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Infection and Immunity, August 2003, p. 4389-4397, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4389-4397.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of Legionella pneumophila

Rolf Köhler,1 Jörg Fanghänel,2 Bettina König,2 Edeltraud Lüneberg,3 Matthias Frosch,3 Jens-Ulrich Rahfeld,2 Rolf Hilgenfeld,4 Gunter Fischer,2 Jörg Hacker,1 and Michael Steinert1*

Institut für Molekulare Infektionsbiologie,1 Institut für Hygiene und Mikrobiologie, Universität Würzburg, D-97070 Würzburg,3 Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, D-06120 Halle,2 Institut für Molekulare Biotechnologie e.V., D-07708 Jena, Germany4

Received 2 December 2002/ Returned for modification 11 February 2003/ Accepted 26 May 2003

The virulence factor Mip (macrophage infectivity potentiator) contributes to the intracellular survival of Legionella pneumophila, the causative agent of Legionnaires' disease. The protein consists of two domains that are connected via a very long {alpha}-helix (A. Riboldi-Tunnicliffe et al., Nat. Struct. Biol. 8:779-783, 2001). The fold of the C-terminal domain (residues 100 to 213) is closely related to human FK506-binding protein (FKBP12), and like FKBP12, Mip exhibits peptidylprolyl cis/trans isomerase (PPIase) activity. The {alpha}-helical N-terminal domain is responsible for the formation of very stable Mip homodimers. In order to determine the importance of the homodimeric state of Mip for its biochemical activities and for infectivity of Legionella, a truncated, monomeric Mip variant [Mip(77-213)] was overexpressed in Escherichia coli and characterized biochemically. In vitro isomerase activity assays revealed that the altered protein exhibits full isomerase activity towards peptide substrates. However, the deletion resulted in a dramatic loss in the efficiency of refolding of reduced and carboxy-methylated RNase T1. By cis complementation of the Mip-negative mutant strain L. pneumophila JR32-2, we constructed the strain L. pneumophila JR32-2.4, which expresses an N-terminally truncated variant of Mip. Infection studies with these strains revealed that the N-terminal part and the dimerization of Mip but not its PPIase activity are necessary for full virulence in Acanthamoeba castellanii. Infection of guinea pigs showed that strains with dimerization-deficient Mip (JR32-2.4) or a very low PPIase activity (JR32-2.2) were significantly attenuated in the animal model. These results suggest a different role of the PPIase activity and the N-terminally mediated dimeric state of Mip in monocellular systems and during the infection of guinea pigs.

* Corresponding author. Mailing address: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-312588. Fax: 49-931-312578. E-mail: michael.steinert{at}mail.uni-wuerzburg.de.

Editor: S. H. E. Kaufmann

Infection and Immunity, August 2003, p. 4389-4397, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4389-4397.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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