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Immune Evasion of the Human Pathogen Pseudomonas aeruginosa: Elongation Factor Tuf Is a Factor H and Plasminogen Binding Protein

Anja Kunert, Josephine Losse, Christin Gruszin, Michael Hühn, Kerstin Kaendler, Stefan Mikkat, Daniela Volke, Ralf Hoffmann, T. Sakari Jokiranta, Harald Seeberger, Ute Moellmann, Jens Hellwage and Peter F. Zipfel
J Immunol September 1, 2007, 179 (5) 2979-2988; DOI: https://doi.org/10.4049/jimmunol.179.5.2979
Anja Kunert
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Josephine Losse
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Christin Gruszin
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Michael Hühn
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Kerstin Kaendler
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Stefan Mikkat
†University of Rostock, Medical Faculty, Rostock, Germany;
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Daniela Volke
‡University of Leipzig, Center for Biotechnology and Biomedicine, Leipzig, Germany;
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Ralf Hoffmann
‡University of Leipzig, Center for Biotechnology and Biomedicine, Leipzig, Germany;
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T. Sakari Jokiranta
§Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; and
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Harald Seeberger
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Ute Moellmann
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Jens Hellwage
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
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Peter F. Zipfel
*Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany;
¶Faculty of Biology, Friedrich-Schiller-University, Jena, Germany
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  • FIGURE 1.
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    FIGURE 1.

    Factor H and FHR-1 bind to intact P. aeruginosa. A, P. aeruginosa strains PAO1, ATCC 27853, NCTC 10662, and SG137 were incubated in HiNHS. Bacteria were washed, and bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western Blotting. Factor H was detected in the eluate as a 150-kDa protein, and FHR-1 was identified as the doublet of 37 and 43 kDa. The mobility of the marker proteins is indicated on the right side. e, elution fraction; w, final wash fraction. B, Binding of Factor H as assayed by flow cytometry. P. aeruginosa strains PAO1 and ATCC 27853 were treated with HiNHS, and after extensive washing bacteria were incubated with Factor H-specific antiserum. Bacteria incubated in PBS served as controls. Representative experiments of five are shown.

  • FIGURE 2.
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    FIGURE 2.

    Factor H and FHR-1 bind to clinical isolates of P. aeruginosa. Clinical isolates of P. aeruginosa derived from patients with pyelonephritis, leukopenia, T cell lymphoma, urosepsis, or myeloma (Is1-Is8) were incubated with human serum. Bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western Blotting. Factor H was detected in the elution fraction as a 150-kDa protein, and FHR-1 was identified as the doublet of 37 and 43 kDa. The mobility of the marker proteins is indicated on the right side. A representative experiment of five is shown. e, elution fraction; w, final wash fraction.

  • FIGURE 3.
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    FIGURE 3.

    Mapping of the binding domains within Factor H and FHR-1. P. aeruginosa strain ATCC 27853 was incubated with recombinant deletion constructs of Factor H and FHR-1. N-terminal (A) and C-terminal fragments of Factor H (B), or recombinant FHR-1 and the C-terminal fragment SCRs 3–5 (C) were used. Bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western Blotting. Arrows indicate the position of the recombinant deletion constructs, which differ in mobility as they include a variable number of SCR domains. The mobility of the marker proteins is indicated on the right side. A representative experiment of five is shown. e, elution fraction; w, final wash fraction.

  • FIGURE 4.
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    FIGURE 4.

    Elongation Factor Tuf of P. aeruginosa is a surface protein. A, Tuf of P. aeruginosa strain PAO1 was recombinantly expressed in E. coli as a His-tagged protein and purified by affinity chromatography. The E. coli lysate, flow through (FT), wash (w), and elution (e) fraction were separated by SDS-PAGE and analyzed by silver staining (lanes 1–4). The elution fraction was analyzed by Western blotting using a mAb detecting the N-terminal histidine tag (lane 5). The band of ∼57 kDa is visualized by silver staining but did not react with the histidine antiserum. B, Tuf is detected at the surface of immobilized P. aeruginosa (strain PAO1) using whole cell ELISA. Tuf was detected using a polyclonal antiserum, which was raised against recombinant Tuf. Data show the mean of four experiments with SD indicated with error bars. ∗, p < 0.001. C, Surface expression of Tuf as identified by immunofluorescence microscopy. P. aeruginosa (strain PAO1) was incubated with a monoclonal anti-Tuf Ab and stained with a Alexa Fluor 647-labeled antiserum (left). Treatment in the absence of the mAb served as control (right). A representative experiment of three is shown. D, Tuf is identified in the surface protein fraction of P. aeruginosa. Purified biotinylated surface proteins of strains PAO1 (lanes 1 and 3) and SG137 (lanes 2 and 4) were separated by SDS-PAGE and analyzed by Western blotting using a monoclonal anti-Tuf Ab or a polyclonal anti-GroEL antiserum. The mobility of the marker proteins is indicated on the right. A representative experiment of three is shown.

  • FIGURE 5.
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    FIGURE 5.

    Factor H, FHL-1 and FHR-1 bind to recombinant Tuf. A, Recombinant Tuf or BSA was applied to a membrane by slot blotting and after incubation with Factor H or FHL-1 bound proteins were detected with specific antisera. BSA was used as a negative control. B, Tuf or BSA were separated by SDS-PAGE, blotted to a membrane, and incubated with FHR-1. Bound FHR-1 was detected with specific antisera. BSA was used as a negative control. C, Binding of Factor H, FHL-1, and FHR-1 to immobilized Tuf was analyzed by ELISA. Bound proteins were detected using specific antisera. BSA was used as a negative control. Data show the mean of three experiments, and SD is indicated by error bars. ∗, p < 0.03; ∗∗, p < 0.001. D, Binding of Factor H to Tuf was analyzed by surface plasmon resonance. The C-terminal Factor H construct SCR 8–20 was immobilized on the chip surface and Tuf was applied as an analyte within the fluid phase. Tuf binding was dose-dependent and resulted in elevated resonance units (RU). E, Localization of the Factor H binding domains by ELISA. Recombinant Tuf was immobilized, and binding of the indicated Factor H deletion constructs was assayed using a polyclonal anti-Factor H antiserum, which reacted similarly to all constructs. A representative experiment is shown. Data show the mean value and SD is indicated by error bars shown.

  • FIGURE 6.
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    FIGURE 6.

    Plasminogen binds to recombinant Tuf. A, Recombinant Tuf was blotted onto a membrane and incubated with purified plasminogen. Bound protein was detected with specific antisera. BSA was used as a negative control. A representative experiment of three is shown. B, Binding of plasminogen to immobilized Tuf was analyzed by ELISA. Bound plasminogen was detected using a specific antiserum. BSA was used as a negative control. Data show the mean and error bars indicate SD. ∗, p < 0.01. C, Plasminogen and Factor H share binding sites for Tuf. Binding of plasminogen to immobilized Tuf was assayed in the presence of increasing molar concentrations of Factor H. Both serum proteins were detected by specific antisera. In human plasma, plasminogen has a concentration of ∼100 nM and Factor H of 140 nM. Thus the overall molar ratio is 1:1.4 (Plasma data), which reflects the in vivo situation. Data show the mean of four independent experiments, and the SD is indicated by error bars. D, Surface plasmon resonance analyses of plasminogen binding to immobilized Tuf. Recombinant Tuf was immobilized to the surface of a sensor chip, and plasminogen was applied at the indicated concentrations. Binding was dose-dependent over a range from 0 to 400 nM.

  • FIGURE 7.
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    FIGURE 7.

    Plasminogen binds to live P. aeruginosa. A, Binding of plasminogen to intact P. aeruginosa (strain AH377) was analyzed by immunofluorescence microscopy. Bound plasminogen was detected using specific antisera (left). Treatment in the absence of plasminogen served as control (right). A representative experiment of three is shown. B, Whole cell ELISA demonstrating plasminogen binding to live bacteria (strain PAO1) that were immobilized to a microtiter plate. Binding of purified plasminogen and of plasminogen derived from HiNHS was detected with specific antiserum. Amino acid analogs ε-aminocaproic acid (εACA) and imidazol (imi) were added to a final concentration of 1 mM. Binding of the antiserum to the bacteria in the absence of a ligand was used as control. Data show the mean with SD indicated with error bars. ∗, p < 0.01 significance relative to the control. Experiments were repeated at least three times. C, Plasminogen attached to P. aeruginosa cleaves fibrinogen. P. aeruginosa strain PAO1 was incubated with plasminogen in the presence of the activator uPA. Following incubation and extensive washing, fibrinogen was added. After 4 and 16 h of incubation of the bacteria, samples were withdrawn, separated by SDS-PAGE, and transferred to a membrane. Fibrinogen was identified using a polyclonal fibrinogen antiserum in combination with HRP-conjugated rabbit antiserum.

  • FIGURE 8.
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    FIGURE 8.

    Tuf-bound Factor H and plasminogen are functionally active. A–C, C3b cleavage due to acquired and endogenous activity. P. aeruginosa strains PAO1 (A), ATCC 27853 (B), or NCTC 10662 (C) were incubated in PBS or HiNHS. Following addition of C3b and Factor I, the reaction was incubated at 37°C for 2 or 18 h. Aliquots were separated by SDS-PAGE and the appearance of C3b degradation products was analyzed by Western blotting. The endogenous C3b cleavage activity was assayed in the absence of serum (PBS). The position of the α′ chain and the β-chain of C3b is indicated. Degradation of C3b is evidenced by appearance of cleavage products and by disappearance of the α′ chain. Degradation products from Factor H-mediated cofactor activity, i.e., α′ 46 and α′ 43, are represented by filled arrows. C3b fragments generated by endogenous strain-specific proteases are indicated by arrowheads and their apparent product size is underlined and shown with boldface. A representative experiment of three is shown. Experiments were repeated at least four times. D, Tuf was immobilized to a microtiter plate and plasminogen was added. After extensive washing, the tissue-type plasminogen activator and the chromogenic substrate S-2251 were added and the absorbance of the converted substrate was measured. Data show the mean and error bars represent SD. E, Tuf-bound Factor H displays cofactor activity. Factor H bound to immobilized Tuf was incubated with C3b and Factor I. The supernatant was removed and separated by SDS-PAGE, and the appearance of C3b cleavage products was analyzed by Western blotting. C3b is represented by the α′ chain and the β-chain, as indicated. C3b degradation is visualized by the appearance of the 46- and 43-kDa bands. In the absence of Factor H, no degradation products were detected (lane 4). As a positive control, immobilized Factor H was directly incubated with C3b and Factor I (lane 5).

  • FIGURE 9.
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    FIGURE 9.

    Survival of P. aeruginosa in Factor H-depleted human plasma. A, P. aeruginosa strain SG137 was incubated for 1 h either in Factor H-depleted complement active human plasma (DHP) or in complement active normal human plasma (NHP). Afterward, bacteria were plated onto agar plates at various dilutions, and CFUs were determined. This experiment was repeated three times, and the mean data and error bars for SD are indicated. ∗, p < 0.005. B, P. aeruginosa strain SG137 was incubated for 1 h in Factor H-depleted human plasma, which was supplemented with the indicated concentrations of purified Factor H. The plasma concentration of Factor H is about 500 μg/ml. CFU obtained under physiological Factor H concentrations was set at 100%. This experiment was repeated four times and data indicate mean values and SD as error bars.

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The Journal of Immunology: 179 (5)
The Journal of Immunology
Vol. 179, Issue 5
1 Sep 2007
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Immune Evasion of the Human Pathogen Pseudomonas aeruginosa: Elongation Factor Tuf Is a Factor H and Plasminogen Binding Protein
Anja Kunert, Josephine Losse, Christin Gruszin, Michael Hühn, Kerstin Kaendler, Stefan Mikkat, Daniela Volke, Ralf Hoffmann, T. Sakari Jokiranta, Harald Seeberger, Ute Moellmann, Jens Hellwage, Peter F. Zipfel
The Journal of Immunology September 1, 2007, 179 (5) 2979-2988; DOI: 10.4049/jimmunol.179.5.2979

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Immune Evasion of the Human Pathogen Pseudomonas aeruginosa: Elongation Factor Tuf Is a Factor H and Plasminogen Binding Protein
Anja Kunert, Josephine Losse, Christin Gruszin, Michael Hühn, Kerstin Kaendler, Stefan Mikkat, Daniela Volke, Ralf Hoffmann, T. Sakari Jokiranta, Harald Seeberger, Ute Moellmann, Jens Hellwage, Peter F. Zipfel
The Journal of Immunology September 1, 2007, 179 (5) 2979-2988; DOI: 10.4049/jimmunol.179.5.2979
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