Abstract
Type VI secretion systems (T6SSs) translocate effector proteins, such as Rhs toxins, to eukaryotic cells or prokaryotic competitors. All T6SS Rhs-type effectors characterized thus far contain a PAAR motif or a similar structure. Here, we describe a T6SS-dependent delivery mechanism for a subset of Rhs proteins that lack a PAAR motif. We show that the N-terminal Rhs domain of protein RhsP (or VP1517) from Vibrio parahaemolyticus inhibits the activity of the C-terminal DNase domain. Upon auto-proteolysis, the Rhs fragment remains inside the cells, and the C-terminal region interacts with PAAR2 and is secreted by T6SS2; therefore, RhsP acts as a pro-effector. Furthermore, we show that RhsP contributes to the control of certain “social cheaters” (opaR mutants). Genes encoding proteins with similar Rhs and PAAR-interacting domains, but diverse C-terminal regions, are widely distributed among Vibrio species.
Highlights
We conducted a systematic genomic analysis on the V. parahaemolyticus strain RIMD2210633 based on our previouslycurated toxin domain profile database for potential toxins contributing to bacterial fitness or pathogenesis[35,36]
Overexpression of the fulllength RhsP tagged by epitope FLAG under the control of the araBAD promoter in E. coli DH5α unexpectedly resulted in no toxicity (Fig. 1b and Supplementary Fig. 1a)
The re-isolated plasmid from E. coli did not contain any mutation in the open reading frame of RhsP, excluding inactivation of its toxicity through spontaneous mutations
Summary
We observed that both RhsP and RhsPi are under the positive regulation of OpaR and the expression of RhsPi was significantly reduced in ΔopaR compared to wild-type in V. parahaemolyticus (Supplementary Fig. 4b & c). VP1518 has T6SS2 was intact and functional (e.g., in wild-type or ΔclpV2 protective effect against RhsPC-mediated killing, because wild- complementation strain ΔclpV2 + clpV2) (Fig. 5c), attesting that a pBAD24 WHH domain pCX340 vp151(r8hsPi) vp1519 vp1520 Positively regulates T6SS2 as well as its toxic pro-effector RhsP/immunity protein RhsPi, QS− produces much less amount of RhsPC and RhsPi than QS+.
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