Genomic Approach for Analysis of Surface Proteins inChlamydia 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 genomicproteomic 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.Chlamydia pneumoniae is an obligate intracellular bacterium and a common human pathogen (48). It is a significant cause of pneumonia in both hospital and outpatient settings, accounting for approximately 7 to 10% of cases of community-acquired pneumonia among adults. C. pneumoniae has also been associated with atherosclerotic and cardiovascular disease, as suggested by results of seroepidemiologic studies, detection of the organism in atherosclerotic plaque specimens, experimental in vitro cell culture studies, animal model studies, and two small secondary prevention antibiotic treatment trials (12,13,15,19,20,28,45).Like all obligate intracellular parasites, for its survival and propagation C. pneumoniae must accomplish several essential tasks which include adhering to and entering host cells, creating an intracellular niche for replication, exiting host cells for subsequent invasion of neighboring cells, and also avoiding host defense mechanisms. To carry out all these functions, C. pneumoniae has developed a unique biphasic life cycle involving two developmental forms, a spore-like infectious form (elementary bodies [EBs]) and an intracelluar replicative form (reticulate bodies [RBs]). Adhesion, host cell colonization capabilities, and the ability to cope with host defense mechanisms when outside the cell presumably rely in large part on EB surface organization.Because of the intrinsic difficulty in working with C. pneumoniae and the lack of adequate methods for its genetic ma...

Chlamydophila pneumoniae

“…Enolase [10] 46 by this pathogen and the wide inter-individual variability in the immune response. Clinical manifestations of atherosclerosis vary from coronary artery disease (CAD) to cerebrovascular disease.…”

“…Thus, another interesting candidate would be low-calcium-response protein E. This seems to be present on the surface of EBs and accessible to antibodies, according to Montigiani et al [10] , inducing activation of T CD4 and CD8 cells with cytokine secretion and antibody neutralization, proving completely effective in the elimination of the infection [14] . It is, therefore, a candidate as a vaccine because it is related to the type III secretion system, and an effective blockade of this system may permanently inhibit the C. pneumoniae infectious process [10] .…”

“…Key studies in the identification of C. pneumoniae proteins include the reports by Vandahl et al [11] and Molestina et al [15] , based on bidimensional electrophoresis analysis and subsequent identification by matrix-assisted laser desorption/ionization-mass spectrometry of a large number of C. pneumoniae proteins. Among studies describing immunogenic proteins, we highlight the identification by Montigiani et al [10] of immunogenic EB surface proteins. They used a genomic and proteomic approach based on in silico prediction, followed by the expression and purification of C. pneumoniae surface antigens, production of mouse immune sera to be used in western blotting and fluorescence-activated cell sorter analyses, confirmed by mass spectro metry analysis of 2D electrophoresis.…”

“…Nevertheless, other studies did not find this association, either by direct detection of the pathogen in atheromatous plaques in coronary arteries [17] or in seroepidemiological studies [18,19] . The possible causal role of C. pneumoniae in the pathogenesis of atherosclerosis is controversial because of the several obstacles in establishing this association, including the difficulty of avoiding contagion Low calcium response E protein [10,14] 43 kDa Regulator type III secretion system Unknown Immunogenic 5…”

“…Its antigens have been widely reported in numerous studies, and the more important are listed in Table 1 [9][10][11][12][13][14] . Although the type and composition of its antigenic proteins are known, further knowledge of its immunogenic antigens is required to improve the diagnosis and prevention of this disease.…”