Chlamydia samples were propagated in Vero cells and collected by washing samples once with ddH2O (Invitrogen), and lysing cells in ddH2O with periodic scraping and pipetting at room temperature for 20 minutes. Following hypotonic lysis, lysates were mixed with 5X SPG buffer (1X concentrations 0.5 g/L KH2PO4, 1.2 g/L Na2PO4, 0.72 g/L L-glutamic acid, 75 g/L sucrose, pH 7.5) and stored at −80°C. Seed preps of Chlamydia strains were used for all infections unless otherwise stated. Bacterial titers, or inclusion forming units (IFUs), were determined by serial dilutions of Chlamydia onto fresh Vero cells.
Infections were performed as described previously (Haldar et al., 2016 (link)). All infections were performed at a multiplicity of infection (MOI) of 2 unless otherwise stated. Diluted Chlamydia was added to fully confluent cell monolayers in cold DMEM supplemented with excess L-tryptophan (100 μg/mL; Sigma). Plates were centrifuged at 4°C, 3000 rpm for 30 minutes and returned to a 37°C incubator until the indicated timepoints for each assay. Time of infection (0 hours post-infection, hpi) was defined as the halfway point (15 minutes) of the centrifugation step, unless otherwise stated.
Co-infections (Figures 3 and 6 ) involving two strains were performed in the same manner, but with an MOI of 3 for garD::GII-GFP and an MOI of 5 for incA− and WT strains.
Infections were performed as described previously (Haldar et al., 2016 (link)). All infections were performed at a multiplicity of infection (MOI) of 2 unless otherwise stated. Diluted Chlamydia was added to fully confluent cell monolayers in cold DMEM supplemented with excess L-tryptophan (100 μg/mL; Sigma). Plates were centrifuged at 4°C, 3000 rpm for 30 minutes and returned to a 37°C incubator until the indicated timepoints for each assay. Time of infection (0 hours post-infection, hpi) was defined as the halfway point (15 minutes) of the centrifugation step, unless otherwise stated.
Co-infections (