Studies on virulence-critical proteins of enteropathogenic Escherichia coli (EPEC)

Abstract

Enteropathogenic Escherichia coli (EPEC) virulence depends on a Type-3 Secretion System (T3SS) that transfers many ‘effector’ proteins into human gastrointestinal cells. The components for the effector-delivery apparatus (T3SS and translocator proteins), virulence-critical surface protein (Intimin) and seven effectors (EspG, EspF, Map, EspH, EspZ, Tir, EspB; latter also a translocator) are encoded on the Locus of Enterocyte Effacement (LEE) pathogenicity island. The EspA translocator protein extends the T3SS and is tipped with EspB and EspD which insert into the plasma membrane enabling effector delivery into the host cytoplasm. Two LEE effectors, Tir and EspZ, have virulence-critical functions with both inserted into the plasma membrane and linked, for Tir, as a receptor for EPEC (via Intimin) and, for EspZ, to prevent a cytotoxic response. It is controversial how Tir becomes inserted into membranes and how EspZ prevent cytotoxicity. Previous work predicted that Tir insertion depends on LEE effector activities. Here, we demonstrate LEE sufficiency for Tir insertion and rule out roles for Intimin and classical LEE (EspG, Map, EspF, EspH, EspZ) effectors. Surprisingly, our data implicated roles for the EspA and EspD translocators in stable Tir-intimin interactions and revealed a new EspZ protective mechanism i.e. prevention of cytotoxicity triggered as a consequence of Tir-Intimin interaction. Furthermore, we provide bioinformatic and experimental support that an unusual Edwardsiella tarda LEE-like region encodes a functional effector-delivery system. The swapping of protein homologues between E. tarda and EPEC has also provided an opportunity to gain insights on the structure/function of ~20 T3SS/translocon components and virulence-critical Tir, Intimin and EspZ proteins.