Polarity of Enteropathogenic Escherichia coli EspA Filament Assembly and Protein Secretion

Abstract

Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic Escherichia coli, two important human enteric pathogens, is unique in that it has a ϳ12-nmdiameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).

Structure of a type III secretion needle at 7-Å resolution provides insights into its assembly and signaling mechanisms

“…The structure also provides insight into needle-length control. Needle subunits, like axial-flagellar proteins, are exported by T3SS through the central channel of the growing structure and assemble at its distal end (27,28). The lengths of the needle and the flagellar hook are both controlled by conserved ruler proteins (29,30).…”

What's the point of the type III secretion system needle?

“…The smaller size of the needle subunits, relative to the flagellin subunit, may mean that the termini of the unfolded subunit remain in sufficiently close proximity to the top of the assembling needle that they correctly associate, without needing to be trapped at the assembly site by a separate capping system. This argument assumes that the needle assembles from the tip as demonstrated for other related systems (32,33).…”

The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis

“…Fluorescent actin staining (FAS) and the EspA immunofluorescent filament staining tests were performed as previously described (26,37). As a negative control, we used an EPEC espA mutant, strain UMD872 (38), and as a positive control, we used UMD872 with plasmid pICC284 expressing the EHEC EspA protein from a pBADMycHis vector (39). Tir translocation assays were performed as previously described (29).…”