title: Genomic approach for analysis of surface proteins in Chlamydia pneumoniae.
abstract: Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.
authors.affiliation: Chiron SpA, 53100 Siena. Sezione di Microbiologia DMCSS, University of Bologna, 40138 Bologna, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Montigiani
authors.forename: Fabiana
authors.initials: F
authors.lastname: Falugi
authors.forename: Maria
authors.initials: M
authors.lastname: Scarselli
authors.forename: Oretta
authors.initials: O
authors.lastname: Finco
authors.forename: Roberto
authors.initials: R
authors.lastname: Petracca
authors.forename: Giuliano
authors.initials: G
authors.lastname: Galli
authors.forename: Massimo
authors.initials: M
authors.lastname: Mariani
authors.forename: Roberto
authors.initials: R
authors.lastname: Manetti
authors.forename: Mauro
authors.initials: M
authors.lastname: Agnusdei
authors.forename: Roberto
authors.initials: R
authors.lastname: Cevenini
authors.forename: Manuela
authors.initials: M
authors.lastname: Donati
authors.forename: Renzo
authors.initials: R
authors.lastname: Nogarotto
authors.forename: Nathalie
authors.initials: N
authors.lastname: Norais
authors.forename: Ignazio
authors.initials: I
authors.lastname: Garaguso
authors.forename: Sandra
authors.initials: S
authors.lastname: Nuti
authors.forename: Giulietta
authors.initials: G
authors.lastname: Saletti
authors.forename: Domenico
authors.initials: D
authors.lastname: Rosa
authors.forename: Giulio
authors.initials: G
authors.lastname: Ratti
authors.forename: Guido
authors.initials: G
authors.lastname: Grandi
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D016993:Chlamydophila pneumoniae / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D016680:Genome, Bacterial; D006801:Humans; D051379:Mice; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins