Salmonella [21]. B. mallei were grown overnight in LBG broth and sub-cultured to OD600 of 0.6 as described above. The bacteria were spun down for 5 min at 1500 RPM onto the macrophages at an MOI of 200 after opsonization in 10% normal mouse serum in RPMI+ medium. Unless specified, extracellular bacteria were removed from the monolayers after a 2 h incubation by washing with pre-warmed RPMI+ medium containing 6 mg/ml of gentamicin (Sigma-Aldrich). After washing the noninternalized bacteria, the MOI was determined to be 10. Burkholderia-infected macrophages were lysed with 1% Triton X-100 in phosphate buffered saline (PBS) at the indicated times after challenge. The intracellular bacteria recovered at various points after infection were enumerated on LB agar plates. The % survival was calculated as (cfu tn/cfu t0)6100. Superoxide anion determination O2N2 was quantified by the superoxide dismutase–inhibitable reduction of ferricytochrome c [22]. The macrophages were infected at an MOI of 40 with Burkholderia resuspended in phenol red–free Earle’s balanced salt solution containing 60 mM ferricy- tochrome c. Because B. mallei is aflagellated, a strain of Salmonella enterica serovar Typhimurium deficient in fliC fljB was used as a positive control. After 1 h incubation in 5% CO2 at 37uC, the OD of the supernatants was determined spectrophotometrically at 550 nm. The concentration of O2N2 was calculated by using an e550 of 2.16103 M21 cm21. All reagents were purchased from Sigma-Aldrich. Nitrite measurement The concentration of nitrite (NO22) produced by Burkholderia- infected macrophages 6 h post-challenge was estimated spectro- photometrically at 550 nm in a Biotek Synergy HT-2 reader after mixing culture supernatants with an equal volume of Griess reagent (0.5% sulfanilamide and 0.05% N-1-naphthylethylenedia- mide hydrochloride in 2.5% phosphoric acid). The NO22 concentration was calculated by regression analysis using a NaNO2 standard curve. Susceptibility to RNS and ROS in vitro Overnight cultures of B. mallei diluted 1:100 in LBG broth were grown at 37uC with shaking to OD600 of 0.6. Bacteria were concentrated by centrifugation and the pellets resuspended in PBS. The bacteria were diluted 1:100 in PBS and incubated with spermine NONOate (Cayman Chemical, Ann Arbor, MI), H2O2 (ThermoFisher Scientific, Waltham, MI), or spermine (Sigma- Aldrich, St. Louis, MO) at 37uC. At the end of the incubation, 200 units of catalase (Sigma-Aldrich, St. Louis, MO) were added to the H2O2-treated samples to consume any remaining H2O2. Serial dilutions were spotted on LBG agar plates and the number of colony forming units counted after 48 h of culture. Microarrays RNA from B. mallei was isolated in Triazol reagent from cultures grown to an OD600 of 0.1 in LB medium. Selected cultures were treated with 1 mM DEANO for 30 min before the isolation of the RNA. Synthesized cDNA was labeled as previously described for Mycobacterium tuberculosis [23] with the following changes. An additional phenol/chloroform extraction was added to clean the RNA after isolation. The resulting RNA was further purified using a Qiagen RNeasy kit. 5 mg RNA were used for cDNA synthesis. After cDNA synthesis, the samples were further purified by phenol/chloroform extraction followed by concentration using a microcon YM-30 column (Millipore, Bedford, MA). Hybridization NO killing of B. mallei reactions contained 5 ml cDNA, 1 ml yeast tRNA (Sigma), and 15 ml hybridization solution (400 ml formamide, 239 ml H2O, 250 ml 206 SSC, 10 ml 10% SDS 10 ml). Samples were heated at 98uC for 2 min, cooled 5 min at 25uC and placed on pre- hybridized Burkholderia array slides (Colorado State University RM-RCE Genomics Core). Hybridization was conducted in standard hybridization chambers overnight in a 42uC water bath and washed with 16 SSC and 0.05% SDS, followed by two washes in 0.066 SSC. The slides were scanned using a Genepix Axon 4000B scanner. Initial data analysis was conducted using Genepix Pro software, followed by analysis using Microsoft Excel as described [23]. Aconitase enzymatic assay 3 mL of stationary phase B. mallei grown in LBG medium were pelleted by centrifugation and resuspended in an equal volume of LB medium. Selected cultures containing 36 OD were treated with either 500 mM or 1 mM spermine NONOate for 30 min. The cultures were pelleted by centrifugation at 4uC, washed with 50 mM Tris-HCl buffer, pH 7.6 and the cytoplasmic proteins extracted in 400 mL B-PER (Pierce, Rockford IL) after vortexing for 30 sec. The samples were diluted 1:2 in 50 mM Tris-HCL buffer, pH 7.6. After removing the insoluble fraction by centrifugation, the supernatants were filter-sterilized. Aconitase activity in the soluble fractions was estimated spectrophotometri- cally at 240 nm by following the formation of cis-aconitate in 50 mM Tris-HCl buffer pH 7.4 containing 30 mM isocitrate. Aconitase activity is expressed as DOD240/min/mg protein. Electron microscopy The phagocytes were plated as described above for killing assays at a density of 46105 per chamber of a 8-well Permanox Labtek chamber slide system (Nalgene Nunc International, Rochester, NY). The macrophages were challenged with B. mallei at an MOI of 10 as described above. Between 2 and 3.5 h after infection, the cells were fixed in 2.5% glutaraldehyde in phosphate buffer, pH 7.4. The specimens were postfixed in 1% osmium tetraoxide, treated with uranyl acetate, dehydrated in ascending ethanol series, and infiltrated with Embed 812. Ultrathin sections were examined in a FEI Technai 62 electron microscope operated at 80 kV. Fluorescence microscopy Macrophages plated on 13 mm glass coverslips in a 24-well plate at a density of 2.56105 cells/well were infected as described above with GFP-expressing B. mallei. The specimens were washed in PBS 3 h after infection and fixed overnight at 4uC in 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) diluted in PBS. After fixation, the coverslips were washed in PBS and the cells were permeablized for 5 min with 0.1% Triton X-100 in PBS. After washing, F-actin was visualized by labeling the infected macrophages with Alexa Fluor 568 phalloidin (Invitrogen). The coverslips were washed with PBS and mounted onto glass slides prepped with Vectashield mounting medium containing DAPI nucleic acid stain (Vectashield Laboratory, Burlingame, CA). The specimens were examined with a Zeiss Axiophot fluorescence microscope equipped with a charge-coupled-device camera controlled by the SlideBook deconvolution image-processing software (Intelligent Imaging Inno- vations, Denver, CO). Statistical analysis Data are presented as mean 6 SEM or in box-and-whiskers plots as median, intraquartile and total ranges. A two-tailed, PLoS ONE | www.plosone.org 3 April 2008 | Volume 3 | Issue 4 | e1976