Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection
Figure 6
C. trachomatis L2 induces a biphasic activation of Erk1/2 which contributes to the induction of fgf2 mRNA expression.
(A) Upper 2 panels: Erk1/2 activation in HeLa cells infected with C. trachomatis L2 for the indicated times was examined by immunoblotting cell lyates with antibodies to phospho-Erk (pERK) and to total Erk. Arrows indicate Erk1/2 (p42/p44). Two peaks of Erk1/2 activation are noted, at 45 min pi and at 10–12 hpi. Lower 2 panels: The same lysates from upper panel were immunoblotted with antibodies to FGF2 or to GAPDH (loading control). In the FGF2 immunoblot, arrows indicate the 24, 22.5/22, 18, and 16 kDa isoforms of FGF2. An abrupt change in FGF2 isoforms is noted at 10 hpi. (B) Total mRNA was isolated from HeLa cells infected with C. trachomatis L2 for the indicated times and the fold change in fgf2 mRNA relative to gapdh mRNA was measured by qRT-PCR. An increase in fgf2 mRNA is detectable by 6 hpi and increases further at 10–12 hpi. **p<0.01, ***p<0.001, compared to uninfected (UI) samples. (C, D) HeLa cells were infected with C. trachomatis L2 for 12 hrs in the presence of 10 µM U0126 (U) between 0–3 hpi or 8–12 hpi. Lysates were immunoblotted with antibodies to pErk or to total Erk. The fold change in fgf2 mRNA relative to gapdh mRNA was quantified by qRT-PCR using total RNA isolated at 12 hpi. Inhibition of either the early peak of Erk activation or the late peak of Erk activation decreased C. trachomatis L2 induction of fgf2 mRNA but did not affect the distribution of FGF2 isoforms. **p<0.01, ***p<0.001 compared to C. trachomatis L2 infected cells. (E, F) HeLa cells were infected with C. trachomatis L2 for 45 min or 12 hrs in the absence or presence of chlorampenicol (CAM; 100 µg/mL) for the indicated times. (E) Erk activation and FGF2 isoforms were detected by immunoblotting cell lyates with the indicated antibody. MOMP serves as a control for bacterial replication. (F) Total RNA was isolated at 12 hpi and the fold change in fgf2 mRNA relative to gapdh mRNA was monitored by qRT-PCR. Bacterial protein synthesis is required for Erk activation and for the change in FGF2 isoforms. *p<0.05, ***p<0.001 compared to uninfected (UI) cells.