Identification and application of T3SS translocation signal in Edwardsiella piscicida attenuated carrier as a bivalent vaccine.

Type III secretion system (T3SS)-dependent translocation has been used to deliver heterologous antigens by vaccine carriers into host cells. In this research, we identified the translocation signal of Edwardsiella piscicida T3SS effector EseG and constructed an antibiotic resistance-free balanced-lethal system as attenuated vaccine carrier to present antigens by T3SS. Edwardsiella piscicida LSE40 asd gene deletion mutant was constructed and complemented with pYA3342 harbouring the asd (aspartate ß-semialdehyde dehydrogenase) gene from Salmonella. Fusion proteins composed of EseG N-terminal 1-108 amino acids and the TEM1-ß-lactamase reporter were inserted in plasmid pYA3342. The fusion protein could secrete into the cell culture, translocate into HeLa cells, and localize in the membrane fraction. Then, the double gene deletion mutant LSE40ΔasdΔpurA was constructed as an attenuated vaccine carrier, and Aeromonas hydrophila GapA (glyceraldehyde-3-phosphate dehydrogenase) was fused with the translocation signal, instead of the TEM1-ß-lactamase reporter. The bivalent vaccine could protect blue gourami (Trichogaster trichopterus) against E. piscicida and A. hydrophila, with the relative per cent survival of 80.77% and 63.83%, respectively. These results indicated that EseG N-terminal 1-108 amino acid peptide was the translocation signal of E. piscicida T3SS, which could be used to construct bivalent vaccines based on an attenuated E. piscicida carrier.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.