Figure 1.

The ApV engages the host ER throughout A. phagocytophilum infection of mammalian host cells. (A) ER markers localize to and within the ApV in mammalian cells. A. phagocytophilum infected RF/6A cells that had been screened with antibodies against calreticulin or PDI were visualized using LSCM. (B) The ER is recruited to the ApV early and the association is retained throughout the course of infection. RF/6A cells that had been synchronously infected with A. phagocytophilum organisms were screened with antibodies against calreticulin and the pathogen derived ApV membrane protein, APH0032, and examined at several post-infection time points using LSCM. (A,B) Host cell nuclei and bacterial DNA were stained with DAPI (blue). The regions that are demarcated by hatched lined boxes indicate the regions magnified in the insets that are demarcated by solid lined boxes. Scale bars, 5 μm. (C) Percentages of calreticulin-positive ApVs, as identified by DAPI stained intravacuolar A. phagocytophilum bacteria, over the course of a synchronous infection. Data are the means and standard deviations for triplicate samples. (D) Calreticulin cofractionates with A. phagocytophilum organisms. Uninfected (U) or A. phagocytophilum-infected HL-60 cells (I) were homogenized and the post-nuclear supernatants were separated by density gradient centrifugation. Successive one-ml fractions were analyzed by Western blot using antibodies against calreticulin and the A. phagocytophilum major surface protein, P44. Results shown are representative of two experiments with similar results.