In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae

Abstract

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

Figure 1. In vivo cross-linking of EpsG and EpsL

(A) Whole cells of V. cholerae TRH7000 wild-type, ΔepsG, ΔepsL, and P_BAD::eps (native eps promoter replaced by arabinose inducible promoter and grown in the presence of 0.01% arabinose) were cross-linked using the indicated concentrations of DSP as described in the experimental procedures, and immunoblotted for EpsG. (B) Whole cells of P_BAD::eps wild-type containing pMMB67 vector, P_BAD::ΔepsG with empty vector or vector encoded epsG, and P_BAD::ΔepsL with empty vector or vector encoded epsL were cross-linked with 0.1 mM DSP and immunoblotted for EpsG. The position of EpsG is indicated. An arrow head designates the EpsG-EpsL complex, and an asterisk represents the putative EpsG dimer. The band directly below the putative EpsG dimer is presumed to be cross-reactive as it is also observed in the absence of DSP. The molecular weight markers are shown in kilo Daltons and lane numbers are indicated.

Figure 2. Mutations in EpsG prevent secretion

Supernatants of over-night cultures of V. cholerae TRH7000 P_BAD::eps wild-type, P_BAD::ΔepsL containing empty vector or vector encoding wild-type epsL, P_BAD::ΔepsG with empty vector, and vector encoded wild-type epsG or mutants were grown over-night at 37°C. Supernatants were separated from the cells and analyzed for the presence of an extracellular protease using the cleavable fluorogenic substrate N-tert-butoxy-canbonyl-Gln-Ala-Arg-7-amido-4-methyl-coumarin as described in the materials and methods. Each sample was assayed in triplicate and the standard error is indicated.

Figure 3. Co-immunoprecipitation of EpsG and EpsL

Triton X-100 cell extracts were prepared from P_BAD::eps wild-type, P_BAD::ΔepsG and complemented strain (Panels A and B), and P_BAD::ΔepsL and complemented strain (Panels C and D) after cross-linking with 0.25 mM DSP. Cleared cell extracts were immunoprecipitated with either anti-EpsG or anti-EpsL antibodies and subjected to SDS-PAGE and immunoblotting with biotinylated anti-EpsG (Panels A and D) or biotinylated anti-EpsL (Panels B and C) antibodies. In all panels the monomer for EpsG or EpsL is indicated with an arrow and the 60 kDa EpsG-EpsL complex is denoted by an arrow head. In panels immunoblotted for EpsG, the putative EpsG dimer is labeled with an asterisk. The molecular weight markers are shown in kilo Daltons. Lane numbers are indicated.

Figure 4. Processing of EpsG is necessary for EpsG-EpsL cross-linking

(A) Whole cells of V. cholerae C6706 wild-type, ΔpilD, and ΔpilD complemented with pACYC184 expressing pilD were cross-linked with the indicated concentrations of DSP, and immunoblotted with antibodies specific for EpsG. (B) Whole cells of P_BAD::ΔepsG expressing wild-type epsG, or epsG with mutation to residue G-1 were cross-linked and analyzed as described above. The processed monomer of EpsG is represented by an arrow, and the EpsG-EpsL complex is labeled with an arrow head. Position of molecular weight markers is shown.

Figure 5. EpsG and EpsL interact in the absence of other T2S components

(A) Whole cells of V. cholerae TRH7000 wild-type, and TRH7000 with the entire eps operon removed (Δeps) expressing plasmid-encoded epsG, or epsG and epsL (pEpsGL) were cross-linked with the indicated concentrations of DSP and immunoblotted for EpsG. (B and C) Whole cells of E. coli MC1061 with pACYC184-pilD and pMMB67 encoding epsG, epsL, or epsG and epsL concomitantly were cross-linked with the indicated concentrations of DSP and probed with antibodies specific for EpsG (B) or EpsL (C). The monomer for EpsG (Panels A and B) and EpsL (Panel C) is indicated by an arrow. An arrow head designates the EpsG-EpsL complex, and an asterisk represents the putative EpsG dimer in Panels A and B. Positions of molecular weight markers are indicated.

Figure 6. Location and interaction between residues Asp91 and Thr112 in EpsG

V. cholerae EpsG (Korotkov et al., 2009; PDB code 3FU1) depicted with the side chains of D91 and T112 colored with yellow carbons and magenta oxygens. The light blue sphere is a bound calcium ion. (A) View approximately perpendicular to the EpsG helix axis with the backbone cartoon in green and the side chains of D91 and T112 as sticks. (B) Surface representation of the same view with the side chains of D91 and T112 with the same color code as in Panel A, the other atoms are colored green for carbons, blue for nitrogens and red for oxygens. A deep cavity is apparent where only a few atoms of D91 and T112 are visible. Figure prepared with PyMOL (DeLano, 2002).

Figure 7. Replacement of residues D91 or T112 alters the cross-linking of EpsG with EpsL

Whole cells of P_BAD::ΔepsG expressing wild-type epsG, or epsG with mutation to either residue D91 or T112 were incubated in the absence or presence of 0.1 mM DSP and subjected to SDS-PAGE and immunoblotting with anti-EpsG antibodies. The EpsG monomer is designated by an arrow, the EpsG-EpsL complex is indicated by an arrow head, and a predicted EpsG dimer is labeled with an asterisk. Molecular weight markers are indicated.