Fig 2.

Differential transcription profiling of OMP candidate genes throughout the A. phagocytophilum infection cycle. DC organisms were used to synchronously infect HL-60 cells, and the infection proceeded for 36 h, a period that allowed the bacteria to complete their biphasic developmental cycle and to reinitiate infection. Total RNAs were isolated from the DC inoculum and from infected host cells at several postinfection time points. RT-qPCR was performed using gene-specific primers. Relative transcript levels for each target were normalized to A. phagocytophilum 16S rRNA gene transcript levels by using the 2^−ΔΔCT method. To determine the relative transcription of OMP candidate genes between RC and DC organisms, normalized transcript levels of each gene at each time point were calculated as fold changes in expression relative to expression at 16 h (encircled in the experimental timeline in panel A), a time point at which the A. phagocytophilum population consists exclusively of RC organisms. (A) Diagram of the experimental design, highlighting the time points at which RNA was isolated, the A. phagocytophilum biphasic developmental and infection stages, and the expression categories into which the genes of interest were classified based on their expression profiles. (B to D) RT-qPCR results for OMP candidate-encoding genes of interest, grouped into early-stage (B), midstage (C), and late-stage (D) genes according to when during the course of infection they are most highly expressed. (E) RT-qPCR results for control genes. The data in panels B to E are the means and standard deviations (SD) of results for triplicate samples and are representative of two independent experiments that yielded similar results.