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J. Bacteriol. doi:10.1128/JB.01889-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Molecular characterization and subcellular localization of macrophage infectivity potentiator, a Chlamydia trachomatis lipoprotein

Division of Rheumatology, Department of Internal Medicine, Geneva University Hospital, Department of Pathology and Immunology, Geneva Medical School, Laboratory of Mass Spectrometry, University of Geneva, and Department of Cell Physiology and Metabolism, Geneva Medical School, Switzerland

^* To whom correspondence should be addressed. Email: sylvette.bas{at}hcuge.ch.

	Abstract

Macrophage infectivity potentiator (MIP) was originally reported to be a chlamydial lipoprotein from experiments showing incorporation of radiolabeled palmitic acid into the native and recombinant MIP, inhibition of post-translational processing of recombinant MIP by globomycin, known to inhibit signal peptidase II, and solubility of native MIP in Triton X-114. However, the detailed structural characterization of the lipidic moiety on MIP has never been fully elucidated. In this study, bioinformatics and mass spectrometry analysis, as well as radiolabeling and immunochemical experiments were conducted to further characterize MIP structure and subcellular localization. In silico analysis showed that the amino acid sequence of MIP is conserved across Chlamydiae species. A potential signal sequence with a contained lipobox was identified and a recombinant variant C20A was prepared by substituting the probable lipobox cysteine for an alanine. Both incorporation of [U-¹⁴C]-esterified glycerol and [U-¹⁴C]-palmitic acid as well as post-translational processing that was inhibitable by globomycin, were observed in wild type recombinant MIP, but not in the C20A recombinant MIP variant. Fatty acid content of native and recombinant MIP were analyzed by gas chromatography-mass spectrometry and the presence of amide-linked fatty acids was investigated by alkaline methanolysis in recombinant MIP. These results demonstrated a lipid modification in MIP similar to that of other procaryotic lipoproteins. In addition, MIP was detected in outer membrane preparation of Chlamydia trachomatis elementary bodies, and was shown to be present at the surface of elementary bodies by surface biotinylation and surface immunoprecipitation experiments.

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