title: Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells.
abstract: Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains exhibiting strongly reduced PPIase activity. These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. pneumophila.
authors.affiliation: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Wintermeyer
authors.forename: B
authors.initials: B
authors.lastname: Ludwig
authors.forename: M
authors.initials: M
authors.lastname: Steinert
authors.forename: B
authors.initials: B
authors.lastname: Schmidt
authors.forename: G
authors.initials: G
authors.lastname: Fischer
authors.forename: J
authors.initials: J
authors.lastname: Hacker
mesh_terms: D000591:Amino Acid Isomerases / Q000502:physiology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000502:physiology; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000502:physiology; D006360:Heat-Shock Proteins / Q000502:physiology; D006801:Humans; D020104:Immunophilins; D016952:Legionella pneumophila / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000382:microbiology; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase