Figure 1. Time course of Dot/Icm dependent translocation.

C. burnetii NM and the icmL::Tn derivative expressing BlaM-77 (A), BlaM-1823 (B) or BlaM-1524 (C) from a plasmid were grown to stationary phase in ACCM-2, enumerated by qPCR, and used to infect HeLa cells at an MOI of 100 (grey bars) and 500 (black bars). At defined times post-infection the BlaM substrate CCF4-AM was added. Low magnification images for each sample were collected and used to calculate the percentage of cells that were translocation positive. This was determined by visual observation of a blue fluorescent emission at 460 nm when the cells were excited at 415 nm. At least 300 cells were quantified per well and each infection was performed in triplicate wells. These experiments were performed at least three independent times. Representative fluorescent micrographs of MOI 100 infections at 24 hours post-infection demonstrate the robust translocation of BlaM-77, BlaM-1823 and BlaM-1524, but not BlaM alone, when expressed by C. burnetii NM (D). Green HeLa cells (emission at 535 nm) represent cells loaded with uncleaved CCF4-AM and blue cells (emission at 460 nm) are indicative of CCF4-AM cleaved by translocated BlaM. The mean ± standard deviation percentage of cells that were translocation positive (blue) is displayed in the bottom right corner of each micrograph. No translocation was detected for C. burnetii expressing BlaM alone or for C. burnetii NM icmL::Tn expressing any of the reporter constructs.