel of gram-negative bacteria such as Chlamydia spp. Salmonella enterica, Shigella flexneri and EPEC.We designed and synthesized a library of 58 salicylidene acylhydrazides and evaluated their activity as virulence-blocking compounds in Y. pseudotuberculosis followed by calculations of quantitative structure activity relationships (QSARs). Four QSAR models were calculated, and when used in consensus they correctly classified between five out of eight compounds for Y. pseudotuberculosis as active or inactive and six out of eight compounds for C. trachomatis.Since the target and mode of action of the salicylidene acylhydrazides were unknown, we used solution and solid phase synthesis to synthesize three different affinity reagents. One of these affinity reagents was used in affinity chromatography experiments, where 19 putative target proteins from an E. coli O157 bacterial lysate were identified. We studied four of the proteins, Tpx, WrbA, FolX, and AdhE, in more detail in Y. pseudotuberculosis and E. coli O157. We believe that the salicylidene acylhydrazides act on multiple targets that together result in down-regulation of T3SS functions. A knockout of AdhE in E. coli O157 showed a similar phenotype as salicylidene acylhydrazide treated E. coli, suggesting that this protein may be particularly interesting as a drug target.Many of the antibiotics used today originate form natural sources. In contrast, most virulence-blocking compounds towards the T3SS are small synthetic organic molecules. Therefore, a prefractionated natural product library with marine and terrestrial biota samples was screened towards the T3SS in Y. pseudotuberculosis. Neohopeaphenol A was identified as a hit and shown to have micromolar activity towards Y. pseudotuberculosis and P. aeruginosa in cell-based infection models.