Cell surface PDI enzymatic activity was assessed by diphtheria toxin (DT) sensitivity assays. Toxin killing of cells requires plasma membrane PDI function, percent survival was determined by comparing the level of cell viability in DT treated and untreated cells. Cells were incubated with 100 ng ml⁻¹ DT, washed, and then incubated at 37°C. 72 h after toxin treatment, cell viability was determined by using the Promega CellTiter 96 Aqueous One Cell Proliferation Assay. CHOK1 cells were sensitive to DT, but CHO6 cells were not. When PDI was expressed in CHO6 (CHO6+PDI), the cells became sensitive to DT, indicating that cell surface PDI activity was restored in CHO6+PDI. Surface PDI activity is not restored in CHO6+PDI-4CS, as CHO6+PDI-4CS cells were relatively resistant to DT. Error bars indicate standard error of the mean (SEM).

(A) Chlamydia attachment to CHOK1 was evaluated by immunofluorescence staining of bacteria (green) 1 h after infection and counter staining with Evans blue. Cells were infected and then maintained in media containing 0 mM or 3 mM bacitracin. Similar levels of bacterial attachment were observed with and without bacitracin. (B) Entry was examined after a 2 h incubation at 37°C. Entry was analyzed by comparing the total number of cell-associated bacteria (permeabilized) and the number of extracellular bacteria (unpermeabilized). 3 mM bacitracin inhibited bacterial entry. (C) The effect of bacitracin on Chlamydia attachment and infection was quantified and is shown as the % attachment and infection of cells not treated with bacitracin. Attachment was analyzed 1 h post-infection and infection was evaluated 24 h post-infection. (D) The effect of bacitracin on Chlamydia development was examined by evaluating cells 24 h post-infection that had not been treated with bacitracin (a′), been treated with 3 mM bacitracin for 20 min prior to infection (b′), been treated with bacitracin for the first 8 h of infection (c′), or been treated with bacitracin only for the last 16 h of the 24 h infection (d′). In all cases the development of bacteria-containing vacuoles was seen, indicative of a normal infection.

(A) CHO6 cells transfected with a vector expressing PDI with a gpi-anchor (PDI-gpi) are marked with a white arrow, untransfected cells are indicated by a yellow arrow. Cells were fixed and stained for PDI using PDI-specific antibody. Expression of PDI-gpi led to a large amount of PDI localized at the cell surface. (B) Cell survival was analyzed 72 h after DT treatment. CHOK1 cells were extremely sensitive to toxin, whereas CHO6 cells were relatively resistant. Expression of PDI-gpi in CHOK1 or CHO6 cells did not alter toxin sensitivity, indicative of an inability of PDI-gpi to properly interact with other cell surface proteins. (C) Chlamydia attachment to CHOK1 and CHO6+PDI-gpi was analyzed, bacteria are shown in green, and PDI is stained red. There was no bacterial attachment to CHO6+PDI-gpi.

(A) Immunoblot of cells transfected with PDI-targeted siRNA or non-targeting siRNA using antibody to PDI. GAPDH expression was evaluated to ensure equivalent loading. (B) Following siRNA treatment, C. psittaci and C. trachomatis attachment were examined. PDI staining is shown in red, and bacteria are stained green. Because of the significant PDI downregulation, cells in PDI siRNA panels are marked by arrows. siRNA downregulation of PDI led to a dramatic decrease in bacterial attachment, but attachment was not affected by treatment with non-targeting siRNA. (C) The number of bacteria attached to cells following treatment with non-targeting siRNA (gray) or PDI siRNA (black) was determined by quantification of the number of cell-associated bacteria by counting eight separate fields of view containing at least ten cells. Error bars indicate standard error of the mean (SEM).

CHO6 cells were transfected with vectors expressing wildtype PDI (CHO6+PDI) or an enzymatic mutant of PDI lacking active site cysteine residues (CHO6+PDI-4CS). 48 h after transfection, cells were infected with Chlamydia for subsequent attachment and entry analysis. Cells were fixed and evaluated by immunofluorescence, bacteria are green and counter staining shown with Evans blue. (A) Bacterial attachment was analyzed 1 h post-infection. Bacterial attachment was recovered in CHO6+PDI as well as in CHO6+PDI-4CS, indicating that PDI enzymatic activity is not necessary for bacterial attachment. (B) The number of bacteria attached to cells was determined by quantification, the number of bacteria associated with each cell in eight separate fields of view containing at least ten cells. Error bars indicate standard of deviation (STDEV). (C) 24 h after infection Chlamydia infectivity was evaluated. Infectivity was restored in CHO6+PDI. No productive infection was observed in CHO6 cells or in CHO6+PDI-4CS. In CHO6+PDI-4CS, the bacteria remained persistently attached to the cell, indicative of the requirement for PDI enzymatic activity for bacterial entry. (D) Infection was quantified by counting the number of inclusions per field of view in eight separate fields of view containing at least ten cells. Error bars indicate the STDEV. (E) Cells were incubated at 37°C for 2 h to allow for bacterial entry. Entry was analyzed by comparing the total number of cell-associated bacteria for staining of permeabilized cells (Total) and the number of extracellular bacteria determined by staining unpermeabilized cells (Extracellular). Bacterial entry was recovered in CHO6 cells expressing PDI (CHO6+PDI) but not in CHO6 cells expressing enzymatically nonfunctional PDI (CHO6+PDI-4CS). (F) Percent internalization represents 1 minus the number of extracellular bacteria divided by the total number of bacteria multiplied by 100. The number of extracellular and total bacteria was determined by quantifying the number of bacteria associated with each cell per field of view in eight separate fields of view containing at least ten cells. Error bars indicate the STDEV.

(A) Bacterial entry into CHOK1 cells treated with 3 mM bacitracin was analyzed after a 2 h incubation at 37°C. Bacteria are shown in green and counter stained with Evans blue. Entry was assessed by comparing the total number of cell-associated bacteria (permeabilized) and the number of extracellular bacteria (unpermeabilized) in cells that were treated with 0 mM or 50 mM TCEP. Percent internalization represents 1 minus the number of extracellular bacteria divided by the total number of bacteria multiplied by 100. The number of extracellular and total bacteria was determined by quantifying the number of bacteria associated with each cell per field of view in eight separate fields of view containing at least ten cells. Error bars indicate the STDEV. Bacitracin-mediated inhibition was overcome by the addition of the membrane-impermeable reducing agent TCEP. (B) Infection was evaluated in CHOK1 cells treated with 3 mM bacitracin 24 h post-infection in cells that had been treated for the initial 2 h of infection with 0 mM or 50 mM TCEP. Development of a normal infection occurred in cells treated with TCEP, whereas the bacteria remained persistently attached to the outside of untreated cells. (C) Bacterial entry into CHO6 cells transfected with a vector expressing enzymatically nonfunctional PDI (PDI-4CS) was analyzed after a 2 h incubation at 37°C. Percent internalization was determined as in (A). Bacterial entry is seen in cells treated with 50 mM TCEP but not in untreated cells.

(A) Cells were incubated with 100 µg ml⁻¹ polyclonal PDI-specific antibody or 100 µg ml⁻¹ irrelevant IgG antibody prior to and during bacterial attachment. Bacteria were allowed to attach for 1 h and binding was analyzed, bacteria are shown in green and counter stain is Evans blue. There was no significant difference in the amount of bacteria that bound to cell with and without PDI antibody treatment. (B) Entry was analyzed after a 2 h incubation at 37°C. Entry was assessed by comparing the total number of cell-associated bacteria (permeabilized) and the number of extracellular bacteria (unpermeabilized) in cells that were treated with100 µg ml⁻¹ polyclonal PDI-specific antibody or 100 µg ml⁻¹ irrelevant IgG antibody.