A binary effector module secreted by a type VI secretion system

Abstract

Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Cargo effectors are secreted by binding non-covalently to the T6SS apparatus. Occasionally, effector secretion is assisted by an adaptor protein, although the adaptor itself is not secreted. Here, we report a new T6SS secretion mechanism, in which an effector and a co-effector are secreted together. Specifically, we identified a novel periplasm-targeting effector that is secreted together with its co-effector, which contains a MIX (marker for type sIX effector) domain previously reported only in polymorphic toxins. The effector and co-effector directly interact, and they are dependent on each other for secretion. We termed this new secretion mechanism “a binary effector module”, and we show that it is widely distributed in marine bacteria.

A DNase T6SS effector requires its MIX domain for secretion

“…Toxicity in E. coli: Bacterial toxicity assays were performed as previously described (Dar et al, 2021), with minor changes. Briefly, to assess the toxicity mediated by VPA1263 or its truncated versions, pBAD K /Myc-His plasmids encoding the indicated proteins were transformed into E. coli MG1655.…”

“…Bacterial competition assays: Bacterial competition assays were performed as previously described (Dar et al, 2021). Briefly, attacker and prey strains were grown overnight in MLB with the addition of antibiotics when maintenance of plasmids was required.…”

“…Protein expression in E. coli: Protein expression in E. coli was performed as previously described (Dar et al, 2021), with minor changes. Briefly, to assess the expression of C-terminal Myc-tagged proteins encoded on arabinose-inducible plasmids, overnight cultures of E. coli MG1655 strains containing the indicated plasmids were washed twice with fresh 2xYT broth to remove residual glucose.…”

“…Vibrio growth assays: Vibrio growth assays were performed as previously described (Dar et al, 2021). Briefly, overnight cultures of V. parahaemolyticus strains were normalized to OD600 = 0.01 in MLB and transferred to 96-well plates (200 μL per well; n = 3 technical replicates).…”

“…The toxins, called effectors, decorate a secreted arrow-like structure comprising an inner tube, made of stacked Hcp hexamers, and a capping spike containing a VgrG trimer and a PAAR repeat-containing protein (Silverman et al, 2013; Hachani et al, 2014; Burkinshaw et al, 2018; Wang et al , 2019); the effector-decorated arrow is propelled outside of the cell by a contracting sheath structure that engulfs the inner tube (Basler et al , 2012). Effectors can be either specialized – Hcp, VgrG, or PAAR proteins containing additional toxin domains at their C-termini - or cargo effectors, which are proteins that non-covalently bind to Hcp, VgrG, or PAAR with or without the aid of an adaptor protein or a co-effector (Jana & Salomon, 2019; Jurėnas & Journet, 2021; Unterweger et al, 2015; Liang et al, 2015; Bondage et al, 2016; Alcoforado Diniz & Coulthurst, 2015; Cianfanelli et al , 2016; Dar et al , 2021). To date, three classes of polymorphic T6SS cargo effectors containing MIX (Salomon et al, 2014a), FIX (Jana et al, 2019), or Rhs (Koskiniemi et al, 2013) domains have been characterized; the Rhs domain is not restricted to T6SS effectors (Koskiniemi et al, 2013).…”