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Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1

  • Molecular and cellular mechanisms of disease
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Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host–cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.

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Abbreviations

CF:

Cystic fibrosis

NHERF1:

Na+/H+ exchanger regulatory factor 1

CFTR:

CF transmembrane conductance regulator

siRNA:

Small interfering RNA

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Acknowledgments

We are grateful to Moira Paroni (Infections and Cystic Fibrosis Unit San Raffaele Scientific Institute, Milano, Italy) and Maria Cristina Dechecchi (Laboratory of Molecular Pathology, University Hospital of Verona, Italy) for helpful discussions and suggestions, Alice Prince (Columbia University, NY, USA) for donating the strains: PAO1, PAK, PAK/NP and PAK/fliC and to Valentina Lovato for excellent technical assistance. This work was supported by grants from the Italian Cystic Fibrosis Research Foundation (grant FFC # 08/2010) with the contribution of “La Bottega delle donne” Delegazione FFC di Treviso – Montebelluna (to A.T and S.J.R), V.B is fellow of the Italian Cystic Fibrosis research Foundation (FFC), M.T is fellow of the University Hospital of Verona and from the Deutsche Forschungsgemeinschaft SFB621-C9 (to U.S.)

Author contributions

R.R., S.J.R., A.T. and G.C. are responsible for conception and design of the research; M.F., A.K.S. and B.R. performed the mice infection experiments, U.S. and A.B... supervised the mouse models studies, V.B. and M.T. performed the cell infection experiments, cell cultures and transfections and contributed to the interpretation and analysis of the data, R.R. and M.F. performed immunoblot experiments, cell cultures and transfections and contributed to the interpretation and analysis of the data, A.I. interpreted the results of the histology of the lung, A.T., U.S. and S.J.R. wrote the manuscript and G.C. provided scientific knowledge and supervised the study.

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Authors and Affiliations

  1. Department of Bioscience, Biotechnology and Pharmacological Science, University of Bari, Via Amendola, 165/A70126, Bari, Italy

    Rosa Rubino, Maria Favia & Stephan J. Reshkin

  2. Laboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Haematology, University Hospital of Verona, Piazzale Stefani, 1, 37126, Verona, Italy

    Valentino Bezzerri, Maela Tebon, Giulio Cabrini & Anna Tamanini

  3. Infections and Cystic Fibrosis Unit, San Raffaele Scientific Institute, Via Olgettina, 58, 20132, Milan, Italy

    Marcella Facchini & Alessandra Bragonzi

  4. Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany

    Anurag Kumar Singh, Brigitte Riederer & Ursula Seidler

  5. Laboratory of Pathology, Department of Pathology and Diagnostics, University Hospital of Verona, Piazzale Stefani,1, 37126, Verona, Italy

    Antonio Iannucci & Giulio Cabrini

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  1. Rosa Rubino
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Correspondence to Ursula Seidler or Anna Tamanini.

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S.J. Reshkin and A. Tamanini contributed equally to this study.

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Rubino, R., Bezzerri, V., Favia, M. et al. Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1. Pflugers Arch - Eur J Physiol 466, 2269–2278 (2014). https://doi.org/10.1007/s00424-014-1474-6

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  • DOI: https://doi.org/10.1007/s00424-014-1474-6

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