S162: Small molecule perimeter defense in entomopathogenic bacteria

The Gammaproteobacterial entomopathogens Xenorhabdus nematophila and Photorhabdus luminescens produce rhabduscin, an amidoglycosyl- and vinyl-isocyanide-functionalized tyrosine analog. By reconstituting the rhabduscin pathway in E. coli, utilizing precursor directed biosynthesis to generate rhabduscin mimics, conducting enzyme inhibitory assays, and visualizing the spatial distribution of rhabduscin with stimulated Raman scattering and confocal fluorescence microscopy, we established rhabduscin’s function as a potent inhibitor of phenoloxidase, an important arm of the insect’s innate immune system, in addition to its placement at the bacterial cell surface. Stimulated Raman scattering microscopy of E. coli heterologously expressing rhabduscin and wildtype X. nematophila showed that rhabduscin binds to the periphery of the bacterial cells. A precursor directed biosynthetic strategy combined with confocal fluorescence microscopy enabled the biosynthesis of an azide-functionalized rhabduscin mimic, which could be accessed at the cell surface by a bioorthogonal extracellular green fluorescent protein probe. Rhabduscin and the cells that produced it were both highly sufficient (low nM inhibition) at inhibiting insect phenoloxidase and the model enzyme, mushroom tyrosinase. We propose a model in which rhabduscin physically binds at the bacterial cell surface to generate a high effective inhibitor concentration in a spatially appropriate manner for phenoloxidase contact.