Fig. 1. Engineering the bacteria-tumor interface.

Engineered bacteria localize their payloads intracellularly [(A) to (D)] or extracellularly [(E) to (H)] and release them by secretion, diffusion, or lysis mechanisms. (A) Bacteria have been engineered to more specifically bind to tumor cells by displaying targeting moieties such as RGD on the external loop of outer membrane proteins such as Omp-A (11). (B) Modified T3SS secretion using the pCASP-HilA vector enables bacterial delivery of macromolecules. hilA and sicP are expressed to improve secretion efficiency and the payload is tagged with a type III secretion signal sequence and chaperone binding domain for cytosolic delivery (15). (C) Expression of invasin, encoded by inv, promotes bacterial uptake into phagosomes and increases invasion efficiency of otherwise extracellular bacteria such as noninvasive E. coli strains, allowing for protected intravacuolar replication. The addition of LLO, encoded by hlyA, increases transfer efficiency of payloads such as plasmids into target cells by forming pores within the vacuolar membrane (17). (D) Bacteria have also been encoded with lysis circuits to enhance the passage of drugs through the bacterial membrane. An arabinose-inducible circuit regulates expression of the flhDC operon, which mediates Salmonella motility and invasion. In turn, bacterial lysis, achieved through the expression of by the bacteriophage-derived lysis gene lysE, is activated upon invasion (23). (E) Neoantigens with an MMP target sequence can be expressed with outer membrane proteins (omp) of S. typhimurium. Tumor-enriched MMPs can then cleave the MMP target sequence, releasing neoantigens locally within the extracellular tumor space (41). (F) Extracellular bacteria such as E. coli have also been encoded with modified T3SS components, whereby expression of the mxi and spa operons are necessary for the expression of T3SS structural components. This construct allows the secretion of therapeutic payloads modified with an N-terminal type III secretion signal sequence to be released outside of the tumor cell (28). (G) Acting as intratumoral bioreactors, E. coli have been engineered to metabolize ammonia, a waste product generated by tumors, into L-arginine. Genomic modifications were made to prevent the negative regulation or inhibition of genes in the biosynthesis pathway by deleting ArgR and integrating ArgA^fbr (42). (H) Bacteria have also been engineered with LuxR-based QS systems that rely on the diffusion of the autoinducer AHL between cells. In this system, luxI produces AHL, which binds to LuxR, engaging the plux promoter for positive feedback regulation. Because AHL is also able to diffuse freely between cells, bacteria can sense when they are at a critical density and drive expression of lysE, inducing quorum-based lysis and repeated intratumoral drug delivery (30).