Proteomic characterisation of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles

Institut für Molekulare Infektionsbiologie, Julius-Maximilians-Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany; Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden; Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität, F.-L.-Jahn-Str. 15, D-17487 Greifswald, Germany; Medizinische Klinik m. S. Infektiologie und Pneumologie, Charité, Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany; Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany

^* To whom correspondence should be addressed. Email: m.steinert{at}tu-bs.de.

	Abstract

Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (SSPs) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here, we present comprehensive proteome reference maps for both fractions, SSPs and OMVs. Protein identification and assignment revealed a total of 181 supernatant proteins, of which 107 were specific to the SSP fraction and 33 to OMVs, respectively. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill but specifically modulate host cells regarding their cytokine response. Binding of immunofluorescence stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggests the delivery of a large and complex group of proteins and lipids within the infected tissue in association with OMVs. According to these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors as well as environmental aspects of this vesicle-mediated secretion.