Exploitation of an iron transporter for bacterial protein antibiotic import

Abstract

Unlike their descendants, mitochondria and plastids, bacteria do not have dedicated protein import systems. However, paradoxically, import of protein bacteriocins, the mechanisms of which are poorly understood, underpins competition among pathogenic and commensal bacteria alike. Here, using X-ray crystallography, isothermal titration calorimetry, confocal fluorescence microscopy, and in vivo photoactivatable cross-linking of stalled translocation intermediates, we demonstrate how the iron transporter FpvAI in the opportunistic pathogen Pseudomonas aeruginosa is hijacked to translocate the bacteriocin pyocin S2 (pyoS2) across the outer membrane (OM). FpvAI is a TonB-dependent transporter (TBDT) that actively imports the small siderophore ferripyoverdine (Fe-Pvd) by coupling to the proton motive force (PMF) via the inner membrane (IM) protein TonB1. The crystal structure of the N-terminal domain of pyoS2 (pyoS2^NTD) bound to FpvAI (K_d = 240 pM) reveals that the pyocin mimics Fe-Pvd, inducing the same conformational changes in the receptor. Mimicry leads to fluorescently labeled pyoS2^NTD being imported into FpvAI-expressing P. aeruginosa cells by a process analogous to that used by bona fide TBDT ligands. PyoS2^NTD induces unfolding by TonB1 of a force-labile portion of the plug domain that normally occludes the central channel of FpvAI. The pyocin is then dragged through this narrow channel following delivery of its own TonB1-binding epitope to the periplasm. Hence, energized nutrient transporters in bacteria also serve as rudimentary protein import systems, which, in the case of FpvAI, results in a protein antibiotic 60-fold bigger than the transporter's natural substrate being translocated across the OM.

Keywords: Pseudomonas aeruginosa; outer membrane receptor; pyocin; transporter.

Fig. 1.

High-affinity binding of pyoS2^NTD to FpvAI mimics that of Fe-Pvd. (A) PyoS2^NTD outcompetes Fe-Pvd for FpvAI binding in analytical gel-filtration chromatography. Protein elution was monitored at A₂₈₀ (solid line), and Fe-Pvd elution was monitored at A₄₀₀ (dashed line). mAu, milliabsorbance units. (B) ITC data for pyoS2^NTD binding FpvAI in the presence of a lower affinity competitor, Δ1–45 pyoS2^NTD (details are provided in Materials and Methods). Data from two independent experiments were fitted to a competitive binding model from which the following thermodynamic parameters were extracted: ΔH = −15.8 ± 1.4 kcal/mol, ΔS = −9.1 ± 4.6 cal⋅mol⁻¹⋅K⁻¹, n = 0.78 ± 0.02 sites, K_d = 0.24 ± 0.004 × 10⁻⁹ M. (C) The 2.8-Å crystal structure of pyoS2^NTD (blue) in complex with FpvAI (gray) highlighting the pyoS2^NTD β-hairpin (yellow), the FpvAI plug (brown) and signaling domains (black), and the TonB1 box (red). Several of the β-strands that make up the β-barrel of FpvAI have been removed to highlight the plug domain. The signaling domain of FpvAI is involved in activating its own expression and that of Pvd (41). (D and E) Comparison of FpvAI plug domain surface when bound by the PRR of pyoS2^NTD and Fe-Pvd, respectively, highlighting the movement of FpvAI Tyr231 from its position in the unliganded state (light blue).

Fig. 2.

PyoS2^NTD translocates across the P. aeruginosa OM. (A) Fluorescence labeling of live FpvAI-expressing P. aeruginosa PAO1 with pyoS2^NTD-AF488. The PAO1 fpvA⁻ transposon mutant PW5036 (fpvA-H02::ISlacZ/hah) exhibits no labeling. (Scale bars, 5 μm.) FRAP experiments on P. aeruginosa PAO1 labeled with pyoS2^NTD-AF488 (B) and pyoS2^NTD-AF488 (C) are shown, where cells were first treated with 100 μM CCCP. The bleached region is highlighted (dashed circle). The absence of FRAP in these experiments suggests pyoS2^NTD remains bound to FpvAI in the OM, whereas FRAP suggests pyoS2^NTD-AF488 has translocated to the periplasm, where it can diffuse laterally. (Scale bars, 1 μm.)

Fig. 3.

Mapping the pyoS2^NTD translocation pathway by photo cross-linking. (A) FRAP experiment on a P. aeruginosa PAO1 cell labeled with pyoS2^NTD-GFP demonstrates stalled import. The bleached region is highlighted (dashed circle). (Scale bar, 1 μm.) (B) Structure of the pyoS2^NTD–FpvAI complex showing the sites of pBpa incorporation in pyoS2^NTD-GFP (yellow diamonds) and the major cross-link sites in FpvAI (yellow circles), most of which are located in the plug domain of the TBDT. The red dotted line indicates the cross-link detected between pBpa184 in pyoS2^NTD and Val197 in FpvAI, which requires this region of the pyocin to translocate at least 76 Å. (C) Ten percent SDS/PAGE gel showing Coomassie-stained, in vivo cross-linked adducts of translocated pyoS2^NTD-GFPpBpa variants following purification by nickel affinity chromatography from OM extracts of P. aeruginosa PAO1 cells. Only cross-links to FpvAI were observed by LC-MS/MS, confirming that GFP traps the translocating pyoS2^NTD within FpvAI (details are provided in Materials and Methods). (D) Fragmentation spectrum of pBpa184-Val197 cross-linked peptides (X = pBpa) with b- and y-ions indicated, along with the E-value, precursor ion mass (MH+), and precursor ion charge.

Fig. 4.

Model for pyoS2^NTD translocation through FpvAI. The three-step model of pyoS2^NTD translocation through FpvAI supported by pBpa cross-linking data (Fig. 3 and Figs. S6–S9) is shown. In step 1, binding of pyoS2^NTD to FpvAI mimics Fe-Pvd, activating the receptor for substrate transport and recruiting the C-terminal domain of TonB1 in the periplasm. In step 2, a PMF-dependent mechanical force, applied via the ExbB–ExbD–TonB1 complex in the IM (not shown), drives unfolding of the labile half of the plug domain. The N terminus of pyoS2^NTD enters the ∼13-Å-wide cavity that is created, allowing it to present its own TonB1 box in the periplasm. In step 3, the pyoS2^NTD TonB1 box is bound by another copy of TonB1; at this time, the mechanical force is used to drive translocation of pyoS2^NTD through the FpvAI lumen. Further translocation is blocked by the force-resistant GFP. The multiple cross-links observed between the labile portion of the FpvAI plug domain and translocated pyoS2^NTD residues are presumed to involve unfolded polypeptide chains in the periplasm.