Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB of Legionella pneumophila Contribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection.

The gammaproteobacterium Legionella pneumophila is the causative agent of Legionnaires' disease, an atypical pneumonia that manifests itself with severe lung damage. L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. In L. pneumophila the surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness of L. pneumophila Furthermore, they contribute to infection of the natural host Acanthamoeba castellanii as well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators of L. pneumophila with compensatory action and pleiotropic effects.

[Muscle efficiency in total shoulder prosthesis implantation: dependence on position of the humeral head and rotator cuff function]. / Muskeleffizienz bei totaler Schulterendoprothetik: Abhängigkeit von Kalottenposition und Rotatorenmanschettenfunktion.

Modern shoulder prostheses permit an anatomic reconstruction of the joint, although the biomechanical advantages are not proven. The goal of this study was to investigate the relationship between position of the humeral head and function of the shoulder prosthesis (muscle efficiency). Shoulder elevation-motion and rotator cuff defects were simulated in vitro in a robot-assisted shoulder simulator. The EPOCA Custom Offset shoulder prosthesis (Argomedical AG, Cham, CH) was implanted in seven normal shoulders (77 +/- 20 kg, 55 +/- 14 years). Active elevation was simulated by hydraulic cylinders, and scapulothoratic motion by a specially programmed industrial robot. Muscle efficiency (elevation-angle/muscle-force of the deltoid muscle) was measured in anatomic (ANA), medialised (MED) and lateralised (LAT) positions of the humeral head, with or without rotator cuff muscle deficiency. Medialisation increased efficiency by 0.03 +/- 0.04 deg/N (p = 0.022), lateralisation decreased it by 0.04 +/- 0.06 deg/N (p = 0.009). Supraspinatus muscle deficiency increased the deltoid force required to elevate the arm, and thus decreased efficiency (ANA p = 0.091, MED p = 0.018, LAT p = 0.028). The data confirm that the position of the humeral head affects the mechanics of total shoulder arthroplasty. Medialisation increases efficiency of the deltoid muscle and may prove useful in compensating isolated supraspinatus muscle deficiency. Lateralisation, in contrast, leads to an unfavorable situation.

Chemical synthesis of acyl thioesters of acyl carrier protein with native structure.

The acyl carrier protein (ACP) of Escherichia coli was converted to acyl-ACP by imidazole-catalyzed S-acylation with N-acylimidazole. The acylation was specific to the sulfhydryl group; no acylation of tyrosine or amino groups of the protein occurred. The acyl-ACP substrates synthesized had a native structure as determined by gel electrophoresis, hydrophobic chromatography, and enzymatic activity. N-Acylimidazoles are readily synthesized and permit preparation of those acyl-ACP substrates that cannot be produced enzymatically.

Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli.

Beta-Ketoacyl-acyl carrier protein synthases I and II of Escherichia coli were purified and characterized. Synthase I was shown to have a molecular weight of 80,000 +/- 5,000 and to be composed of two similarly sized subunits. Synthase II had a molecular weight of 85,000 +/- 5,000 and also was apparently homodimeric. Gel electrophoresis of partial proteolytic digests demonstrated that synthases I and II share few if any common peptides. Synthases I and II also were shown to be unrelated by immunological criteria. An improved assay for beta-ketoacyl-acyl carrier protein synthase activity gave kinetic parameters for synthases I and II at both 27 degrees C and 37 degrees C using five long chain acyl-acyl carrier protein substrates. The properties of synthase II are consistent with the proposed role of this enzyme in the modulation of fatty acid synthesis by temperature. fabF mutants of E. coli lack synthase II. The fabF locus was mapped at min 24.5 of the E. coli genetic map and the clockwise map order was found to be pyrC, fabD, fabF, purB.

Beta-ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis.

Cvc- mutants of Escherichia coli are deficient in the synthesis of cis-vaccenic acid and in the temperature control of fatty acid synthesis. In this communication, it is demonstrated that these mutants lack beta-ketoacyl-acyl carrier protein synthase II. The deficiencies in cis-vaccenate synthesis and synthase II are shown to be due to a lesion in the same gene, fabF. Lesions in the fabF gene are found to affect growth only when the strain also carries a lesion in the fabB gene, the structural gene for beta-ketoacyl-acyl carrier protein synthase I.