NO killing of B. mallei Table 1. Bacterial strains and Plasmids. Strains/Plasmids Relevant properties B. mallei ATCC 23344 Wild type B. mallei Mo126.2 iscR mutant S. Typhimurium BC696 fliC2 fljB2 DH5a JM109 pRK2013 pMO79 pMO90 pMO126 DH5a harboring pRK2013 Cloning strain harboring pMO79 & pMO126 Helper plasmid Mobilizable replicative plasmid Mobilizable siucide plasmid Mobilizable disruption plasmid Description bma1709::pMO126 SL1344 DfliC DfljB Isolated in 1944 from a human case of glanders: Gms, Pbr F2 ø80lacZM15 endA1 recA1 hsdR17(rK2 mK+) supE44 thi-1 DgyrA96 relA1D (lacZYA-argF)U169] e142(McrA2) recA1 endA1 gyrA96 thi-1 hsdR17 (rK2 mK+) supE44 relA1 D(lac-proAB) [F9 traD36 proAB lacIqZDM15] RK2 derivative, Kanr mob+ tra+ ColE1 oripBBR1 Kanr mob+ gfp rfp ColE1 Kanr mob+ gfp xyle Source [50] [51] This study Laboratory stock Stratagene [16] Hamad & Voskuil Hamad & Voskuil pMO90 carrying an internal fragment of the B. mallei iscR homologue This study Gms = gentamicin sensitive. Pbr = polymixin B resistant. Kmr = kanamycin resistant. doi:10.1371/journal.pone.0001976.t001 membrane-bound or cytosolic components of the NADPH oxidase [13]. The clinical importance of NADPH oxidase in resistance to B. cepacia has been recreated in p47phox-deficient mice [14]. The NADPH oxidase has also been linked to the anti-B. pseudomallei arsenal of macrophages [12]. In contrast to ROS, current investigations on NO-related anti-Burkholderia activity have been rather controversial. Experimental animal models and macro- phage cell cultures have shown that iNOS is dispensable for innate host defense against B. cepacia and B. pseudomallei [12,14]. Moreover, others have shown that the greatest Burkholderia growth occurs during maximal NO synthesis [8]. Inhibition of iNOS mRNA transcription appears to underlie the lack of a role of RNS against B. pseudomallei [9,10]. On the other hand, IFNc can enhance NO-mediated intracellular killing of B. pseudomallei [11]. Recently, it has been reported that B. mallei is susceptible to NO generated by macrophages [15], although the mechanisms of antimicrobial activity remain unclear. Similarly, it remains unclear whether the NADPH oxidase plays a role in resistance to the intracellular pathogen B. mallei. The major goal of this study was to characterize the contribution of NADPH oxidase and iNOS hemoproteins to the macrophage antimicrobial arsenal against B. mallei. Methods Bacterial Strains and genetic manipulations B. mallei strain ATCC 23344 was used in these studies (table 1). Bacteria grown overnight in 10 ml of Luria Bertani broth supplemented with 4% glycerol (LBG) at 37uC in a 315 RPM shaking incubator were sub-cultured into pre-warmed LBG and grown to an OD600 of 0.6. The B. mallie iscR homologue, locus BMA1709, was disrupted by inserting a 400 bp internal region of the BMA 1709 using homologous recombination. The 400 bp internal region was amplified by PCR using an iscR forward primer containing a BamHI restriction site (59-tgactacg- gatccgcccggtgacgcttgcaggcatcag-39) and the iscR reverse primer containing an SphI restriction site (59-aagcccgtcgcatgcgc- gacgggctccggcttgcgcttg-39). After double digestion with BamHI and SphI, the PCR product was directionally cloned into the suicide vector pMO90, creating the disruption plasmid pMO126. pMO126 was introduced into B. mallei using triparental mating [16] in the presence of the helper E. coli DH5a pRK2013 strain [17]. Mating reactions were plated on LB agar plates supple- mented with 30 mg/ml kanamycin to select for transconjugants, and 20 mg/ml polymyxin B to select against the helper and donor E. coli strains. The resulting colonies were re-streaked for isolation on LB plates supplemented with 30 mg/ml kanamycin and 20 mg/ ml polymyxin B. Insertion of pMO126 into the B. mallei genome and the disruption of BMA 1709 was confirmed by PCR. The replicative plasmid pMO79 encoding a green fluorescence protein was conjugated into B. mallei strain ATCC 23344 by triparental mating as described above. The presence of pMO79 in B. mallei was confirmed by fluorescence microscopy. Macrophages C57BL/6 and congenic iNOS2/2 [18] or gp91phox2/2 [19] mice were bred in our animal facility according to Institutional Animal Care and Use Committee guidelines. Peritoneal macro- phages were harvested from mice 4 days after intraperitoneal inoculation of 1 mg/ml sodium periodate as described [20]. The peritoneal exudate cells were resuspended in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% heat- inactivated fetal bovine serum (BioWhittaker, Walkersville, MD), 15 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma-Aldrich), and 100 U ml21/100 mg ml21 of penicillin/ streptomycin (Cellgro) (RPMI+ medium). The RPMI+ medium used to culture the THP-1 cell line was supplemented with 50 mM 2-mercaptethanol. The peritoneal exudate cells were seeded in flat-bottom 96-well plates for macrophage killing assays at a density of 26105 cells per well. The macrophages were selected by adherence after 24 h of culture at 37uC in a 5% CO2 incubator. Murine J774 (clone ATCC TIB-67) and AMJ2-C11 (clone ATCC CRL-2456) and human U937 (clone ATCC CRL-1593.2) and THP1 (clone ATCC TIB-202) macrophage-like cells grown in RPMI+ medium supplemented as described above were used as additional sources of mononuclear phagocytes. Selected groups of macrophages were treated with 200 U/ml of IFNc (Life Technologies, St. Paul, MN) overnight prior to Burkholderia infection. Just prior to infection the macrophages were washed with pre-warmed RPMI without antibiotics. Macrophage killing assays The macrophage killing capacity was quantified by a gentami- cin protection assay following a modified protocol described for PLoS ONE | www.plosone.org 2 April 2008 | Volume 3 | Issue 4 | e1976