Spore preparations and fluorescent labeling
Spores were prepared from B. anthracis Sterne 7702 and enumerated using a hemacytometer (Thermo Fisher Scientific, Waltham, MA), as described previously [46]. As quality control, spore preparations were tested for both heat resistance and the capacity to germinate, as described [46].
Mammalian cell culture
Abelson murine leukemia virus-transformed murine macrophages derived from ascites of BALB/c mice (RAW 264.7 macrophage-like cells; CRL-2278; ATCC, Manassas, VA) were maintained within a humidified environment at 37°C and under 5% CO2 in complete DMEM, (Thermo Scientific, Waltham, MA) containing penicillin (100 U; Gibco BRL, Grand Island, NY), streptomycin (0.1 mg/ml; Gibco BRL), L-glutamine (2 mM; Sigma, St. Louis, MO), and FBS (10%; JRH Biosciences, Lenexa, KS). MH-S cells (CRL-2019; ATCC) were maintained within a humidified environment at 37°C and under 5% CO2 in complete RPMI medium (Thermo Scientific) containing penicillin-streptomycin (100 U, Gibco BRL), L-glutamine (4 mM), and FBS (10%). JAWSII (CRL-11904; ATCC) were maintained within a humidified environment at 37°C and under 5% CO2 in complete MEMα (Thermo Scientific) containing penicillin-streptomycin (100 U), L-glutamine (4 mM), and FBS (20%). All tissue culture plasticware was purchased from Corning Incorporated (Corning, NY).
Evaluation of B. anthracisspore germination in cell culture media
Using 96 well plates, spores prepared from B. anthracis 7702 (1.0 × 108 spores/mL) were incubated at 37°C and under 5% CO2 in BHI (BD Biosciences, San Jose, CA), LB (0.1% tryptone, BD Biosciences; 0.05% yeast extract, BD Biosciences; 0.05% NaCl, Fisher Chemical, Fairlawn, NJ), PBS pH 7.2 (Mediatech, Manassas, VA), or germinating amino acids (10 mM L-alanine, 10 mM L-inosine, both from Sigma) in PBS pH 7.2. In other studies, spores were incubated in 96 well plates (108 spores/mL) and at 37°C and under 5% CO2 in the following cell culture media without or with FBS (10%, unless otherwise indicated; Mediatech): DMEM (0.1, 0.5, 1, 5 or 10% FBS), RPMI-1640, MEMα modification (10 or 20% FBS), MEM (Mediatech), AMEM (Gibco), EMEM (Mediatech), BME (Sigma), CIM (Gibco), Ham's F-12 (Mediatech), McCoy's 5A (M5A, ATCC), or DMEM with 10% FBS and 10 mM D-alanine (Sigma) and D-histidine (Sigma). In some assays, FBS obtained from Mediatech was substituted with FBS purchased from Invitrogen or Sigma. As described previously [39], spore germination was evaluated by measuring loss in spore refractility or loss of heat resistance, while outgrowth was monitored by monitoring the elongation of bacilli using a Delta Vision RT microscope (Applied Precision; Issaquah, WA), outfitted with an Olympus Plan Apo 100 × oil objective. DIC images were collected using a Photometrics CoolSnap HQ camera; (Photometrics, Tucson; AZ), and processed using SoftWoRX Explorer Suite (version 3.5.1, Applied Precision Inc).
Pre-conditioning of cell culture media
To pre-condition cell culture medium, monolayers of RAW264.7 or MH-S cells in 24-well plates (80 to 95% confluency) were washed three times with Hanks' balanced salt solution (HBSS) and then incubated in DMEM (for RAW264.7 cells) or RPMI-1640 (for MH-S cells) without FBS and penicillin-streptomycin in a humidified environment at 37°C and under 5% CO2. After 4 or 24 h, the medium was withdrawn, centrifuged (600 × g for 5 min), and the supernatant was filter sterilized using a 0.22 μm filter (Corning). To evaluate heat sensitivity, some of the filter-sterilized pre-conditioned medium was incubated at 95°C for 10 min or, alternatively, 65°C for 30 min Alternatively, some of the filter-sterilized pre-conditioned medium (3 mL) was dialyzed four times against PBS pH 7.2 (500 mL), using dialysis tubing with 12,000-14,000 molecular mass cutoff (Spectrum Laboratories, Inc., Rancho Dominguez, CA), each time for 6 h.
Mammalian cell viability
To evaluate the viability of RAW264.7, MH-S, or JAWSII cells, alterations in membrane permeability, as indicated by relative PI (1 μg/mL; Invitrogen Molecular Probes, Eugene, OR) uptake, were measured using flow cytometry, as previously described [46].
Flow cytometry
Analytical flow cytometry was carried out using a Beckman Coulter EPICS XL-MCL™ flow cytometer equipped with a 70-μm nozzle, 488 nm line of an air-cooled argon-ion laser, and 400 mV output. The band pass filter used for detection of Alexa Fluor 488 spores was 525/10 nm. The long pass filter used for cell cycle phase determination assays and mammalian cell viability assays was 655 nm/LP. Cell analysis was standardized for side/forward scatter and fluorescence by using a suspension of fluorescent beads (Beckman Coulter Inc., Fullerton, CA). At least 10,000 events were detected for each experiment (>2000 events per min). Events were recorded on a log fluorescence scale and evaluated using FCS Express 3.00.0311 V Lite Standalone. Sample debris (as indicated by lower forward and side scatter and a lack of PI staining) represented a small fraction (1 to 2%) of the detected events and was excluded from analysis.
Cell cycle assay
To compare the cell-cycle profiles of RAW264.7 cells cultured in FBS-containing medium or FBS-free medium, relative PI uptake was measured using flow cytometry. At 4 or 24 h, as indicated, cells were incubated at room temperature with Cellstripper™ (Mediatech). After 15 min, the cells were further diluted with PBS pH 7.2 containing 10% FBS (800 mL). The cell suspensions were centrifuged for 5 min at 500 × g at room temperature. The pellets were resuspended in 300 μL of PBS pH 7.2 at room temperature, fixed by adding anhydrous ethanol (100%, 700 μL prechilled to -20°C, Fisher Scientific) with continuous vortexing, and then further incubated for at least 2 h at -20°C. The cells were centrifuged for 5 min at 500 × g at room temperature, and the pellets were resuspended in 1 mL of PBS pH 7.2, and then incubated at room temperature for 30 min. The cells were centrifuged 5 min at 500 × g at room temperature. The cell pellets were resuspended in 300 μL PBS pH 7.2, 0.1% Triton X-100 (MP Biomedicals, Solon, OH), DNase-free RNase A (100 mg/mL; Sigma), and PI (10 μg/mL), and further incubated at room temperature for 60 min. The stained cells were analyzed by flow cytometry.
Mammalian cell metabolism assay
To compare the metabolic activities of RAW264.7 cells cultured in FBS-containing medium or FBS-free medium, the relative conversion of tetrazolium 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (tetrazolium; 5 mg/mL, Sigma) to formazan over 30 min and at 37°C was measured at 570 nm with a Synergy 2 plate reader (BioTek Instruments, Inc., Winooski, VT), as described [39, 52].
In vitro infection of mammalian cells with B. anthracis
Mammalian cells (5.0 × 105 total cells/well) were incubated in the appropriate complete medium, as indicated above under "Mammalian cell culture," for two days in a humidified environment at 37°C and under 5% CO2, resulting in 80-95% confluency. To calculate the number of spores needed to achieve MOI 10, cells from several wells were detached using Cellstripper™ and enumerated using a hemacytometer. The cells were used only if greater than 90% of the cells excluded trypan blue; generally, greater than 95% of the cells within the monolayer excluded trypan blue. Prior to the addition of labeled spores, cells were washed three times with HBSS and then incubated in DMEM (RAW264.7 and JAWSII) or RPMI-1640 (MH-S), without or with FBS, as indicated. To synchronize the exposure of cells to spores, spores were immediately and gently centrifuged (600 × g for 5 min) onto the surfaces of cells. The plates were incubated within a humidified environment at 37°C and under 5% CO2 for the indicated times prior to analysis.
Quantification of B. anthracisuptake by mammalian cells
Internalization of B. anthracis spores by mammalian cells was quantified using a previously described flow cytometry based assay [46]. Briefly, the indicated mammalian cell lines were seeded into 48-well plates (Corning) in order to achieve 80-95% confluency after two days of incubation. As previously described [46], B. anthracis spores were labeled using an amine reactive Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Molecular Probes-Invitrogen). Alexa Fluor 488-labeled B. anthracis spores were quantified using a hemacytometer, added to cells at the desired MOI, and immediately but gently centrifuged (300 xg for 5 min) onto the surface of cells. The plates were incubated within a humidified environment at 37°C and under 5% CO2 for the indicated times prior to analysis using flow cytometry, as previously described [46]
To discriminate intracellular spores from those which remain surface-associated during infection, cells were analyzed in the presence of trypan blue, a membrane-impermeable, Alexa Fluor 488® fluorescence quenching agent [53]. Previously, 0.5% trypan blue was demonstrated to completely quench the fluorescence emission of Alexa Fluor 488-labeled spores bound to the surface of mammalian cells, while having no affect the fluorescence emission of internalized spores [46]. From these data, the percentage of cells with internalized B. anthracis was calculated by dividing the number of viable cells with greater than background auto-fluorescence by the total number of viable cells. For spore internalization experiments, viable mammalian cells (typically 90-98% of the total events) were readily identified by their high forward scatter and lack of propidium iodide (PI) staining. A second distinct population, (2-10%) of dead cells was routinely detected with relatively lower forward scatter (which indicates a smaller size) and positive PI staining (indicating non-viable cells; data not shown). Over the course of 60 min, we observed no detectable increase in cell death in the presence of labeled spores, as indicated by PI uptake (data not shown). Finally, sample debris (as indicated by relatively lower forward and side scatter and a lack of PI staining) represented a small fraction (1-2%) of the detected events. Based on these data, the data from subsequent experiments were gated to include only viable cells, while excluding non-viable cells, cellular debris, and spores not associated with cells. Alternatively, the time dependent total uptake of spores was determined by plotting the geometric mean of the fluorescence intensity (MFI).
Quantification of viable, intracellular B. anthracis
Cells were incubated with dormant B. anthracis spores, as indicated above. For germinated B. anthracis spore infections, B. anthracis spore were germinated with 10 mM L-alanine and L-inosine in 1 × PBS pH 7.2 for 30 min and washed twice with 1 × PBS pH 7.2 to remove germinants and enumerated as described above. After 30 min, cells were washed three times with HBSS, and further incubated in the indicated medium with FBS (10%) and gentamicin (100 μg/ml) to kill all external germinated spores. After 15 min, the cells were washed three times with HBSS, and further incubated in the indicated appropriate medium supplemented with FBS (10%). At the indicated times, the cells were lysed by incubating with sterile tissue culture grade water (Mediatech) for 5 min at 25°C. Serial dilutions of the lysates were plated on LB agar plates and incubated overnight at 37°C. CFU were enumerated by direct counting of visible colonies and correcting for the appropriate dilution.
Statistics
All data are representative of those from three or more independent experiments. The Q-test was performed to eliminate data that were statistical outliers [54]. Error bars represent standard deviations. P values were calculated with Student's t test using paired, one-tailed distribution. P < 0.05 indicates statistical significance. Statistical analyses to calculate means, standard deviations, and Student's t tests, were calculated using Microsoft Excel (version 11.0).