Cell culture and infection

HEK 293 (ATCC CRL-1573, human embryonic kidney epithelial) cells were cultured in Delbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS). THP-1 (ATCC TIB-202, human monocytic) and Vero (ATCC CCL-81, African green monkey epithelial) cells were cultured in RPMI medium 1640 (Life Technologies) supplemented with 10% FBS. WASH conditional knockout mouse embryonic fibroblasts (MEFs) (Daniel Billadeau, Mayo Clinic) were grown in DMEM containing 10% FBS. Generation and characterization of these cells are described in Gomez et al. [81]. MEFs were grown in DMEM containing 10% FBS, and knockout of WASH was obtained with two sequential 24 hr treatments of 3 μM 4-OHT (Sigma, H7904). After treatment, MEFs were washed and cultured for an additional 6–7 days before use in experiments [81]. All cell lines were incubated at 37°C with 5% CO₂. Infection of Vero cells with C. trachomatis was at a multiplicity of infection (MOI) of 50 based on inclusion forming units.

C. burnetii Nine Mile phase II, RSA439 (NMII) was propagated in Vero cells and purified as described [82]. The C. burnetii dotA mutant was propagated in the synthetic medium ACCM-2 as described [17]. C. trachomatis (LGV-434, serotype L2) was propagated in HeLa cells and purified as described [83]. For infection, HEK 293, Vero, or MEF cells were seeded on coverslips in 24-well plates at a density of 4 x 10⁴ cells per well. THP-1 cells were seeded on coverslips in 24-well plates at a density of 3 x 10⁵ cells per well and stimulated with 200 nM phorbol myristate acetate (Sigma) for 24 hr for differentiation into macrophage-like cells and attachment to coverslips. For immunofluorescence staining, cells were infected at an MOI of 100 based on genome equivalents (GE) quantified by TaqMan qPCR using a StepOnePlus Real-Time PCR system (Applied Biosystems) and primers specific to C. burnetii groEL [38]. For C. burnetii growth analysis, cells were infected at an MOI of 10.