INFLUENCE OF SITE-SPECIFICALLY ALTERED MIP PROTEINS ON INTRACELLULAR SURVIVAL OF LEGIONELLA-PNEUMOPHILA IN EUKARYOTIC CELLS

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocyt es, macrophages, and protozoan organisms. The Mip (macrophage infectiv ity potentiator) protein represents a factor of L. pneumophila necessa ry for optimal intracellular survival, Interestingly, Mip belongs to t he substance class of FK 506-binding proteins and exhibits peptidyl-pr olyl cis/trans isomerase (PPIase) activity that can be inhibited by th e immunosuppressant FK 506. In order to identify amino acids most like ly to be involved in the enzymatic activity of Mip, site-directed muta genized Mip proteins were constructed and characterized, It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitut ion resulted in strongly reduced PPIase activity of the recombinant Mi p proteins (5.3 and 0.6% of the activity of the wild-type Mip, respect ively). Genes coding for the wild-type and for site-directed-mutageniz ed Mip proteins were used to complement three different Mip-negative m utants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. Whi te Mip protein expression could be restored in the corresponding compl ementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investig ate the influence of the PPIase activity of Mip on intracellular survi val of L. pneumophila, invasion assays were performed using the macrop hage-like cell line U937, human blood monocytes, and Acanthamoeba cast ellanii. The Mip-negative mutants were approximately 50- to 100-fold l ess infective for A, castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains, T he wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains, In addition, no differ ences in intracellular survival were observed between the wild-type is olates and the Legionella strains exhibiting strongly reduced PPIase a ctivity, These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. pneumophila.