Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Injection of ExoS-Bla into differentiated HL-60 cells is sensitive to pharmacological agents.

<p><b>A/</b> VD3-differentiated HL-60 cells were exposed to 10 µM of cytochalasin D (CytoD), 10 µM of latrunculin B (LtrB) or 50 nM of Wortmannin for 30 min prior and during infection, or to 2 µM of TAT-C3 toxin, 100 µM of LY-294002, 12 µM of genistein, 10 µM of PP2, and 10 µM of PF-573-228 for 120 min prior and during a 3 h infection period at MOI 10 with the PAO1ΔSTY-SBlaR146A strain. The percentage of injection-positive cells was evaluated by flow cytometry. <b>B/</b> Eukaryotic plasma membranes were purified by fractionation on sucrose gradient, after infection of cells pre-treated with inhibitors as above. Proteins were analysed by immunoblotting using anti-PopB. Ni : non-infected VD3-differentiated HL-60 cells, CTRL: infected VD3-differentiated HL-60 cells without inhibitor. <b>C/</b> The ability of PAO1ΔSTY-SBlaR146A strain to secrete ExoS-Bla <i>in vitro</i> was assayed for each inhibitor modifying injection by immunoblotting of total secreted ExoS-Bla protein using the anti-ß-lactamase antibody. Pa: supernatant of T3SS non-induced <i>P.aeruginosa</i>, IPa: supernatant of T3SS induced <i>P. aeruginosa</i> without inhibitor.</p

Efficiency of ExoS-Bla and ExoY-Bla translocation in different cell lines.

<p><b>A/</b> A549 epithelial cells, BJAB, Jurkat, non-differentiated HL-60 cells (HL-60), or HL-60 differentiated into neutrophils, macrophages and monocytes by DMSO, PMA or Vitamin D3 (VD3), respectively, were infected at MOI of 10, for 3 h, with CHAΔS expressing ExoS-BlaR146A and analyzed by flow cytometry. <b>B/</b> HL-60 and U937 were differentiated in monocytes with VD3 (black bars) or not (white bars) and infected at MOI 10 with PAO1ΔSTY strains expressing either ExoS-BlaR146A or ExoY-Bla as described above. The error bars indicate standard deviation (n = 3). <b>C/</b> Non-differentiated HL-60 (thin lane) or HL-60 differentiated in neutrophils (dot line), monocytes (dash line) or macrophage (thick line) were labelled with a FITC conjugated antibody specific for CD11b and analysed by flow cytometry.</p

Switching T3SS resistant HL-60 cells to T3SS permissive cells by serum starvation or panning to anti-CD43.

<p>Non-differentiated HL-60 cells were treated as follows: maintained during 15 min in medium supplemented (+ FCS) or not (− FCS) (a), incubated during 5 min in either naive (− anti CD43) or coated wells with a monoclonal anti CD43 (+ anti CD43), first panned in a anti CD43-coated well during 5 min and then transferred to a naïve well, (transferred) (b) and cells were next incubated at MOI 10 with the CHAΔS-SblaR146A strain and analysed by flow cytometry. Non-differentiated HL-60 were incubated during 5 min in a well coated with anti CD43 in the absence (− CytoD) or the presence (+ CytoD) of 10 µM of cytochalasine D for 30 min prior and during infection at MOI 10 with the CHAΔS-SblaR146A strain (c). The error bars indicate standard deviation (n = 3). Insert: schematic drawing of the experiments presented in the histogram (part <b>b</b>).</p

Characterization of ExoS-Bla and ExoY-Bla reporters.

<p><b>A/</b> Secretion profiles of <i>P. aeruginosa</i> strains carrying the reporter fusion ExoS-Bla. Fifteen µL of culture supernatants of either wild-type CHA strain or mutant CHA strains expressing ExoS-Bla grown under T3SS-inducing conditions were analyzed by immunoblotting with antibodies directed against ExoS, PopB, PopD and PcrV. <b>B/</b> Co-cultures of A549 cells with <i>P. aeruginosa</i> strains for 3 h at MOI 10. Injection of either ExoS-Bla or ExoY-Bla fusions by strains CHA and PAO1ΔSTY was detected after incubating cells with β-lactamase substrate CCF2-AM either by fluorescence microscopy using a 20× objective (upper panel) or by flow cytometry (lower panel). The horizontal bar in flow-cytometry histograms indicates the gating used to determine the percentage of β-lactamase positive cells revealing ExoS-Bla injection. Mean Fluorescence Intensity is indicated in each panel. Scale bar, 100 µm.</p

Oligomerization of PcrV and LcrV, protective antigens of Pseudomonas aeruginosa and Yersinia pestis.

Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higher-order species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8-10 nm, respectively. The C-terminal helix, alpha12, of PcrV and notably the hydrophobic residues Val(255), Leu(262), and Leu(276) located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials