TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha

Abstract

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.

Keywords: Agrobacterium tumefaciens; Fha; PppA; Pseudomonas aeruginosa; TagF; antibacterial activity; bacterial genetics; gene regulation; post-translational regulation; protein phosphorylation; protein secretion; protein-protein interaction; type VI secretion system.

Figure 1.

A. tumefaciens C58 t6ss gene clusters and TagF-pppA domain organization. A, the imp operon (atu4343 to atu4330), hcp operon (atu4344 to atu4352), and vgrG2 in A. tumefaciens strain C58 were designated tss or tag based on nomenclature proposed by Shalom et al. (34) and specific names derived from Lin et al. (30) and Bondage et al. (31). B, TagF-PppA domain organization according to information from the NCBI conserved domain database. TagF-PppA is predicted as a cytoplasmic protein (aa 1–471) with an N-terminal conserved TagF (DUF2094) domain (aa 11–219) and a C-terminal PppA (PP2Cc) domain (aa 244–470).

Figure 2.

Both TagF and PppA domains can repress type VI secretion and antibacterial activity at post-translational levels in A. tumefaciens. A, type VI secretion analysis. Shown is Western blot analysis of total (T) and secreted (S) proteins isolated from WT C58 harboring the vector pTrc200 (V) or various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The nonsecreted protein ActC and RNA polymerase α subunit RpoA were internal controls. The proteins analyzed and sizes of molecular weight standards are shown on the left and right, respectively, and indicated with arrowheads when necessary. FL, full-length TagF-PppA protein. B, A. tumefaciens antibacterial activity assay against E. coli. The A. tumefaciens WT C58 harboring the vector pTrc200 (V) or various overexpressed plasmids or ΔtssL mutant harboring the vector pTrc200 (V) was co-cultured on LB agar with E. coli strain DH10B cells harboring the plasmid pRL662. C, A. tumefaciens intraspecies competition in planta. The A. tumefaciens WT C58 harboring the vector pTrc200 (V) or various overexpressed plasmids or ΔtssL mutant harboring the vector pTrc200 (V) was used as attacker strain to mix with the target strain Δ3TIs harboring pRL662 and infiltrated into N. benthamiana leaves. B and C, data are mean ± S.D. of at least three biological replicates. Different letters above the bar indicate statistically significantly different groups of strains (p < 0.01 for B; p < 0.05 for C) based on cfu of the surviving target cells.

Figure 3.

Both TagF and PppA domains repress T6SS activity independently of the PpkA-mediated TssL phosphorylation pathway in A. tumefaciens. A, Phos-tag SDS-PAGE analysis to detect the phosphorylation status of TssL-His. Shown is Western blot analysis of the same volumes of Ni-NTA resins (10 μl) associated with TssL-His from different strains treated with (+) or without (−) CIAP and examined by a specific antibody against His₆. Total protein isolated from ΔtssL was a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His (p-TssL-His) and lower band indicating unphosphorylated TssL-His. B and C, Western blot analysis of the endogenous phosphorylation status of TssL (pTssL). Shown is Western blot analysis of total proteins isolated from WT C58, ΔppkA, ΔtssL, or C58 harboring the vector pTrc200 (V) or various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The specific antibody for pTssL was generated against the 15-mer peptide (⁷SSWQDLPpTVVEITEE²¹), with phosphorylated Thr-14 of TssL underlined. RNA polymerase α subunit RpoA was an internal control. The proteins analyzed and molecular weight standards are on the left and right, respectively, and are indicated with an arrowhead when necessary. FL, full-length TagF-PppA proteins.

Figure 4.

TagF represses T6SS activity independent of the TPP pathway in A. tumefaciens. A and B, type VI secretion analysis. Shown is Western blot analysis of total (T) and secreted (S) proteins isolated from WT C58 harboring the vector pTrc200 (V) or ΔtssL harboring the vector pTrc200 (V) or ΔppkAΔtagF-pppA harboring various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The nonsecreted protein ActC and RNA polymerase α subunit RpoA were internal controls. The proteins analyzed and molecular weight standards are shown on the left and right, respectively, and indicated with an arrowhead when necessary. C, A. tumefaciens antibacterial activity assay against E. coli. The A. tumefaciens WT C58 harboring the vector pTrc200 (V), ΔtssL harboring the vector pTrc200 (V), or ΔppkAΔtagF-pppA harboring various overexpressing plasmids was co-cultured on LB agar with E. coli strain DH10B cells harboring pRL662. Data are mean ± S.D. (error bars) of at least three biological replicates. Different letters above the bar indicate statistically significantly different groups of strains (p < 0.01) based on cfu of the surviving target cells.

Figure 5.

TagF directly interacts with Fha of A. tumefaciens and P. aeruginosa. A and B, yeast two-hybrid protein–protein interaction results. SD−WL medium (SD minimal medium lacking Trp and Leu) was used for the selection of plasmids. SD−WLHA medium (SD minimal medium lacking Trp, Leu, His, and Ade) was used for the auxotrophic selection of bait and prey protein interactions. The positive interaction was determined by growth on SD−WLHA medium at 30 °C for at least 2 days. The positive control (+) showing interactions of SV40 large T-antigen and murine p53 and negative control (vector) are indicated. C, bacterial two-hybrid analysis. Various combinations of recombinant pKT25 and pUT18C plasmids harboring P. aeruginosa TagF^Pa or Fha1^Pa proteins were co-transformed into E. coli. A graphical representation of the β-gal activity from co-transformants is shown, the plasmid combinations are indicated below, and images of corresponding E. coli spots on LB agar plates containing X-gal are displayed at the top. The strength of the interaction was investigated by measuring the β-gal activity of cells. The average activity in Miller units is indicated. Experiments were carried out in duplicate, and data are mean ± S.D. (error bars) Different letters above the bar indicate statistically significantly different groups (p < 0.01). T18, empty vector pUT18C; T25, empty vector pKT25.

Figure 6.

Conserved amino acid residues of TagF are critical for TagF–Fha interaction in A. tumefaciens. A, amino acid sequence alignment of TagF or TagF domain orthologs from selected bacterial species. Conserved amino acid residues are highlighted in black, and those used for mutagenesis are indicated with an asterisk. Sequences were aligned and highlighted by use of ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.) Part of the aligned result is shown here, and the fully aligned result and full information for bacterial strains and protein accession numbers are shown in Fig. S3A. B, Agrobacterium TagF protein is present as a monomer on gel filtration analysis in vitro. Purified His-tagged TagF domain (aa 1–214) was analyzed by SDS-PAGE. The proteins analyzed and molecular weight standards are shown on the right and left, respectively. His-tagged TagF proteins were further analyzed by use of a Superdex 75 16 × 60 column, and the elution profiles were recorded as absorbance at 280 nm showing that His-tagged TagF elutes as a single peak (∼26 kDa monomer). C, relative positions of the conserved amino acid residues in P. aeruginosa TagF^Pa protein revealed as a monomer with crystal structural information according to the X-ray crystal structure of P. aeruginosa TagF monomer (Protein Data Bank entry 2QNU). The corresponding conserved amino acid residues of A. tumefaciens TagF are indicated in parenthesis. D, yeast two-hybrid protein–protein interaction results with Fha and various TagF proteins. SD−WL medium (SD minimal medium lacking Trp and Leu) was used for selecting plasmids. SD−WLHA medium (SD minimal medium lacking Trp, Leu, His, and Ade) was used for the auxotrophic selection of bait and prey protein interactions. The positive interaction was determined by growth on SD−WLHA medium at 30 °C for at least 2 days. The positive control (+) showing interactions of SV40 large T-antigen and murine p53 and negative control (vector) are indicated.

Figure 7.

Conserved amino acid residues of TagF are required for repressing type VI activity in A. tumefaciens. A, Western blot analysis of total (T) and secreted (S) proteins isolated from WT C58 harboring the vector pTrc200 (V) or various TagF-Strep–overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The nonsecreted protein ActC and RNA polymerase α subunit RpoA were internal controls. The proteins analyzed and molecular weight standards are shown on the left and right, respectively. B, A. tumefaciens antibacterial activity assay against E. coli. The A. tumefaciens WT C58 or ΔtssL or chromosomally encoded tagF-pppA variants, including tagF-pppA with substitutions in the tagF domain (tagF^GK-pppA, tagF^DW-pppA, and tagF^SDR-pppA), were co-cultured on LB agar with E. coli strain DH10B cells harboring the plasmid pRL662. Data are mean ± S.D. (error bars) of at least three biological replicates. Different letters above the bar indicate statistically significantly different groups of strains (p < 0.05) based on cfu of the surviving target cells.

Figure 8.

Conserved amino acid residues of TagF^Pa critical for TagF^Pa–Fha1^Pa interaction are required for repressing H1-T6SS activity in P. aeruginosa. A, yeast two-hybrid protein–protein interaction results with P. aeruginosa Fha1 and various P. aeruginosa TagF proteins. SD−WL medium (SD minimal medium lacking Trp and Leu) was used for selecting plasmids. SD−WLHA medium (SD minimal medium lacking Trp, Leu, His, and Ade) was used for auxotrophic selection of bait and prey protein interactions. The positive interaction was determined by growth on SD−WLHA medium at 30 °C for at least 2 days. The positive control (+) showing interactions of SV40 large T-antigen and murine p53 and negative control (vector) are indicated. B, P. aeruginosa H1-T6SS secretion analysis. Shown is Western blot analysis of total (T) or secreted (S) proteins isolated from P. aeruginosa PAKΔretS (H1-T6SS–induced) harboring the vector pRL662 (V) or PAKΔretS harboring various overexpressed plasmids grown in TSB with specific antibodies. The nonsecreted RNA polymerase β subunit (RNAP) was an internal control. The proteins analyzed and molecular weight standards are shown on the left and right, respectively, and indicated with an arrowhead when necessary. C, P. aeruginosa H1-T6SS–mediated antibacterial assay against E. coli. Overnight cultures of P. aeruginosa PAKΔretS or PAKΔretSΔtssB1 (T6SS-defective strain) harboring the vector pRL662 (V) or various tagF-Strep–overexpressing plasmids were mixed with equivalent numbers of E. coli DH5α carrying a plasmid (pCR2.1) expressing β-gal. Data are mean ± S.D. of at least three biological replicates. Different letters above the bar indicate statistically significantly different groups of strains (p < 0.05) based on cfu of the surviving target cells. D, the presence of Fha1^Pa increases the stability of TagF^Pa protein in P. aeruginosa. Shown is Western blot analysis of total (T) proteins isolated from P. aeruginosa PAKΔretS (H1-T6SS–induced) or PAKΔretSΔH1 (deletion of retS and H1-T6SS cluster) harboring various plasmid combinations grown in TSB with specific antibodies. All protein samples were analyzed by SDS-PAGE followed by Coomassie Blue staining (CBR) and served as an internal control. The proteins analyzed and molecular weight standards are shown on the left and right, respectively, and indicated with arrowheads when necessary.

Figure 9.

Proposed models of TPP activation and TagF-mediated post-translational repression of type VI secretion in A. tumefaciens and P. aeruginosa. Proposed models of TPP activation (T6SS ON) and TagF-mediated repression (T6SS OFF) in A. tumefaciens (top) and P. aeruginosa (bottom) are illustrated. Key activation or repression events are summarized at the bottom of each model. Protein names are indicated in or near the designated molecules. IM, inner membrane; OM, outer membrane. For a detailed description of the proposed models, see “Discussion.”