The delivery of bacterial proteins into host tissue is particularly relevant for bacterial pathogens, enabling them to spread, cause infections and disease. Bacteria can directly deliver proteins into the host cells using large protein complexes (T3SS, T4SS and T6SS) or secrete proteins that can direct their own entry into host cells. Here we have investigated the major toxin from Enteropathogenic E.coli (EPEC) responsible for severe and frequently fatal diarrhea in infants1. The EspC toxin belongs to a group of autotransporters (T5SS) that are able to direct their own bacterial secretion along with their subsequent targeting and penetration into host epithelia2,3. Upon cellular entry, EspC targets the cell cytoskeleton to cause damage to the epithelium, a hallmark of diarrheal infections.
Aims: We sought to investigate the features of EspC that allow its cellular entry and ability to cause tissue destruction.
Methods: We employed a comprehensive array of methods to explore EspC function including X-ray crystallography, enzyme assays, mutational analysis, along with specialised cell invasion assays.
Results/Discussion: Here we show the first X-ray crystal structure of the EspC toxin, that reveals that it forms an almost 1000 residue b-helical stalk with an additional 3 functional domains. We dissect how these domains affect toxicity and cellular entry, including how its serine protease cleaves intracellular fodrin associated with the cellular cytoskeleton. Importantly, in a novel twist we show how this unique T5SS EspC hijacks the complex T3SS machinery for more efficient cellular delivery, which results in higher mortality in a model of infection. Finally we develop a range of nanobody inhibitors that target these EspC functional domains to inhibit cellular entry and toxicity.