Figure 3. Contribution of reactive oxygen and nitrogen species to the anti-Burkholderia activity of macrophages. The contribu- tion of ROS and RNS to host defense against B. mallei was assessed by comparing the antimicrobial activity of primary macrophages isolated from immunocompentent C57BL/6 mice (control) or congenic phox2/2, iNOS2/2 or phox2/2iNOS2/2-immunodeficient mice. Selected groups of macrophages were treated with 200 U/ml IFNc 16 h before infection. The number of B. mallei recovered from the macrophages was estimated 5 h after the infected macrophages were treated with gentamicin. Data from 16 independent observations gathered on 3 separate experiments are represented in box-and-whiskers plots as median, intraquartile and total ranges. *P,0.05 compared to unstimu- lated control macrophages. **P,0.05 compared to IFNc-primed macrophages from C57BL/6 mice. doi:10.1371/journal.pone.0001976.g003 macrophages, while neither RNS nor ROS appear to add to the early innate host defenses of macrophages against B. mallei. B. mallei is susceptible to RNS The poor anti-Burkholderia activity of IFNc-primed macrophages lacking iNOS suggest that RNS generated by activated phagocytes exert early antimicrobial activity against B. mallei. Consequently, the ability of macrophages to produce NO in response to B. mallei NO killing of B. mallei infection was tested by measuring the accumulation of NO22. Supernatants of B. mallei-infected J774 cells contained about 200 mM NO22 after 5 h of infection (fig. 4A). The concentration of NO22 seen in B. mallei-infected cells closely paralleled the NO32 levels contained in RPMI medium, raising the possibility that a bacterial nitrate reductase activity is responsible for the observed NO22 burst. This idea was substantiated by the fact that the putative B. mallei nitrate reductase activity could be inhibited by the flavoprotein inhibitor diphenyleneiodonium (fig. 4A). To determine whether host cell iNOS activity can be stimulated upon B. mallei challenge, the capacity of macrophages to generate NO22 was therefore estimated in NO32-free medium. As shown in figure 4B, IFNc-primed macrophages, but not unstimulated controls, generated copious amounts of NO congeners in response to B. mallei. Accumulation of NO22 in the supernatants reflects a functional iNOS enzymatic complex, since the amount of NO22 generated by iNOS-deficient macrophages was negligible. Figure 4C shows that B. mallei are extraordinarily susceptible to RNS. Spermine NONOate reduced the viability of B. mallei in a dose and time dependent manner (fig. 4C & D). The viability of B. mallei was significantly reduced after a 4 h exposure to as low as 10 mM of the NO donor spermine NONOate (fig. 4C). A time course indicated that the killing of B. mallei by 100 mM spermine NONOate was already evident after 30 min and continued unabated for at least 2 h (fig. 4D). The anti-B. mallei effects associated with spermine NONOate are specific to RNS generated by this NO donor, since the viability of B. mallei was similarly unaffected upon culture in PBS or exposure to the spermine base control. NO enhances transcription of genes associated with [Fe-S] cluster assembly To gain insight into the targets of RNS antimicrobial activity, global transcriptional profiles were compared between B. mallei cultures untreated or treated with sublethal NO concentrations. Microarray expression analyses showed the transcriptional up- regulation of gene products involved in iron acquisition and [Fe-S] cluster assembly and repair (table 2). This pattern of transcription suggests that [Fe-S] clusters of dehydratases are critical B. mallei targets of RNS-mediated cytotoxicity. Accordingly, the enzymatic Figure 4. Antimicrobial activity of reactive nitrogen species generated by Burkholderia-infected macrophages. The ability of B. mallei to generate NO22 in RPMI+ medium is shown in A. Selected samples were treated with 10 mM of the flavin inhibitor diphenyleneiodonium (DPI). The NO22-producing capacity of macrophages responding to B. mallei was assessed in low NO32 DMEM medium (B). The survival of B. mallei to increasing concentrations of the NO donor spermine NONOate is shown in C. The effects of 100 mM spermine NONOate on the viability of B. mallei was studied overtime (D). Untreated or spermine-treated B. mallei were used as controls. Data in panel A are represented in box-and-whiskers plots as median, intraquartile and total ranges. The rest of the data represent the mean 6 sem. doi:10.1371/journal.pone.0001976.g004 PLoS ONE | www.plosone.org 5 April 2008 | Volume 3 | Issue 4 | e1976