A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition

Abstract

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.

Fig 1. H2-T6SS is required for Azu secretion.

(A) Deletion of retS enables expression and secretion of Hcp2. A mini-CTX plasmid directing the expression of Hcp2-Flag chimera was integrated into the P. aeruginosa derivative strains, respectively. Western blot analysis of Hcp2-Flag in the cell-associated (Cell) and concentrated supernatant (Sup) protein fractions from the indicated strains grown in M9 minimal medium. For the pellet fraction, an antibody against RNA polymerase α (α-RNAP) was used as a loading control in this and subsequent blots. (B) Azu is secreted by H2-T6SS. The cultured condition is the same as described in A. EV represents the empty vector pAK1900. (C) Total cell (Total), Cytoplasmic (Cyto) and periplasmic (Peri) fractions of P. aeruginosa expressing Azu, PA0943, XcpP or CtpA were examined by Western blot. The tagged proteins were detected using a Flag antibody. RNA polymerase (RNAP) and β-lactamase (β-lac) were used as cytoplasmic or periplasmic fraction controls, respectively. Data are representative of three independent replicates. (D) Interactions of Azu and VgrG2b. Cell lysates of P. aeruginosa containing pMMB67H-VgrG2a-Flag or pMMB67H-VgrG2b-Flag were incubated with GST or GST-Azu protein individually, and protein complex were captured by glutathione beads. The single and double asterisks represent GST and GST-Azu, respectively. GST-SiaD was a negative control. (E) Azu secretion is VgrG2b dependent but not VgrG2a. The cultured condition is the same as described in A.

Fig 2. Copper (Cu²⁺) regulates H2-T6SS expression and promotes a growth advantage of P. aeruginosa for bacterial competition.

(A-B) The expression of hcp2 and tssA2 was induced by low Cu²⁺. A mini-CTX plasmid directing the expression of Hcp2-Flag or TssA2-Flag chimera was integrated into the P. aeruginosa background strain, respectively. (A) Bacteria was cultured in LB medium supplemented with either 0.25 mM EDTA or 0.25 mM EDTA with the indicated concentrations of CuSO₄. Total protein was probed for the presence of the fusion protein. (B) Bacteria was cultured in LB medium supplemented with either 0.05 mM, 0.1 mM, or 0.25 mM CuSO₄. Total protein was probed for the presence of the fusion protein. (C) Cu²⁺ influences H2-T6SS assembly. Chromosomally encoded ClpV2-sfGFP localization in the P. aeruginosa measured by fluorescence microscopy. Cells were grown in the indicated conditions to OD₆₀₀ = 1.0 and H2-T6SS activated were analyzed. N = total number of cells analyzed for each strain. (D) Interbacterial growth competition assays between P. aeruginosa and E. coli. The competition assays were examined under LB conditions supplemented with either 0.25 mM EDTA or 0.25 mM EDTA and 0.1 mM CuSO₄ for 6 h. Quantification of cfu before (initial) and after (final) growth competition assays between the indicated organisms. Note that wild-type PAO1 displayed a growth advantage against the competitors than the ΔclpV2 and Δazu mutant under the present conditions. Error bars represent the mean ± s.d. of three independent experiments. **P<0.01, ****P<0.0001 based on two-way ANOVA Dunnett’s multiple comparison test; NS, not significance. EV represents the empty vector pAK1900.

Fig 3. CueR regulates the expression of H2-T6SS directly and is required for H2-T6SS-mediated antibacterial activity.

(A) Deletion of cueR increases the levels Hcp2 (upper) and TssA2 (down) relative to wild-type PAO1. Western blot analysis of Hcp2-Flag and TssA2-Flag in the cell-associated fractions from wild-type PAO1, ΔcueR, and the complemented strain (ΔcueR/p-cueR) cultured in LB medium containing 0.25 mM EDTA with or without 0.1 mM CuSO₄. (B) EMSAs showing that CueR binds to the promoter region of hcp2 and tssA2. PCR products containing hcp2, tssA2, cueA, and PA1374 promoter fragments were added to the reaction mixture at a concentration of 2.0 ng. The protein concentration of each sample is indicated above its lane. As a positive control, CueR can efficiently bind to the cueA promoter region. However, no band shift was observed when CueR protein incubates with PA1374 promoter region. (C) CueR affects H2-T6SS assembly. Chromosomally encoded ClpV2-sfGFP localization in the wild-type PAO1 and ΔcueR mutant measured by fluorescence microscopy, respectively. Cells were grown in LB to OD₆₀₀ = 1.0 and H2-T6SS activated were analyzed. N = total number of cells analyzed for each strain. (D) Quantification of recovered prey cells (E. coli) after co-incubation with strains PAO1, ΔcueR, or ΔcueRΔclpV2. Error bars indicate the mean ± s.d. of three biological replicates. **p<0.01 by student′s t-test; NS, not significant when compared to prey recovery after co-incubation with the parent strain. EV represents the empty vector pAK1900.

Fig 4. Azu interacts with a TBDT family transporter involved in Cu²⁺ transport.

(A) OprC was retained by agarose beads coated with GST-Azu. GST-binding beads coated with GST-Azu or GST was incubated with cell lysates. After washing with PBS buffer, the proteins separated by SDS/PAGE gels were visualized using silver staining, and bands retained by the GST-Azu coated beads were identified by mass spectrometry. The asterisk band represents the OprC protein. (B) Co-IP assays showing Azu binds to OprC. Cell lysates of P. aeruginosa containing pMMB67H-oprC-His with either p-gacA-Flag or p-azu-Flag were individually incubated with Flag beads, and then the beads retained protein complexes were detected by western blot against Flag antibody or His antibody. The double asterisks denote a nonspecific signal in the soluble protein fraction. Beads only sample was a blank control, GacA-Flag was a negative control. (C) The expression of oprC was repressed by CueR and high Cu²⁺. Cells of the indicated strains were grown in LB medium containing 0.25 mM EDTA with or without 0.1 mM CuSO₄. The expression of oprC was measured by qRT-PCR. (D) OprC is involved in Cu²⁺ acquisition in P. aeruginosa. Strains were cultured at OD₆₀₀ = 1.0 in M9 medium containing 1.0 mM EDTA. Cu²⁺ associated with bacterial cells was measured by ICP-MS. (E) Interbacterial growth competition assays between P. aeruginosa and E. coli. The competition assays were examined in LB or LB medium containing 0.25 mM EDTA with or without 0.1 mM CuSO₄ for 6 h. Quantification of cfu before (initial) and after (final) growth competition assays between the indicated organisms. (C-E), error bars represent the mean ± s.d. of three biological replicates, **P<0.01, ***P<0.001, ****P<0.0001 based on two-way ANOVA Dunnett’s multiple comparison test. NS indicates not significance. EV represents the empty vector pAK1900.

Fig 5. H2-T6SS-dependent Cu²⁺ transport system is important for competitive growth and virulence.

(A) OprC and Azu mediate H2-T6SS-dependent intrabacterial competition. The indicated strains were grown in LB medium supplemented with 0.25 mM EDTA for 6 h. Quantification of cfu before (initial) and after (final) growth competition assays between the indicated organisms. The cfu of the relevant P. aeruginosa versus competitor was plotted. Error bars represent the mean ± s.d. of three biological replicates. *P<0.05, **P<0.01, ***P<0.001 based on two-way ANOVA Dunnett’s multiple comparison test. (B) The clpV2, azu, and cueR deletion reduced the virulence of P. aeruginosa. C57BL6 mice were intranasally challenged with wild-type PAO1, ΔclpV2, Δazu, or ΔcueR at 1×10⁷ cfu in 50 μl PBS, moribund mice were killed to obtain survival data (n = 6/strain). (C-D) Mice were infected with 1×10⁷ wild-type PAO1, ΔclpV2, Δazu, or ΔcueR strain intranasally (n = 6/strain). At 12 h, serums and lungs from mice infected with no bacteria (PBS), or the indicated strains were recovered. Bacterial loads were determined by serial dilution and plating. **P<0.01 compared to wild-type PAO1 by Student's t test. The strains contain either the empty vector pAK1900 or the complemented plasmid.

Fig 6. A proposed model of H2-T6SS-mediated Cu²⁺ transport in P. aeruginosa.

Under Cu²⁺ rich conditions, the copper homeostasis regulator CueR represses the expression of H2-T6SS genes. The Cu²⁺ transport is mainly dependent on the known systems, such as CopY, CueA and CusR/S. However, the repression of H2-T6SS by CueR is relieved under the Cu²⁺-limited conditions, and the Cu²⁺-binding protein Azu is subsequently secreted by the activated H2-T6SS. Secreted Azu scavenges extracellular Cu²⁺ and delivers its load via direct interaction with OprC.