The β-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion

Abstract

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs− mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro−Ala−Ala−Arg (PAAR) repeat domains, which bind the β-spike of trimeric valine−glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact β-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs+ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the β-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact β-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Structure of a bacterial Rhs effector exported by the type VI secretion system

“…In contrast to a strain expressing wild-type RhsA, strains expressing the D1324N or D1346N variants of the protein were unable to outcompete RhsA-sensitive recipient bacteria indicating that C-terminal autocleavage is required for T6SS-dependent killing by this effector (Figure 2C). These findings mirror what has been observed for T6SS-exported Rhs proteins in Aeromonas dhakensis and Enterobacter cloacae suggesting that toxin domain liberation is a universal property of Rhs effectors 18, 31 .…”

“…These findings mirror what has been observed for T6SS-exported Rhs proteins in Aeromonas dhakensis and Enterobacter cloacae suggesting that toxin domain liberation is a universal property of Rhs effectors 18,31 .…”

“…Based on the collective structural and functional data presented in this work combined with the findings of other recently published 18, 31 work on T6SS-exported Rhs proteins, we propose a model for cytoplasmic delivery of RhsA and suggest a possible release mechanism for the toxin domain of the effector (Figure 6).…”

Diversification of the Type VI Secretion System in Agrobacteria

“…Modularity is an important property of T6SSs, as it allows components to organize at multiple levels and evolve semiautonomously, preserving functionality while conferring flexibility ( 50 ). For example, RHS and specialized effectors, also in agrobacteria, allow for reshuffling of toxin domains downstream of a conserved sequence while maintaining functions necessary for T6SS assembly and activation ( 51 , 52 ). In addition, genes necessary for effector loading, a final checkpoint that regulates T6SS assembly and activation, are consistently clustered as a functional unit in vgrG loci ( 39 , 53 ).…”

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms

“…These authors also suggested that the T6SS-2 was implicated in biofilm formation and cell adherence and that it contributed to bacterial colonization of the mouse gut in vivo 41 . The finding on the system's functionality should be taken with caution, however, as Donato and colleagues showed that the T6SS-2 of E. cloacae strain ATCC 13047 was defective due to a large deletion and the insertion of an IS903 element, which led to the pseudogenization of several T6SS-2 genes (clpV2, vgrG3, PAAR, and tssF2) 42 . Notably, the sequence of the T6SS-2 cluster in the here-described sequencing data of strain DSM 30054/ATCC 13047 was 100% identical to the one previously reported (accession number CP001918 43 ), confirming these pseudogenes.…”