Proteins complexed to the P1 adhesin of Mycoplasma pneumoniae

• Published in: Microbiology (Society for General Microbiology) 2000-03, Vol.146 (3), p.741-747
• Main Authors: Layh-Schmitt, Gerlinde, Podtelejnikov, Alexandre, Mann, Matthias
• Format: Article
• Language: English
• Subjects: adhesin P1; Adhesins, Bacterial - metabolism; Bacterial Adhesion; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Chromatography, Affinity - methods; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Formaldehyde; Fundamental and applied biological sciences. Psychology; Immunoblotting; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Microbiology; Mycoplasma pneumoniae; Mycoplasma pneumoniae - growth & development; Mycoplasma pneumoniae - metabolism; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Polymers; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-146-3-741

Hygiene-Institut, Universität Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany 1 EMBL, Meyerhofstraße 1, 69123 Heidelberg, Germany 2 Author for correspondence: Gerlinde Layh-Schmitt. Tel: [phone]. Fax: [phone]. e-mail: Layhschmitt.G{at}pg.com Adherence of Mycoplasma pneumoniae to host cells requires several mycoplasmal membrane proteins and cytoskeleton-like proteins in addition to the adhesin P1, a transmembrane protein of 170 kDa. To analyse interactions of the P1 adhesin with other membrane proteins or with cytoskeleton-like proteins, cross-linking studies were performed in vivo using the permeant reagent paraformaldehyde. The cross-linked protein complex was isolated by immunoaffinity chromatography, and proteins complexed to the P1 protein were identified by immunoblot analysis followed by high mass accuracy tryptic peptide mapping using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). In addition to the P1 protein and a truncated form of the same protein, the adhesin-related 30 kDa protein, two membrane proteins of 40 and 90 kDa, the cytoskeleton-associated 65 kDa protein and two cytoskeleton-forming proteins, HMW1 and HMW3, were found to be components of the isolated protein complex. Furthermore, the cross-linked complex contained the chaperone DnaK and the E1 subunit of pyruvate dehydrogenase. In summary, it was shown that cytadherence-associated membrane proteins are located in close proximity to cytoskeleton-like proteins, suggesting a functional interaction between membrane and cytoskeleton-like proteins. DnaK might be involved in translocation of proteins from the cytoplasm to the membrane and pyruvate dehydrogenase might be a structural protein of the attachment organelle. Keywords: Mycoplasma pneumoniae , P1 adhesin, paraformaldehyde cross-linking Abbreviations: MALDI MS, matrix-assisted laser desorption/ionization mass spectrometry a Present address: Procter & Gamble Pharmaceuticals, Health Care Research Center, Anti-Infective Research, PO Box 8006, 8700 Mason-Montgomery Road, Mason, OH 45040, USA. b Present address: Protein Interaction Laboratory, The University of Southern Denmark, Campusvej 55, DK 5230, Denmark.