Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin

Abstract

Infection of epithelial cells by two biovars of Chlamydia trachomatis results in the tyrosine phosphorylation of several host proteins. The most prominent change in host protein tyrosine phosphorylation involves a complex of proteins with molecular masses of 75 to 85 kDa (pp75/85) and 100 kDa (pp100). The C. trachomatisinduced tyrosine phosphorylation of pp75/85 and pp100 is observed in several cell lines, including epithelial cells, fibroblasts, and macrophages. Subcellular fractionation and detergent solubility properties of pp75/85 are consistent with its association with the cytoskeleton. Phosphoamino acid analysis demonstrates that the pp75/85 complex is phosphorylated on both tyrosine and serine residues. Immunofluorescence studies of chlamydia-infected cells by using fluorescein isothiocyanate-phalloidin and antibodies to phosphotyrosine and cortactin demonstrate that tyrosine-phosphorylated proteins, as well as cortactin, are localized to the chlamydial vacuole and that this process is facilitated by actin. MATERIALS AND METHODS Cell culture. The human epithelial HeLa cell line (ATCC CCL2; American Type Culture Collection, Rockville, Md.) was grown in Dulbecco's modified essential medium H16 (DME-H16 medium) supplemented with 5% (vol/vol) fetal calf serum (FCS) (DME-5). The mouse fibroblast L929 cell line (ATCC CCL1) was grown in RPMI 1640 medium containing 25 mM HEPES and 10% (vol/vol) FCS. The murine macrophage cell lines RAW 264.7 (ATCC TIB 71) and J774A.1 (ATCC TIB 67) were cultured in DME-H21 medium supplemented with 5% (vol/vol) calf serum and 5% (vol/vol) FCS. All cell lines were grown at 37°C in 5% CO 2. Medium was obtained from the Cell Culture Facility, University of California, San Francisco. FCS was obtained from Gibco (Bethesda, Md.). Growth and purification of chlamydiae. The MoPn biovar of C. trachomatis was routinely grown in HeLa cells for 48 h, after which it was purified as described previously (10). The LGV L2 serovar was obtained from Richard S. Stephens (University of California, Berkeley) and propagated in L929 cells. In some experiments, the MoPn biovar was purified by centrifugation on a discontinuous Renografin (Squibb Diagnostics, BMS Pharmaceutical Group, Irvine, Calif.) gradient (17). Infections were carried out at a multiplicity of infection (MOI) of 20 to 100 inclusion-forming units. Despite the high-MOI inocula used, no immediate cytotoxicity (prior to 18 h) was observed. Chlamydial infection and protein extraction. Subconfluent monolayers of HeLa, L929, RAW, or J774 cells (2 ϫ 10 5 cells) in 100-mm-diameter plates were infected for 1 h at 37°C with a purified preparation of MoPn or LGV in sucrosephosphate-glutamine buffer (10) diluted in DME-5. The inoculum was removed and replaced by fresh medium, and the infection was allowed to proceed for different periods of time. For mock infections, the cells were incubated with a preparation of uninfected cells purified by the protocol used for chlamydiae. After the medium was removed, the cells were washed with and scraped into cold

Chlamydia pneumoniae Infection of Human Endothelial Cells Induces Proliferation of Smooth Muscle Cells via an Endothelial Cell-Derived Soluble Factor(s)

“…Furthermore, our results demonstrate that viable C. pneumoniae is not required for HUVEC stimulation, suggesting that cell surface binding and/or phagosomal entry events are important in transducing the signals involved in upregulating cytokine or chemokine gene expression in the HUVEC. Such early events capable of inducing host cell signal transduction pathways via host protein tyrosine phosphorylation have been described for C. trachomatis (4,5) and are activated also by heat-or UV-treated C. trachomatis. A common signal transduction pathway may be activated in C. pneumoniae-infected HUVEC.…”

Involvement of cystic fibrosis transmembrane conductance regulator in infection‐induced edema

“…C. trachomatis infection as well as infection due to other bacteria leads to the production of cytokines and inflammatory mediators that also cause increase in CFTR expression and activity as reviewed by Ajonuma et al (2002) and subsequent fluid formation as demonstrated in this study by IL-1b. Secondly, C. trachomatis infection is known to induce protein tyrosine phosphorylation even as early as 15 min (Bliska et al, 1993;Birkelund et al, 1994;Fawaz et al, 1997;Hosseinzadeh et al, 2000) and upregulation (Xia et al, 2003) of several proteins. Interestingly, in this study, C. trachomatis infection also increased the expression of CFTR.…”

Endocytosis ofCandida albicansby vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins

“…The identity of the 80 and 82 kDa proteins that are phosphorylated in response to C. albicans remains to be determined. Known tyrosine kinase substrates with similar molecular masses include, cortactin, the 85 kDa regulatory subunit of class IA phosphatidylinositide 3-kinase, and the ezrin, radixin and moesin family of proteins (Takeuchi et al, 1994;Dehio et al, 1995;Fawaz et al, 1997;Lowry et al, 1998;Wymann and Pirola, 1998;Skoudy et al, 1999). Experiments are in progress to determine whether any of these candidate proteins are phosphorylated in endothelial cells that are infected with C. albicans.…”