Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in Agrobacterium tumefaciens

Abstract

The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.

Serine/threonine kinase PpkA coordinates the interplay between T6SS2 activation and quorum sensing in the marine pathogen Vibrio alginolyticus

“…DotU (TssL) proteins localize to the inner membrane and serve as a critical component of the T6S anchor module (Durand et al ., ; Zoued et al ., ). A. tumefaciens TssL is phosphorylated solely at T14 by PpkA (Lin et al ., ) (Supporting information Fig. S7A).…”

“…In A. tumefaciens phosphorylated TssL (DotU) interacts with Fha, a forkhead‐associated domain‐containing protein, and this phosphorylation dependent interaction is required for T6S activity (Mougous et al ., ; Lin et al ., ). Indeed, we found that EPGS mutants harbouring either a deletion of fha2 (Δ fha2 ) or the substitution of conserved Fha2 residues implicated in phosphopeptide binding (Arg 36 and Ser 52) with alanine (R36A S52A) (Supporting information Fig.…”

“…D). Taken together, these findings strongly suggest that phosphorylation of DotU2's Thr4 and Thr28 residues enable its interation with Fha2 and T6SS2 function, a manner similar to that reported in A. tumefaciens (Lin et al ., ),…”

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

“…A mechanism of posttranslational activation, named the threonine phosphorylation pathway (TPP), has been described in P. aeruginosa and Agrobacterium tumefaciens. It requires sensing and signaling of the attack by the TagQRST proteins, leading to the PpkA-dependent phosphorylation of a forkhead-associated protein (FHA) or of the TssL T6SS core component and, ultimately, to the activation of the late stages of T6SS assembly (93)(94)(95)(96)(97). However, no homologue subunits to the TagQRST-PpkA-PppA-FHA threonine phosphorylation pathway are encoded within the S. Typhimurium SPI-6 T6SS gene cluster, suggesting that the mechanism of sensing and posttranslational activation is different from that described in P. aeruginosa or A. tumefaciens.…”

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

“…Agrobacterium tumefaceins C58 harbors one T6SS that is activated transcriptionally by an ExoR-ChvG/ChvI signaling cascade and posttranslationally via threonine phosphorylation when sensing acidity (34)(35)(36). Three type VI effectors, namely type VI amidase effector (Tae) and type VI DNase effectors (Tde1 and Tde2), confer antibacterial activity of this bacterium.…”