Discussion

We present a novel method for fast, synchronized, and efficient delivery of proteins and peptides into eukaryotic cells. It relies on the Yersinia enterocolitica bacterial type 3 secretion system (T3SS), a nanomachine which injects functional polypeptides produced in bacteria directly into eukaryotic cells. The system's advantages include rapid, tunable and homogenous delivery, making it attractive for various applications, such as the study of transient signaling and kinetics, or the investigation of toxic proteins.
A pro-apoptotic factor (PAF) was selected to explore the technology in a high-throughput screening setup. The 15kDa PAF was expressed in bacteria and delivered to cells, where it accumulated to high levels, as measured one hour post-inoculation, ultimately leading to apoptotic cell death. Induction of apoptosis, assessed 16 hours post-inoculation, was observed in a reproducible and dose-dependent manner. A 1.6kDa peptide corresponding to the active domain present in the PAF, expressed in bacteria and delivered to cells, likewise resulted in reproducible and dose-dependent induction of apoptosis.
Pre-incubation of cells with an inhibitor of downstream signaling (caspase 3/7 inhibitor) protected cells from apoptosis upon protein or peptide delivery, demonstrating the selectivity of pathway activation. Using the caspase inhibitor as positive control under high-throughput screening conditions, wells with different levels of controls were identified with high confidence.
Different target cell lines were inoculated with Y. enterocolitica expressing the PAF, with HeLa and CHO most susceptible and HEK293 to a lesser extent, while Jurkat appeared to be resistant to apoptosis induction.
These results indicate that the Y. enterocolitica bacterial protein delivery nanomachine permits targeted, efficient and fast protein or peptide delivery into various cell lines, facilitating the investigation of difficult target proteins, including toxic proteins like pro-apoptotic factors. The technology has to date been validated with a large variety of proteins which elicited effects in different pathways. This approach will benefit exploration of cell signaling and enable novel avenues in drug discovery.

Authors: Laurent Brault(1), Emelie Guevarra(1), Serge P. Parel(1), Stephan Fasler(1), Christoph Kasper(2), Marlise Amstutz(2), Simon Ittig(2), Daniela Brodbeck(1)

(1)Exquiron Biotech AG, Reinach, Switzerland, and (2)Type 3 Technologies, Basel, Switzerland