Bacteria and cell culture. C. trachomatis
lymphogranuloma venereum (LGV) serovar L2/434/Bu elementary bodies (EBs) were purified from infected L929 cells using a 30% Renografin density gradient (21
) and stored in sucrose phosphate glutamate (SPG) at −80°C until use. C. trachomatis
serovar D/UW-3/Cx was isolated from infected HeLa 229 cells and C. muridarum
strain Nigg was purified from infected L929 cells as described previously (21
). L929 cells and HeLa 229 cells were routinely cultured in RPMI 1640 tissue culture medium (Mediatech, Inc., Manassas, VA) supplemented with 5% fetal bovine serum (FBS) (Thermo Fisher Scientific, Liverpool, NY) and 10 μg/ml gentamicin (MP Biomedicals, Santa Ana, CA) at 37°C in a humidified atmosphere of 5% CO2
. Cells were seeded in 96-well plates (Bioexpress, Kaysville, UT) at a density of 7 × 104
cells/ml, 200 μl/well, and incubated overnight prior to infection. In order to minimize the edge effect (well-to-well variations in the number of cells), plated cells were incubated for 1 h at room temperature prior to incubation at 37°C in an atmosphere of 5% CO2
, as previously described (14
Microbicide and antibiotic preparation.
Polymyxin B sulfate (Enzo Life Science, New York, NY) and tetracycline hydrochloride (USB Corporation, Cleveland, OH) were obtained in powder form. Stock solution of polymyxin B sulfate was prepared in Hanks buffered salt solution (HBSS) (Mediatech, Inc., Holly Hill, FL) to a concentration of 10 mM. Tetracycline was dissolved in sterile water to a stock concentration of 1 mg/ml. Hydroxyethyl cellulose (HEC) (90 kDa; Sigma-Aldrich, St. Louis, MO) was prepared as a stock solution at a concentration of 12% (wt/vol) in water. The HEC gel (polymeric liquid) was adjusted to pH 4.5 using 10 M NaOH and stored at 4°C. At the time of the assay, a 12% HEC gel was diluted to a final concentration of 2.8% (wt/vol) in phosphate-buffered saline (PBS). The mixture was shaken for 1 h at 37°C to achieve a uniform solution, centrifuged for 1 min at 300 × g to remove any bubbles, adjusted to pH 7.0, and stored at 4°C until use.
EB dilution assay.
Purified C. trachomatis L2 EBs were serially diluted 2-fold in HBSS and transferred to ~70% confluent L929 cell monolayers (90 μl/well) in a 96-well plate. Ninety microliters of HBSS alone was added to mock-infected control cells. Cells were incubated for 2 h at room temperature. Following the incubation period, chlamydial inocula were removed and cells were washed once with 200 μl of HBSS. Two hundred microliters of fresh medium (RPMI–5% fetal bovine serum–10 μg per ml gentamicin) was added to each well, and cells were incubated at 37°C in an atmosphere of 5% CO2 for 24 h.
Antichlamydial compound inhibitory assay.
C. trachomatis L2 EBs were diluted in HBSS. Polymyxin B and hydroxyethyl cellulose were serially diluted 2-fold in HBSS containing EBs to the concentration range of 1 mM to 15.625 μM and 2.8% to 0.175%, respectively. Compound-EB mixtures were incubated for 60 min at room temperature. Compound-treated EB inocula were mixed using a multichannel pipette (LabNet Excel, Edison, NJ) at the start and end of the incubation period to ensure uniformity of the inocula. The mixtures containing the compound and EBs were then transferred to ~70% confluent L929 cell monolayers in a 96-well plate and incubated at room temperature for 2 h. Following the incubation period, the inocula were removed and cells were washed once with 200 μl of HBSS. Two hundred microliters of fresh medium (RPMI–5% fetal bovine serum–10 μg per ml gentamicin) was added to each well. For tetracycline treatment, fresh medium containing an appropriate concentration (0.1 to 0.7 μg/ml) of tetracycline was added following 2-h inoculation period. Cells were incubated at 37°C in an atmosphere of 5% CO2 for 24 h.
C. trachomatis serovar D and C. muridarum infection.
Purified C. trachomatis
serovar D or C. muridarum
was serially diluted 4-fold in HBSS and transferred to ~70% confluent HeLa 229 or L929 cell monolayers (90 μl/well) in a 96-well plate, respectively. Ninety μl of HBSS alone was added to mock-infected control cells. The plate was centrifuged at 900 × g
for 1 h at room temperature (21
). Following the incubation period, chlamydial inocula were removed and cells were washed once with 200 μl of HBSS. Two hundred microliters of fresh medium (RPMI–5% fetal bovine serum–10 μg per ml gentamicin) was added to each well, and cells were incubated at 37°C in an atmosphere of 5% CO2
for 48 h for serovar D and for 24 h for C. muridarum
before they were analyzed.
Guinea pig polyclonal serum against Chlamydia
L2 EBs was purchased from Abcam (Cambridge, MA). Rabbit polyclonal anti-RpoA antibody was affinity purified using the AminoLink Plus immobilization kit (Thermo Scientific, Rockford, IL). The protein concentration of purified antibody was determined to be 1.01 mg/ml by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Sera and purified antibody were serially diluted 5-fold in HBSS. Prior to infection, purified C. trachomatis
L2 EBs were added to each serum or purified antibody sample and incubated for 30 min at 37°C to allow interaction between EBs and the antibody to take place (24
). HBSS alone was used as a negative control. Following the incubation period, 90 μl of each sample containing antibody-EB mixture was added to a monolayer of L929 cells in a 96-well plate and incubated at room temperature for 2 h. After the incubation period, inocula were removed and cells were washed once with HBSS. Two hundred microliters of fresh medium (RPMI–5% fetal bovine serum–10 μg per ml gentamicin) was added to each well, and cells were incubated at 37°C for 24 h.
Immunofluorescence assay (IFA).
At 24 or 48 h postinfection (hpi) for serovar L2 or D, respectively, infected cells were fixed with 100% methanol (200 μl/well) for 10 min at room temperature and washed once with PBS. Cells were stained using the MicroTrack C. trachomatis culture confirmation test (Syva Co., Palo Alto, CA) with dilution to 1:40 in PBS (50 μl/well) for 60 min in the dark, followed by a 5-min staining with 1 μM 4′,6-diamidino-2-phenylindole (DAPI) in PBS (50 μl/well). DAPI was removed, and 1 ml of PBS was added. Plates were stored in the dark at 4°C until imaging.
Images were automatically captured with a BD Pathway BioImager 855 microscope (Becton, Dickinson and Company, Franklin Lakes, NJ) with a 20×, 0.4-numerical-aperture objective, fully equipped for multicolor capture, and an Orca ER camera (Hamamatsu Photonics, Bridgewater, NJ). The system was controlled by Attovision collection software (Becton, Dickinson and Company, Franklin Lakes, NJ), with automated infrared and image autofocus capture for 15 fields per well for a 96-well plate. Multiple plate capture was enabled by a Twister II robotic arm (Caliper Lifesciences Inc., Hopkinton, MA), which was integrated with the Attovision software.
Image analysis using CellProfiler and CellProfiler Analyst.
Images acquired by automated microscopy were loaded into the open-source software program CellProfiler (Broad Institute, Cambridge, MA), followed by segmentation of images and identification of objects. The process had four main steps: segmentation of nuclei, identification of whole cells, segmentation of chlamydial inclusions, and tabulation of measurements (see Fig. S1 in the supplemental material). The first task, performed by CellProfiler, was identification of the objects. Using CellProfiler modules, the nuclei, referred to as primary objects within CellProfiler, were identified first from the DNA-stained images. Once the nuclei had been identified, the subsequent module used the whole-cell-stained images and previously identified nuclei to identify the cell boundaries (whole cell), which were referred to as secondary objects. The area of the identified nuclei was subtracted from the whole cells to identify the cytoplasm, referred to as tertiary objects. For identification of chlamydial inclusions, CellProfiler expanded each area of nuclei by 15 pixels and used this expanded region to search for inclusions. Inclusions were then identified and segmented. The second task performed by CellProfiler was generating a cytological profile of each cell. CellProfiler measured a large number of cellular and subcellular features once all objects (nuclei, cells, cytoplasm, and identified chlamydial inclusions) had been identified. These quantitative measurements consist of apparent cellular and subcellular features, as well as an extensive amount of less-evident details, such as intensity of the stain, size, shape, and pixel correlation between objects. Once these measurements were taken, the cytological profile was created for each cell to be utilized by CellProfiler Analyst (Broad Institute, Cambridge, MA) and exported to a spreadsheet. Both nuclei and chlamydial inclusions were related to the identified cells so that each inclusion was assigned to only one nucleus. For enumeration of inclusion-forming units (IFU), the last module within the pipeline (ExportToDatabase) was modified to allow for classification of individual inclusion rather than cells by CellProfiler. To enumerate infected cells, the cytological profiles were loaded into CellProfiler Analyst. Random images, each containing a single cell, were presented to initiate the training process. Approximately 20 images from the positive (infected) and negative (uninfected) controls were manually classified by dragging and dropping the images into the appropriate bins within the CellProfiler Analyst interface (see Fig. S2a in the supplemental material). Based on the cytological profiles of these classified cells, CellProfiler Analyst identified the parameters necessary to accurately distinguish between uninfected and infected cells by generating a set of 50 rules (i.e., parameters). The software then presented a new set of cells that had been classified as positive or negative based on the rules generated. Any cells classified inaccurately were manually sorted into the appropriate bin to continue the training process. To assess the accuracy of the training, images from selected wells were opened and individual cells were automatically marked as uninfected or infected by CellProfiler Analyst based on the established rules (see Fig. S2b in the supplemental material). Any cell that had been classified incorrectly was manually reassigned to the appropriate bin. After the correction, a set of refined rules was generated and used to score every cell in every image. These steps were repeated until the system was able to accurately classify greater than 95% of cells into the correct category. Upon completion of scoring, a spreadsheet containing the numbers of positive and negative cells for each well was generated and exported.