Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli

Abstract

SummaryMany mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III-secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)-EPEC strains exhibit a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3± 5 nm. The pores induced by type III-secreted proteins of DA-EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55±65 nm and to be raised 15±20 nm above the membrane plane.We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro. These results, together with the homologies of EspB and EspD to proposed functional domains of other poreforming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.

Identification of a Third EspA-binding Protein That Forms Part of the Type III Secretion System of Enterohemorrhagic Escherichia coli

“…From the same transcript as that for EspA, two additional translocators (EspD and EspB) are translated to form the transmembrane pore that facilitates effector translocation across the host membrane (14). Secretion of the TTS translocators requires the assistance of cytoplasmic chaperones that have some common properties, namely that they have a low molecular mass (Ͻ15 kDa), have an acidic pI, and contain a C-terminal amphipathic helix (41).…”

“…EspA polymerizes into the filamentous structure, and its polymerization is dependent on coiled-coil interactions between the C-terminal region of the molecule (11) in a way similar to flagella assembly (9). EspB has been reported to interact with EspA (12,13) and EspD (14), and the complexes that form at the distal portion of the needle structure may act as a pore at the contact site with the host cell membrane and participate in the initial step of bacterial adherence (15).…”

Mecanismos de virulencia de Escherichia coli enteropatógena

“…De forma simultánea a la unión de T3SS al enterocito y a la entrada de las proteínas efectoras a través de los poros, la bacteria ingresa la proteína Tir (receptor) que facilita la adherencia a la intimina bacteriana y es indispensable para la formación del pedestal y la lesión intestinal 35 . Una vez dentro de la célula hospedera, la cabeza de Tir se proyecta a la superficie de la membrana del enterocito, donde actúa como receptor para la intimina y para la transmisión de señales después de la interacción 36 .…”

Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme

“…In EPEC, the needle in turn is capped by a filamentous extension composed of EspA (13)(14)(15)(16)(17) that facilitates the span of the T3SS across the microvilliated surface of the infected host gut epithelial cells. In the final step of assembly, the translocon (EspB and EspD) assembles at the tip of the EspA filament and inserts into the host membrane to form a pore (18) for direct delivery of virulence effectors into the host cell.…”