Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA

Abstract

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energize their transport into cells and catalyze their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; bacteriocin; pyocin.

Figure 1.. Amino acid sequence similarity of pyocins SX1 and SX2 with pyocins S2 and G and proposed domain architecture of pyocins SX1 and SX2.

(A) Percentage amino acid similarity for pyocins SX1/SX2 and their immunity protein (Im) with pyocin S2 and G. (B) Proposed domain architecture of pyocins SX1 and SX2 consisting of five domains including N-terminal unstructured domain (UD), transporter-binding domain (TBD), CPA-binding domain (CBD), inner membrane translocation domain (IMT) and cytotoxic domain (CD). Numbers above the boxes indicate amino acid positions.

Figure 2.. Purification and in vivo activity of pyocins SX1 and SX2.

(A) SDS–PAGE gel (12%) of purified pyocins SX1 and SX2 (74–76 kDa) and their immunity proteins (10 kDa). The dashed line indicates splicing of gels. (B) Survival plot for groups of larvae injected with PBS or pyocins alone and groups of larvae infected with P. aeruginosa P7 and treated with PBS or pyocins. Groups of 30 larvae were injected with ∼10⁴ CFU of P. aeruginosa P7 followed by either PBS (control), pyocin SX1 or SX2 (10 µg). The numbers of survivor were observed at 24 and 48 h after pyocin treatment. (C) Killing activity of pyocins SX1 and SX2 after injection into G. mellonella larvae. Groups of three larvae were injected with PBS (control) or the pyocins and were collected at different time point. Three larvae were pooled, homogenized in cold PBS and centrifuged. Five microliters of the clear fraction were spotted onto P. aeruginosa P7 cell lawn on a LB plate.

Figure 3.. Molecular activities of pyocins SX1 and SX2. Plasmid nicking activity of pyocin SX1 and SX2.

One microgram of pUC18 DNA was incubated with 200 ng of pyocin SX1 (A) or SX2 (B) (immunity protein removed) in the presence different divalent metals. S, supercoiled DNA; L, linear DNA and O, open circle DNA. (C) In vitro transcription-translation of Renilla luciferase in the presence of 200 ng of different pyocins/colicin. (D) In vitro transcription-translation of Renilla luciferase in the presence of pyocin SX2 at different concentrations. The experiment was done with three replications. Error bars represent standard deviation of the mean. * indicate significant difference comparing to the untreated control (student's t-test, P < 0.05).

Figure 4.. The TonB-dependent receptor PA0434 is required for pyocins SX1 and SX2 killing.

Five microliters of pyocin SX1 or SX2 (1 mg/ml) were spotted on to cell lawn of wild type P. aeruginosa PAO1 (PAO1 WT), PAO1 with transposon insertion in PA0434 gene (PAO1ΔPA0434) and complement strain of PAO1Δ PA0434 (PAO1Δ PA0434::PA0434).

Figure 5.. PA0434 expression specifically responds to Cu(II) availability.

(A) Overlay spot plate assay of pyocins SX1/SX2 with different metal compounds spotted adjacent to the pyocin. 5 µl of 1 mM metal solution were spotted onto LB plate overlaid with P. aeruginosa PAO1 alongside 5 µl of 1 mg/ml pyocin SX1 or SX2 (position of metal compound spot indicated by added black circle). The overlapping areas between CuSO₄ and pyocin spots showed decreased killing activity while other metals did not affect the killing zone indicating that Cu. (B) Overlay spot plate assay of pyocins SX1 and SX2 under normal condition (LB agar), copper-enriched conditions (LB agar + 1 mM CuSO₄) and copper-deficient conditions (LB agar + 2 µM TETA). (C) MIC of pyocins SX1, SD2 and AP41 against P. aeruginosa on LB agar supplemented with different metal compounds or metal-chelators. Increasing and decreasing MICs compared with the controls are highlighted in yellow, blue and gray.

Figure 6.. Analysis of the changes in the transcription of pa0434 and fptA genes in P. aeruginosa cells grown with different metals or metal-chelators.

RT-qPCR was performed on P. aeruginosa PAO1 cells grown with different metals (0.5 mM CuSO₄, 0.5 mM FeCl₃ or 0.5 mM ZnCl₂) or metal-chelators (2 mM TETA, 200 µM bipyridine or 30 µM TPEN) for 8 h. Results are given as the relative normalized expression in a log₂ scale. The data were normalized relative to the reference gene rpsL and are representative of three independent experiments. Error bar indicates standard error of the mean. * indicates significantly difference compared with the untreated control (t-test, P < 0.010).

Figure 7.. Predicted structure and entry mechanism of pyocin SX2.

(A) Predicted structure of the pyocin SX2-ImSX2 complex. (B) Proposed mechanism of pyocin SX2 (and SX1) translocation into P. aeruginosa. (i) The pyocin CBD domain (cyan) binds to CPA on the cell surface. (ii) The UD and TBD (blue) bind to PA0434 transporter and activate unfolding of the transporter's plug domain. (iii–iv) The UD passes through PA0434 barrel and presents its TonB-box in periplasm. Then, TonB1 binds to the pyocin TonB-box and imports the pyocin via PMF generated by TonB1–ExbB–ExbD complex. The immunity protein (Im) is released during the translocation. (v) The pyocin is proteolytically processed and only the cytotoxic domain (CD) is thought to be transported into cytoplasm via FtsH.