FIG 4.

PDI-mediated disulfide bond reduction of the A. phagocytophilum surface but not the host cell surface is key for infection. HL-60 cells were incubated with antibody BD34 to catalytically inhibit cell surface PDI or with isotype control antibody. The HL-60 cells were then treated with TCEP for 30 min to chemically reduce host cell surface disulfide bonds, followed by PBS washing and subsequent incubation with A. phagocytophilum organisms. Alternatively, A. phagocytophilum DC organisms were treated with TCEP for 30 min to reduce bacterial cell surface disulfide bonds, followed by PBS washing and subsequent incubation with BD34- or isotype control-treated HL-60 cells. At 24 h, the HL-60 cells that had been exposed to A. phagocytophilum (Ap) under each condition were examined by immunofluorescence microscopy for the percentages of infected cells (A) and numbers of ApVs per cell (B). Data are presented as the mean values ± SD from triplicate samples and are representative of individual experiments performed three times. Statistically significant values are indicated. *, P < 0.05; **, P < 0.01.