Engineering Probiotic E. Coli With a Type III Secretion System for Targeted Delivery of Therapeutic VHH

To combat severe disease manifestations in the gut from both pathogenic and autoimmune insults, we are engineering probiotic microbes to release therapeutics into the gut as a novel method of treatment delivery. A bacterial protein delivery system was designed based upon the Shigella flexneri type III secretion system (T3SS) to secrete therapeutic proteins both inside and in the vicinity of mammalian cells. The T3SS is a nanomachine that traditionally delivers effector proteins into the host cell to mediate pathogenesis. Fusion of a type III effector secretion sequence to therapeutic proteins results in variants that are recognized as secreted substrates. We recently developed probiotic E. coli strains that express a T3SS. These probiotic T3SS competent E. coli strains can deliver therapeutic proteins modified with type III secretion signals into host cells. This work describes the development of engineered probiotic T3SS competent E. coli that can secrete VHH proteins. VHH are therapeutic antibody fragments that effectively bind and block activity of target antigens. We demonstrate that DH10B E. coli can use the T3SS to directly secrete VHH, modified with the OspC3 effector secretion sequence, into mammalian cells. These bacteria successfully deliver a variety of VHHs, including those that bind to inflammasome ASC protein and a heterotetrameric VHH-based neutralizing agent (VNA) designed to neutralize Shiga toxin (Stx). A remaining question is whether VHH or VNA secreted via the T3SS maintain their function. If evidence indicates αASC VHH inhibit inflammasome activation, we envision this VHH as a potential anti-inflammatory therapeutic. Whereas, a functional secreted αStx VNA could serve as a therapeutic against enterohemorrhagic E. coli infections. We envision modifying our T3SS platform to secrete the αStx VNA into the intestinal lumen as well as the cytosol. Finally, we engineered probiotic E. coli strains MP, HS and Nissle 1917 for future use in VHH delivery. This research pursues a novel approach of utilizing bacterial type III secretion systems to deliver therapeutic VHH and VNA to the gut.