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.