New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

Grant English,William N Hunter,Sarah J Coulthurst,Velupillai Srikannathasan,Katharina Trunk,Vincenzo A Rao

doi:10.1111/mmi.12028

Abstract

Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self-resistance (‘immunity’) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.

Highlights

Protein secretion systems and their substrates are central to bacterial virulence and interaction with other organisms (Gerlach and Hensel, 2007)
Protein secretion systems are critical to bacterial virulence and interactions with other organisms
Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS

Summary

Introduction

Protein secretion systems and their substrates are central to bacterial virulence and interaction with other organisms (Gerlach and Hensel, 2007). Six different secretion systems (Types I–VI) are used by Gram-negative bacteria to transport specific proteins to the exterior of the bacterial cell or further inject them into target cells. T6SSs are complex multi-protein assemblies that span both bacterial membranes and inject effector proteins directly from the bacterial cytoplasm into target cells (Bonemann et al, 2010; Cascales and Cambillau, 2012). Hcp and VgrG, form the extracellular part of the secretion machinery and depend on a functional T6SS apparatus for their movement to the outside of the bacterial cell (the presence of Hcp in the secreted fraction has provided a useful assay for basic T6SS assembly and activity; Pukatzki et al, 2009).

Results

Discussion

Conclusion