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

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.

Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair

“…PP and Tgi2 PP is consistent with the affinities measured for other T6SS E-I pairs, including P. aeruginosa Tae1-Tai1, Enterobacter cloacae Tae4-Tai4, and two Tae4-Tai4 family members from S. marcescens, which all bind with low nanomolar to high picomolar dissociation constants (12,13,15).…”

“…Cell wall-degrading T6S effectors distribute into two groups: those that act as amidases, cleaving the peptidoglycan molecule within its peptide stems and cross-links and those that act as glycoside hydrolases, cleaving the glycan backbone of the molecule. T6S amidase effectors have been studied extensively (3,9,10,(12)(13)(14)(15). The enzymes are broadly distributed among Proteobacteria and form four phylogenetically distinct families that constitute the Tae (type IV secretion amidase effector) superfamily.…”

“…This approach was used for the identification of Tae4 family members from Serratia marcescens (12). Alternatively, mass spectrometry-based methodologies have been successful in the identification of T6S effectors from P. aeruginosa, Burkholderia thailandensis, and S. marcescens (2,9,18).…”

The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

“…Gram-negative bacteria through the delivery of different toxins which target the peptidoglycan of susceptible bacterial species (30)(31)(32)(33)(34). A heuristic informatic approach was recently developed for large-scale identification of T6SS antibacterial effectors (35).…”

Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae

“…T6SS dueling activity has also been recently shown to be critical to the selection and targeting of T6SS + prey by P. aeruginosa (17), suggesting immunity proteins may also be important for resistance to heterologous attack by related T6SS + organisms. Despite the large number of T6SS-possessing bacteria, T6SS effector-immunity pairs have only been experimentally identified in P. aeruginosa (15), Burkholderia thailandensis (18), and Serratia marcescens (19), and they share little homology in primary sequence. A heuristic approach based on physical and contextual properties of known effector-immunity pairs predicts that they are widespread in T6SS-possessing bacteria (18).…”