Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

Sean Miletic,Jiri Wald,Nikolaus Goessweiner-Mohr,Thomas C Marlovits,Maurice Pantel,Oliver Vesper,Vadim Kotov,Dirk Fahrenkamp

doi:10.1038/s41467-021-21143-1

Abstract

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.

Highlights

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection
We previously showed that the Salmonella late effector protein SptP fused to a green fluorescent protein (GFP) tag can be visualized as a subtracted density in the needle complex, confirming that the filament functions as the conduit for effector proteins[18]
To obtain molecular snapshots of an injectisome needle complex engaged with a substrate, we applied cryogenic electron microscopy to purified needle complexes, which had been enriched for trapped SptP3x-GFP by immunoprecipitation (Supplementary Figs. 1 and 2)

Summary

Introduction

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. Many important human pathogens including Salmonella, Shigella, Yersinia, and enteropathogenic Escherichia coli employ a conserved, virulent type III secretion system (T3SS), commonly referred to as the injectisome, to deliver a pleiotropic arsenal of proteins into target eukaryotic cells[1] These proteins modulate host cell signal transduction processes to establish a biological niche within the host, making T3SSs crucial virulence determinants[2]. In all structures known to date, the proposed translocation channel through the EA is sealed by a gasket with an above loop, making comprehension of substrate transport through the needle complex difficult It remains unclear how the EA achieves selective effector protein transport given the multitude of proteins that are present in the bacterial cytoplasm. Direct visualization of a substrate throughout the complete secretion conduit has remained challenging, leaving questions as to how and where the EA would eventually open to allow passage of effector proteins, while maintaining the integrity and composition of compartments separated by a biological membrane, unresolved

Methods

Results

Conclusion