Determination of viable infectious yield.

The viable infectious yield of Chlamydia was evaluated by calculating the number of IFU per milliliter in HEp-2, BK, and McCoy cells. Confluent host cells were infected with harvested cell lysate in serial dilutions in 96-well cell culture plates (Sigma-Aldrich, Australia). At 30 hpi, the host cell monolayer was washed with phosphate-buffered saline (PBS) and fixed with 100% methanol for 10 min. The fixed cells were permeabilized with Triton X-100 for 15 min and then blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, Australia) in PBS. Primary antibody (anti-Chlamydia HtrA) (10, 58) was incubated for 1 h at 1:500 dilution in 1% BSA, followed by three washes in 0.2% Tween 20 in PBS. Secondary antibody [goat anti-rabbit IgG(H+L)-Alexa Fluor 488; Invitrogen, Australia] was added at 1:600 dilution in 1% BSA for 1 h, followed by 4 washes in 0.2% Tween 20 in PBS. Triplicate samples at appropriate serial dilutions were counted in nine fields of view per dilution using fluorescence microscopy (Nikon Eclipse TiS fluorescence microscope). The viable infectious yield was determined as previously described (22). Graphical images were made and statistical analysis was conducted using GraphPad Prism (version 7).