Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection
Figure 10
C. trachomatis serovar E co-opts the FGF2 pathway.
(A) HeLa cells were treated with heparinase for 2 hr in SFM and infected with C. trachomatis serovar E either in SFM or in SFM supplemented with FGF2 (100 ng/mL) for 1 hr. Shown is the representative mean binding (± SEM) from three independent experiments. **p<0.01. (B) HeLa cells were serum starved for 2 hrs and then infected for 1 hr with C. trachomatis serovar E either in SFM or in SFM supplemented with FGF2 (100 ng/mL) in the presence or absence of heparin (1 mg/mL). Shown is the representative mean binding (± SEM) from three independent experiments. ***p<0.001 (C) Renograffin-purified serovar E EBs were incubated with 100 ng/mL of FGF1 or FGF2 in SFM containing 0.1% BSA for 1 hr at 37°C. The EB-FGF mixture was centrifuged onto coverslips, fixed, and stained with DAPI (to visualize the EBs) and FGF1 or FGF2 antibody. Co-localization of EBs with FGF1 or FGF2 was quantified from at least 8 different fields. The data are expressed as a mean percentage of bacteria associated with FGF (± SEM) compared to total bacteria. ***p<0.001. (D) Upper 2 panels: Erk1/2 activation in HeLa cells infected with C. trachomatis serovar E was examined by immunoblotting cell lyates with antibodies to pERK or to total ERK. The 42 and 44 kDa forms of ERK are indicated by the arrows. Middle panel: Cell lysates were immunoblotted with C. trachomatis MOMP antibody. Lower 2 panels: The change in cell-associated FGF2 isoforms in HeLa cells infected with C. trachomatis serovar E for the indicated times was assessed by immunoblotting cell lysates with antibodies to FGF2. GAPDH serves as a loading control. 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. (E) Total mRNA was isolated from HeLa cells infected with C. trachomatis serovar E for the indicated time and the fold change in fgf2 mRNA relative to gapdh mRNA was measured by qRT-PCR. An increase in fgf2 mRNA is detectable by 9 hpi and increases further at 12 hpi. ***p<0.001 compared to mock-infected cells. (F) HeLa cells were mock-infected for 72 hrs or infected with C. trachomatis serovar E in 5% FBS for 24, 48, or 72 hrs. Conditioned media were collected, filtered, immunoprecipitated with FGF2 antibody, and immunoblotted with FGF2 antibody. (G) Renograffin-purified serovar E EBs were resuspended in mock-CM or CM from serovar E infected (CT-CM). Binding to HeLa cells was measured at 1 hpi. Shown is the representative mean binding (± SEM) from three independent experiments. ***p<0.001 compared to mock-CM.