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 FK 506. 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.

Microbial Peptidyl-Prolyl cis / trans Isomerases (PPIases): Virulence Factors and Potential Alternative Drug Targets

“…The purified recombinant proteins exhibited a pronounced loss of PPIase activity in in vitro peptide folding assays (5.3% for the Asp142Leu mutant and 0.6% for the Tyr185Ala mutant). However, when the same variants of mip were used to complement ⌬mip mutants, they behaved in infection studies of amoeba or human macrophages and blood monocytes as if wild-type mip had been used (243). It was concluded that either additional properties other than PPIase activity are important for Mip action during intracellular infection or the residual enzymatic activity of the mutant proteins was still sufficient for exerting PPIase-dependent phenotypes.…”

Legionella: from environmental habitats to disease pathology, detection and control

“…Within this ecological niche, L. pneumophila alters its gene expression and expresses various virulence-related genes in response to starvation and signalling by (p)ppGpp (Abu Kwaik et al, 1998). The Mip (macrophage infectivity potentiator) protein, which belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase, represents a factor of L. pneumophila necessary for optimal intracellular survival (Wintermeyer et al, 1995). L. pneumophila is able to replicate within the phagosomes of mammalian cells, because it is not traf®cked through the endosomal±lysosomal pathway and is surrounded by the rough endoplasmic reticulum (Abu Kwaik et al, 1998).…”

Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix

“…The expression of a number of specific virulence traits could be linked to the particular growth phases (for review see Heuner and Steinert, 2003). Among these is the Legionella Mip (macrophage infectivity potentiator) protein which is known to contribute to the survival of Legionella in different cell lines and to virulence in guinea pigs (Engleberg et al, 1989;Cianciotto et al, 1990;Cianciotto and Fields, 1992;Wintermeyer et al, 1995;Swanson and Hammer, 2000). The Mip promoter is temporarily repressed immediately after invasion of human monocytes and regains activity after 24 h of intracellular replication (Wieland et al, 2002).…”

“…The fold of the C-terminal domain (residues 100-213) is closely related to the human FK506-binding protein FKBP12 (Fischer et al, 1984;Schmid, 1993). The binding of the immunosuppressants FK506 and rapamycin to a hydrophobic site of this C-terminal domain inhibit the PPIase activity of the Mip protein. Infection studies in Acanthamoeba castellanii, Hartmannella vermiformis, human mononuclear phagocytes, lung epithelial cells and guinea pigs revealed that Mip-negative mutants of Legionella are less infective when compared with their isogenic Mip-positive parental strains (Cianciotto and Fields, 1992;Cianciotto et al, 1995;Wintermeyer et al, 1995). The determination of the three-dimensional structure of Legionella Mip enabled a rational starting point for functional analysis of this protein (Köhler et al, 2003).…”