Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus.

Tobias Dietsche,Mirita Franz-Wachtel,Jun Yan,Mehari Tesfazgi Mebrhatu,Matthias J Brunner,Samuel Wagner,Oliver Kohlbacher,Susann Zilkenat,Boris Macek,Charlotta Schärfe,Patrizia Abrusci,Iwan Grin,Susan Lea,Carol V Robinson,Thomas C Marlovits,Jorge E Galán,Brian K Coombes

doi:10.1371/journal.ppat.1006071

Abstract

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.

Highlights

Type III secretion systems (T3SSs) are used by many Gram-negative bacterial pathogens and symbionts to translocate effector proteins in one step across the bacterial envelope and into eukaryotic host cells [1] where they modulate host cell physiology to promote bacterial survival and colonization [2]
We further show that one subunit each of the proteins SpaQ, SpaR, and SpaS are closely associated to the SpaP gate and may function in the gating mechanism, and that the protein PrgJ is attached to this gate on the outside to connect it to the hollow needle filament projecting towards the host cell
We further show that a complex of SpaP, SpaQ, SpaR, and SpaS assembles in vivo before incorporation into the needle complex base, and that these four export apparatus components form a compact assembly with multiple reciprocal interactions at TM helices three and four of the SpaP pentamer

Summary

Introduction

Type III secretion systems (T3SSs) are used by many Gram-negative bacterial pathogens and symbionts to translocate effector proteins in one step across the bacterial envelope and into eukaryotic host cells [1] where they modulate host cell physiology to promote bacterial survival and colonization [2]. The base of the injectisome, consisting of an outer membrane secretin ring and two inner membrane ring components, anchors the system to the bacterial cell envelope [3]. A filamentous needle projects away from the base towards the host cell and serves as conduit for translocated effectors [4,5]. Five cytoplasmic proteins select and unfold the substrates, which are handed over to the actual export apparatus [6,7] housed in a membrane patch at the center of the inner ring [8,9]. The five export apparatus components are thought to facilitate the actual secretion function of T3SSs, including energy coupling, membrane translocation, and substrate specificity switching [1]. Needle filament, and export apparatus are together termed needle complex

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Conclusion