Figure 1
Figure 1. Expression of Fpn limits the growth of Chlamydia. (A) HEK293Fpn-GFP cells were incubated with FAC for 24 hours and induced to express Fpn-GFP (green) using ponasterone A. Eighteen hours after induction, cells were incubated with C psittaci at an MOI of 100 bacteria/cell for 2 hours, extracellular bacteria removed by extensive washing, and cells placed in growth medium for specified times. Twenty-four hours after infection, cells were fixed and processed for immunofluorescence using a mouse anti–C psittaci antibody followed by an Alexa 594–conjugated goat anti–mouse IgG (red). Cells that were successfully induced showed Fpn-GFP (green) at the plasma membrane and had small inclusions. Cells that did not express Fpn-GFP had large inclusions. Arrowheads denote large inclusions and arrows denote small inclusions. Bar represents 10 μm. (B) Bone marrow macrophages isolated from C57/B6 mice were iron loaded and infected with C psittaci for 2 hours as in panel A. Following infection, cells were incubated with or without 1 μg/mL hep25 for 18 hours. Cells were methanol fixed and processed for immunofluorescence using rabbit anti-Fpn and mouse anti–C psittaci followed by an Alexa 594–conjugated goat anti–rabbit IgG (red) and an Alexa 488–conjugated goat anti–mouse IgG (green). Arrowheads denote C psittaci and arrows denote plasma membrane Fpn. Bar represents 10 μm. (C) Cells treated as in panel B were solubilized in lysis buffer and applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Chlamydial infection was assessed by Western blot using mouse anti–C psittaci followed by peroxidase-conjugated goat anti–mouse IgG. Fpn levels were assessed using rabbit anti-Fpn followed by peroxidase-conjugated goat anti–rabbit IgG. Tubulin levels were detected using mouse antitubulin followed by peroxidase-conjugated goat anti–mouse IgG as a loading control. (D) Large (greater than 1 μ) and small inclusions were quantified from cells infected with C psittaci or C trachomatis. More than 200 cells were analyzed for inclusion size for each sample. Error bars represent the standard deviation of the mean of 3 independent experiments. (E) Macrophages were incubated with C psittaci for 2 hours, extracellular bacteria removed, and cells incubated in growth media for an additional 18 hours. Cells were solubilized in lysis buffer and applied to SDS-PAGE, and Fpn levels assessed as in panel D.

Expression of Fpn limits the growth of Chlamydia. (A) HEK293Fpn-GFP cells were incubated with FAC for 24 hours and induced to express Fpn-GFP (green) using ponasterone A. Eighteen hours after induction, cells were incubated with C psittaci at an MOI of 100 bacteria/cell for 2 hours, extracellular bacteria removed by extensive washing, and cells placed in growth medium for specified times. Twenty-four hours after infection, cells were fixed and processed for immunofluorescence using a mouse anti–C psittaci antibody followed by an Alexa 594–conjugated goat anti–mouse IgG (red). Cells that were successfully induced showed Fpn-GFP (green) at the plasma membrane and had small inclusions. Cells that did not express Fpn-GFP had large inclusions. Arrowheads denote large inclusions and arrows denote small inclusions. Bar represents 10 μm. (B) Bone marrow macrophages isolated from C57/B6 mice were iron loaded and infected with C psittaci for 2 hours as in panel A. Following infection, cells were incubated with or without 1 μg/mL hep25 for 18 hours. Cells were methanol fixed and processed for immunofluorescence using rabbit anti-Fpn and mouse anti–C psittaci followed by an Alexa 594–conjugated goat anti–rabbit IgG (red) and an Alexa 488–conjugated goat anti–mouse IgG (green). Arrowheads denote C psittaci and arrows denote plasma membrane Fpn. Bar represents 10 μm. (C) Cells treated as in panel B were solubilized in lysis buffer and applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Chlamydial infection was assessed by Western blot using mouse anti–C psittaci followed by peroxidase-conjugated goat anti–mouse IgG. Fpn levels were assessed using rabbit anti-Fpn followed by peroxidase-conjugated goat anti–rabbit IgG. Tubulin levels were detected using mouse antitubulin followed by peroxidase-conjugated goat anti–mouse IgG as a loading control. (D) Large (greater than 1 μ) and small inclusions were quantified from cells infected with C psittaci or C trachomatis. More than 200 cells were analyzed for inclusion size for each sample. Error bars represent the standard deviation of the mean of 3 independent experiments. (E) Macrophages were incubated with C psittaci for 2 hours, extracellular bacteria removed, and cells incubated in growth media for an additional 18 hours. Cells were solubilized in lysis buffer and applied to SDS-PAGE, and Fpn levels assessed as in panel D.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal