<i>In situ</i> deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic <i>E. coli</i>

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic <i>Escherichia coli</i> (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. <i>Citrobacter rodentium</i> (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the <i>C. rodentium</i> genome, we generated Cr_Tir-M^EHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse precolonization with HS-PROT₃EcT-TD4, a human commensal <i>E. coli</i> strain (<i>E. coli</i> HS) engineered to efficiently secrete an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-M^EHEC(ΦStx2a). This study suggests that commensal <i>E. coli</i> engineered to deliver payloads that block essential virulence determinants can be developed as a new means to prevent and potentially treat infections including those due to antibiotic resistant microbes.