A disordered region in the EvpP protein from the type VI secretion system of Edwardsiella tarda is essential for EvpC binding.

Wentao Hu,Ka Yin Leung,Yu-Keung Mok,Ganesh Anand,J Sivaraman,Shannon Wing-Ngor Au

doi:10.1371/journal.pone.0110810

Abstract

The type VI secretion system (T6SS) of pathogenic bacteria plays important roles in both virulence and inter-bacterial competitions. The effectors of T6SS are presumed to be transported either by attaching to the tip protein or by interacting with HcpI (haemolysin corregulated protein 1). In Edwardsiella tarda PPD130/91, the T6SS secreted protein EvpP (E. tarda virulent protein P) is found to be essential for virulence and directly interacts with EvpC (Hcp-like), suggesting that it could be a potential effector. Using limited protease digestion, nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa) contains a substantial proportion (40%) of disordered regions but still maintains an ordered and folded core domain. We show that an N-terminal, 10-kDa, protease-resistant fragment in EvpP connects to a shorter, 4-kDa protease-resistant fragment through a highly flexible region, which is followed by another disordered region at the C-terminus. Within this C-terminal disordered region, residues Pro143 to Ile168 are essential for its interaction with EvpC. Unlike the highly unfolded T3SS effector, which has a lower molecular weight and is maintained in an unfolded conformation with a dedicated chaperone, the T6SS effector seems to be relatively larger, folded but partially disordered and uses HcpI as a chaperone.

Highlights

The type VI secretion system (T6SS) was first discovered as an essential bacterial secretion system for bacterial pathogenesis [1,2], and its activity is tightly controlled by both environmental and bacterial regulatory factors [3]
Nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa) contains a substantial proportion (40%) of disordered regions but still maintains an ordered and folded core domain
Accumulated evidence shows that the T6SS of pathogenic bacteria have dual roles in both pathogenicity and inter-bacterial competition, and that the T6SS is an effective weaponry against other Gram-negative bacteria in the living habitat [4]

Summary

Introduction

The type VI secretion system (T6SS) was first discovered as an essential bacterial secretion system for bacterial pathogenesis [1,2], and its activity is tightly controlled by both environmental and bacterial regulatory factors [3]. The T6SS core apparatus assembles from 13 ‘‘core components’’ proteins, from TssA-M (type six sub-units), in addition to Hcp (haemolysin corregulated protein), VgrG (valine-glycine repeat protein G), the ClpV AAA+ ATPase and the T4SS IcmFand IcmH-like proteins [6,7,8]. These proteins are arranged in the form of two sub-assemblies: a dynamic bacteriophage-like structure and a cell-envelope-spanning membrane-associated assembly [9]. TssK is a trimer that connects the two systems by directly interacting with TssL, HcpI and TssC [17]

Methods

Results

Conclusion