Peptide linkage mapping of the Agrobacterium tumefaciens vir-encoded type IV secretion system reveals protein subassemblies

Abstract

Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens vir-encoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane pore-forming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.

Fig 1.

Minimal interacting domains of VirB7 and VirB9 proteins. The N-terminal residues of VirB9 and VirB7 peptide prey isolated from the high-resolution library were determined. The position of the peptide termini are plotted versus the codon position of the full-length prey protein (x axis) and the number of isolates identified (y axis). The lines below each plot represent the minimal prey domain determined to interact with the bait used. (A) VirB9 prey isolated with full-length VirB7 bait. (B) Total VirB7 prey isolated with two VirB9 baits, VirB9(14–293) and VirB9(183–293).

Fig 2.

Minimal interacting domains of VirB proteins. The N-terminal and C-terminal residues of prey isolated from the high-resolution library are plotted versus codon position (x axis) and the number of isolates identified (y axis). Data are color-coded for each bait used (see Inset). Prey that have determined N and C termini and that were isolated by a unique bait are aligned to define regions in common, indicating minimal interacting domains (horizontal blue lines below the amino acid numbers). The prey represented are as follows VirB9 (A), VirB1 (B), VirB4 (C), VirB8 (D), and VirB10 (E).

Fig 3.

VirB protein interactions. The pairwise interactions identified among the VirB proteins are summarized. The horizontal lines represent the peptide sequence of each VirB protein with the length in amino acids indicated to the right. Blue shaded regions indicate the minimal domains that contribute to each pairwise interaction. The numbers indicate the specific residue in the coding sequence that delimits each domain. The heavy arrows indicate the direction in which the interaction was observed, from bait to prey. The data most easily sort into two main complexes; the first consists of VirB1, VirB7, VirB9, and VirB11, and the second consists of VirB1, VirB4, VirB8, VirB10, and VirB11. VirB4–VirB4 interactions are presented as a third group. VirB1–VirB1, VirB1–VirB4, VirB8–VirB8, VirB8–VirB10, and VirB10–VirB11 interactions are not represented but are addressed in the text.

Fig 4.

Model for assembly of the Agrobacterium T4SS. Protein interactions determined in this work are depicted in A and B. Previously determined interactions are shown in C. The double lines at the top and bottom of each panel represent the inner and outer membranes, and the shaded region represents the periplasmic space and peptidoglycan. (A) VirB8 functions as a “founding member” of the T4SS and recruits VirB1 to the site of assembly. VirB1 locally remodels the peptidoglycan (represented by decreased shading of the periplasm). (B) VirB1 activity allows recruitment of other T4SS components such as VirB4, VirB7, VirB9, VirB10, and VirB11 by clearing the peptidoglycan or by recruiting components via direct interactions. VirB7–VirB9 heterodimers are critical for stability of T4SS proteins. (C) As the assembly matures, the remaining components are recruited, including VirB3, the pilus (VirB2, VirB5), and inner membrane components (VirB6, VirD4). VirB1* may function extracellularly, and its loose association with the surface is indicated. VirB7 and VirB5 contribute to pilus assembly. VirB4, VirB6, VirB11, and VirD4 all have been postulated to create channels for substrate secretion (blue arrows). (D) A model for assembled T4SS based on interactions described in A–C. VirB7–11 and VirB4 form a functional core of the T4SS that spans both bacterial membranes. Known substrates for export include VirE2 and, perhaps, its specific chaperone VirE1, VirF, and the T-complex (VirD2–ssDNA). VirD4 is likely the recognition protein coupling VirD2 export to the T4SS and may function as a DNA-helicase to liberate the T-strand from the Ti plasmid. VirB11 forms a pore and may facilitate substrate export.