title: 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.
abstract: 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 T(1). 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.
authors.affiliation: Institut für Molekulare Infektionsbiologie, Universität Würzburg, D-97070 Würzburg, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Köhler
authors.forename: Jörg
authors.initials: J
authors.lastname: Fanghänel
authors.forename: Bettina
authors.initials: B
authors.lastname: König
authors.forename: Edeltraud
authors.initials: E
authors.lastname: Lüneberg
authors.forename: Matthias
authors.initials: M
authors.lastname: Frosch
authors.forename: Jens-Ulrich
authors.initials: JU
authors.lastname: Rahfeld
authors.forename: Rolf
authors.initials: R
authors.lastname: Hilgenfeld
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
authors.forename: Jörg
authors.initials: J
authors.lastname: Hacker
authors.forename: Michael
authors.initials: M
authors.lastname: Steinert
mesh_terms: D000048:Acanthamoeba / Q000382:microbiology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002918:Chymotrypsin; D004195:Disease Models, Animal; D006168:Guinea Pigs; D006801:Humans; D020104:Immunophilins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016952:Legionella pneumophila / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D007877:Legionnaires' Disease / Q000209:etiology; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014774:Virulence / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002918:Chymotrypsin; D020104:Immunophilins; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase