05 are reported as statistically significant. Bactericidal assays The method used to examine the effect of bpaC mutations on the ability of Burkholderia

to resist the bactericidal activity of complement is outlined elsewhere [9, 77, 81]. We used final concentrations Ralimetinib supplier of 50% and 25% serum in assays with B. pseudomallei and B. mallei, respectively. Protein preparations, western blot, purification of recombinant BpaC protein, and antibody production Sarkosyl-insoluble OM protein preparations were obtained as described by Carlone et al. [82]. The methods used to prepare whole cell lysates and perform western blot H 89 supplier experiments are described elsewhere [8, 53, 54, 57, 83, 84]. His-tagged recombinant BpaC was obtained from cultures of E. coli TUNER carrying the plasmid pELHisBPSL1631-BMA1027, as previously outlined by our laboratory [67]. To obtain polyclonal Abs directed against BpaC, Selleck PLX3397 the purified His-tagged protein was emulsified in Freund’s adjuvants (SIGMA-ALDRICH®) and used to immunize female BALB/c mice as reported by Lafontaine and colleagues [85]. Immunofluorescence labeling of E. coli and microscopy Expression of BpaC on the surface of E. coli recombinant bacteria was visualized by immunofluorescence microscopy as outlined by Balder et al. [55]. Briefly, paraformaldehyde-fixed E. coli cells were spotted onto glass slides. These bacteria were probed with α-BpaC polyclonal

Abs, followed by incubation with a goat α-mouse antibody labeled with Alexa Fluor 546® (Life Technologies™) and the nucleic acid dye DAPI

(Life Technologies™). Slides were examined by microscopy using a Zeiss LSM 510 Meta confocal system. ELISA Duplicate wells of Immulon™ 2HB plates (Thermo Scientific Nunc) were coated overnight at 4C° with 1 μg of His-tagged BpaC. Excess unbound antigen was removed by washing the wells with PBS + 0.05% Tween 20 (PBST), and the wells were then blocked with PBS + 0.05% containing 3% dry milk (blocking buffer) for 1 hour at Oxymatrine room temperature. After washing with PBST, the wells were probed overnight at 4°C with sera from mice that survived acute aerosol infection with B. mallei ATCC 23344 and B. pseudomallei 1026b [67] diluted in blocking buffer. After this incubation, the wells were washed with PBST and incubated overnight with a goat α-mouse antibody conjugated to Horse Radish Peroxidase (SouthernBiotech) diluted in blocking buffer. After washing off the excess secondary antibody with PBST, 100 μL of the SureBlue™ TMB Microwell Peroxidase Substrate (KPL) was added to the wells. Color development, which is indicative of Abs binding to BpaC, was measured spectrophotometrically by determining the absorbance of well contents at a wavelength of 650 nm. Animal experiments Female BALB/c mice (6–8 weeks of age) were purchased from Frederick National Laboratory for Cancer Research.