Proteomic analysis ofChlamydia pneumoniae-infected HL cells reveals extensive degradation of cytoskeletal proteins

Cytoskeletal proteins of HL cells, following Chlamydia pneumoniae infection, were studied by two-dimensional gel electrophoresis and two-dimensional difference gel electrophoresis. Proteome analyses of HL cells at 48 and 72 h postinfection revealed significant changes in important constituents of the intermediate filament and microtubulin networks. These cytoskeletal proteins, identified as keratin K8, keratin K18, vimentin and beta-tubulin, were represented by several distinct spots with different pI and molecular weight values, implying that they have undergone posttranslational modifications stimulated by the infection. According to MS analyses, these proteins appeared to be N- and/or C-terminally truncated. Additional immunoblot analyses suggested that inhibiting the activity of the chlamydial protease-like activity factor (CPAF) by lactacystin results in increased stability of keratin K18, vimentin and beta-tubulin in infected HL cells. Interestingly, primary amino acid sequence analyses revealed potential recognition/cleavage sites for CPAF in each of these cytoskeletal proteins. These results provide an insight into the pathogenic mechanisms exploited by chlamydia, and suggest that proteolytic modification of the indicated proteins may be involved in establishing a productive infection.