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. 2017 Mar 28:8:14888.
doi: 10.1038/ncomms14888.

A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition

Jinshui Lin  1   2 , Weipeng Zhang  1   3 , Juanli Cheng  1   2 , Xu Yang  1 , Kaixiang Zhu  1 , Yao Wang  1 , Gehong Wei  1 , Pei-Yuan Qian  3 , Zhao-Qing Luo  4 , Xihui Shen  1
Affiliations

A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition

Jinshui Lin et al. Nat Commun. .

Abstract

Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell-cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell-cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. A gene proximal to H3-T6SS is required for the growth of a mutant missing known iron acquisition systems.
(a) The organization of H3-T6SS and its neighbouring genes. Structural genes are depicted as colour arrows that indicate the transcriptional directions. The gene numbers are only indicated at the end of the cluster. Gene PA2374 (tseF) identified in the screening is marked by an inverted triangle, indicating transposon insertion. (b) Domain structure of TseF. The positions and regions of the predicted membrane occupation and recognition nexus motifs are shown. (c) The growth of the mutants in iron-deficient media. Relevant bacterial strains were grown in TSB or succinate MM containing EDDHA (0.5 μg ml−1). Cell growth was monitored by measuring OD600. Note that the deficiency of growth by mutant PAΔ3FeΔtseF in iron-deficient media can be restored by expressing either pvdA or tseF. The curves represent three biological replicates; error bars are s.d.
Figure 2
Figure 2. TseF is a substrate of H3-T6SS.
(a) A plasmid directing the expression of TseF-VSV-G chimera was introduced into the indicated P. aeruginosa strains. Total protein or proteins in culture supernatant was probed for the presence of the fusion protein. The cytosolic RNA polymerase (RNAP) was similarly detected as a control. Note that deletion of H3-T6SS component genes clpV3, hsiB3-C3 or hcp3 drastically reduced the release of TseF-VSV-G into extracellular milieu. (b) TseF interacts with VgrG3 and VgrG1b. GST-TseF was incubated with three different VgrG proteins, and protein complexes were captured by glutathione beads. Note that TseF can be co-purified with VgrG3 or VgrG1b but not VgrG1a. The unrelated protein PA0533 cannot be co-purified with any of the tested proteins. Full blots are shown in Supplementary Fig. 14.
Figure 3
Figure 3. TseF interacts with PQS.
(a) Culture supernatant of P. aeruginosa causes an increase in the mobility of TseF. GST or GST-TseF was incubated with culture supernatant of P. aeruginosa or E. coli prior to native-PAGE or SDS–PAGE analysis. (b) The quinolone signal is required for causing TseF mobility shift. Culture supernatant of a series of P. aeruginosa mutants was tested for the ability to cause TseF mobility shift. Note that deletion of genes involved in the PQS signal production abolished the activity. (c–e) PQS binds TseF with high affinity. TseF but not PA0533 was retained by immobilized PQS (c); His6-TseF but not His6-Fur was retained by PQS spotted on membranes; under this condition, a higher amount of HHQ was required to retain similar amount of His6-TseF (d); PQS binds to TseF with a Kd of 0.33 μM, which was reduced to 0.15 μM when PQS-Fe was used (e). Neither PQS nor PQS-Fe3+ interacted with GST. (f) Iron supplied by PQS requires TseF. The growth of the indicated bacterial strains was assessed in iron-deficient succinate MM (EDDHA: 5 μg ml−1) without (left panel) or with 20 μM PQS-Fe3+ (right panel). Note that only the expression of tseF rescued the growth of PAΔ3FeΔtseF in the presence of PQS-Fe3+ after >2 days of incubation. The curves represent three biological replicates; error bars are s.d. Full blots are shown in Supplementary Fig. 14.
Figure 4
Figure 4. Porin OprF and the siderophore receptor FptA are essential for TseF-mediated iron uptake.
(a) The identification of OprF and FptA as the binding partners of TseF. Total cell lysates of the indicated bacterial strains were incubated with beads coated with GST or GST-TseF. After removing unbound proteins by extensively washing, proteins retained were resolved by SDS–PAGE followed by silver staining. Proteins specifically retained by GST-TseF were identified by mass spectrometry. (b,c) TseF directly interacts with OprF and FptA. GST or GST-TseF was incubated with His6-tagged OprF or AtpA and the potential protein complex was captured by glutathione beads (b). Note that only His6-OprF was retained by GST-TseF. A similar procedure was used to demonstrate direct binding between TseF and FptA1–186 but not PA4426 (c). Full blots are shown in Supplementary Fig. 14. (d) The role of OprF and FptA in the uptake of iron supplied by PQS-Fe3+. The growth of the indicated bacterial strains was assessed in succinate MM with EDDHA (5 μg ml−1) without (left panel) or with (20 μM) PQS-Fe3+ (right panel). Note that only in the presence of both TseF and one of the receptors were the bacteria able to use PQS-Fe3+ to support its growth. The curves represent three biological replicates; error bars are s.d.
Figure 5
Figure 5. OMV complex is involved in TseF-mediated iron acquisition.
(a) TseF is incorporated into the OMV complex. OMVs prepared from the ΔtseF mutant expressing TseF-VSV-G, FliC-VSV-G or VgrG1b-VSV-G and the proteins of interest were probed. The cytosolic RNA polymerase was detected as a control. Full blots are shown in Supplementary Fig. 14. (b) TseF is required for supplementing the bacterium the iron by OMVs. Mutant PAΔ3FeΔtseF grown in the presence of EDDHA (5 μg ml−1) was supplemented with OMVs (0.01 OD ml−1) prepared from bacterial strain with the indicated genotypes and bacterial growth was monitored by measuring the absorbance at OD600. (c) Iron acquisition via OMVs requires OprF or FptA. OMVs from wild-type, ΔtseF or its complemented strain were used to supply iron for derivatives of mutant PAΔ3FeΔtseFΔfptAΔoprF. Note that only those expressing pvdA or one of the binding partners (FptA and OprF) for TseF can use this iron source. (d,e) The TseF iron acquisition system is required for bacterial survival in a host. Equal amounts of the indicated bacterial strains and wild-type bacteria were co-inoculated onto silkworms and the survival of the bacteria was determined. Note that the strains lacking all the four iron acquisition systems are defective in competing against the wild-type bacteria in the host, which can be fully complemented by expressing the corresponding genes. Data shown were the average of three independent experiments and error bars indicate s.d. Student's t-test: *P<0.05; **P<0.01.
Figure 6
Figure 6. A model for TseF-mediated iron acquisition via the OMV complex.
We propose that TseF is exported by H3-T6SS and then incorporated into OMVs containing PQS-Fe3+. Recognition of TseF by the cell surface receptors FptA or OprF would facilitate the transport of iron into the cell by a yet unidentified mechanism.
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