Development of transgenic Caenorhabditis elegans expressing human transthyretin as a model for drug screening.

Familial amyloid polyneuropathy is a hereditary systemic amyloidosis caused by a mutation in the transthyretin (TTR) gene. Amyloid deposits in tissues of patients contain not only full-length TTR but also C-terminal TTR fragments. However, in vivo models to evaluate the pathogenicity of TTR fragments have not yet been developed. Here, we generated transgenic Caenorhabditis elegans strains expressing several types of TTR fragments or full-length TTR fused to enhanced green fluorescent protein in the body wall muscle cells and analyzed the phenotypes of the worms. The transgenic strain expressing residues 81-127 of TTR, which included the ß-strands F and H, formed aggregates and caused defective worm motility and a significantly shortened lifespan compared with other strains. These findings suggest that the C-terminal fragments of TTR may contribute to cytotoxicity of TTR amyloidosis in vivo. By using this C. elegans model system, we found that (-)-epigallocatechin-3-gallate, a major polyphenol in green tea, significantly inhibited the formation of aggregates, the defective motility, and the shortened lifespan caused by residues 81-127 of TTR. These results suggest that our newly developed C. elegans model system will be useful for in vivo pathological analyses of TTR amyloidosis as well as drug screening.

Novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone DnaK.

Biofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of the dnaK gene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness of Escherichia coli biofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation of E. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 µM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype of E. coli wild-type cells to make them resemble those of the isogenic dnaK deletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs.