ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury

Authors

  • Viviane Finck-Barbançon,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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  • Joanne Goranson,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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    • Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver CO 80262, USA

  • Lei Zhu,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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  • Teiji Sawa,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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    • Department of Anesthesia and Critical Care, University of California, San Francisco, California 94143, USA

  • Jeanine P. Wiener-Kronish,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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    • Department of Anesthesia and Critical Care, University of California, San Francisco, California 94143, USA

  • Suzanne M. J. Fleiszig,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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    • School of Optometry, University of California, Berkeley, California, 94720, USA

  • Christine Wu,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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    • School of Optometry, University of California, Berkeley, California, 94720, USA

  • Liane Mende-Mueller,

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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  • Dara W. Frank

    1. Department of Microbiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, USA.
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Dara W. Frank E-mail frankd@post.its.mcw.edu; Tel. (414) 456 8766; Fax (414) 266 8522.

Abstract

The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::Ω and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::Ω. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

Footnotes

  1. Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver CO 80262, USA

  2. Department of Anesthesia and Critical Care, University of California, San Francisco, California 94143, USA

  3. School of Optometry, University of California, Berkeley, California, 94720, USA

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