Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa

Abstract

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.

Keywords: Crystal Structure; Host-Pathogen Interactions; Membrane Proteins; Pseudomonas aeruginosa; Type III Secretion System.

FIGURE 1.

PopB and PopD interact with artificial membranes. A, scheme of expression clones used in this work. Both Pop proteins are co-expressed with their common chaperone PcrH, which carries a thrombin-cleavable 6His tag. rbs, ribosomal binding site. B, liposomes containing PopB (left) or PopD (right) were treated with different agents that destabilize the association of proteins to membranes (1 m KCl, 6 m urea, 2.5 × CMC DDM) to characterize their association with membranes. After 4 h of incubation and sucrose gradient centrifugation, fractions were recovered from the top to the bottom of the gradient and were analyzed by Western blotting using anti-PopB and/or anti-PopD rabbit antibodies.

FIGURE 2.

Trypsin digestion of PopB and PopD inserted into liposomes. A and C, purified PopD (in solution or inserted into liposomes) was digested with trypsin at RT at a molar ratio of (1:500), and samples were loaded on SDS-PAGE after different digestion times. n.d, non-digested sample. B, PopB proteoliposomes were digested with trypsin at the same ratio as described above; aliquots were loaded on SDS-PAGE, and the gel was developed by Western blotting using purified anti-PopB antibodies. D, representation of PopB and PopD with the positions of the residues recognized by trypsin and identified by N-terminal sequence are highlighted. The black rectangles represent the two transmembrane (TM) regions. Asterisks indicate bands analyzed by N-terminal sequencing for proteins in liposomes, and the diamond indicates a band seen for PopD only in solution. E, one-dimensional NMR spectrum of PopB (1–149) recorded at 25 °C on an 800 MHz spectrometer.

FIGURE 3.

CCF2 and nitrocefin assays used to determine the orientation of the C terminus of PopB on membranes. PopB could potentially be inserted in host cell membranes with its N and C termini pointing toward the inside (panel A) or the outside (panel B) of the infected cell. To discriminate between these two orientations, a β-lactamase reporter gene was fused to the C terminus of PopB, and its activity was followed using two β-lactamase substrates: CCF2 (upper parts), that penetrates eukaryotic cells and turns from green to blue upon cleavage, and nitrocefin (bottom parts), a non-permeable substrate that turns from yellow to red upon hydrolysis of the β-lactam ring. The two transmembrane regions of PopB are shown as yellow boxes.

FIGURE 4.

Characterization of the PopB-β-Lac fusion expressed in P. aeruginosa. A, P. aeruginosa CHA wild type or mutant strains were grown in T3SS-inducing conditions. When the cultures reached an A_{600 nm} value of 1.0 absorbance units, 100 μl of culture were centrifuged, and the supernatants were analyzed by Western blotting with rabbit anti-β-Lac and anti-PopB antibodies. B, cytotoxicity of PopB-β-Lac mutants. Macrophages were infected with P. aeruginosa CHA wild type, popBpopD mutant (ΔBD) or mutant complemented with either PopBwt/PopDwt (ΔBD/BD) or PopB-β-Lac/PopDwt (ΔBD/B-β-Lac/D). Non-infected (NI) cells were used as negative control. After 3 h of infection, LDH released was measured to determine the % of cell death. C, intracellular β-Lac activity was determined after infection of A549 cells with the indicated strains using CCF2. A P. aeruginosa strain expressing the fusion protein ExoS-β-Lac (ΔS-S-β-Lac) was used as a positive control. D, the complemented cytotoxic P. aeruginosa strain CHAΔBD/B-β-Lac/D, the non-secreting strain CHAΔF/B-β-Lac/D, and the constitutively secreting strain CHAΔV/B-β-Lac/D were used to infect A549 epithelial cells in triplicate to an MOI of 30. β-Lactamase activity was assayed by measuring the absorbance at 486 nm using nitrocefin as a substrate. M, molecular mass standards.

FIGURE 5.

PopB ⁵¹TGVALTPPS⁵⁹ binds within the concave region of PcrH. A, sequences of N-terminal peptides of translocon proteins that interact with type II chaperones as well as of the mutants tested in Fig. 7. B, schematic representation of the structure of PcrH (in gray) displaying most residues that interact with PopB ⁵¹TGVALTPPS⁵⁹. D, a close-up view of the established hydrogen bonds. The omit density map is contoured at 1.0 σ. C, electrostatic surface representation of PcrH in complex with the PopB peptide. Peptide residues are labeled, and carbon atoms are represented as yellow sticks (and in cyan for PcrH), with oxygen atoms in red and nitrogen atoms in blue.

FIGURE 6.

Comparison of structures of chaperone-translocator peptide complexes: PcrH-PopB (gray), PcrH-PopD (cyan; Ref. 8), IpgC-IpaB (yellow; Ref. 50), and SycD-YopD (blue; Ref. 52). The seven (eight for IpgC) helices of the different chaperones are shown. The structures were aligned with Coot. The translocator peptides bind in a very similar fashion to the concave region of their respective chaperones, especially within the region corresponding to the conserved P′/V′XL′XXP′ motif.

FIGURE 7.

Characterization of PopB mutants in P. aeruginosa. A, P. aeruginosa strains carrying the two different sets of mutations in PopB, L55D/P58D (PopB-2D) and V53D/L55D/P57D/P58D (PopB-4D), were grown in T3SS-inducing conditions. When the cultures reached an A_{600 nm} value of 1.0 absorbance units, 100 μl of cultures were centrifuged, and the pellet (Expression) and the supernatant (Secretion) were analyzed by Western blotting with anti-PopB, anti-PopD, and anti-PcrH antibodies. B, cytotoxicity of PopB mutants. Macrophages were infected with cultures of P. aeruginosa CHA depleted for popB and popD genes (ΔBD) or complemented with PopDwt and PopBwt, PopB-2D, or PopB-4D using a multiplicity of infection of 5. A non-infected culture was used as negative control. After 1–4 h of infection, LDH release into the supernatant was measured to determine the % of cell death. C, stability of PopB mutants was followed for up to 3 h after the addition of chloramphenicol, a protein synthesis inhibitor. The cell contents were analyzed by Western blotting with anti-PopB antibodies and anti-PcrV as a control.