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S-nitrosothiols was suggested by the demonstration of an ˙NO dioxygenase activity of the E. coli flavohemoglobin that required NADPH, FAD, and O2 (66, 79). Expression of hmp also protected E. coli from nitrosative stress under anaerobic conditions. In this case, the mechanism appears to involve formation of a nitrosyl-heme intermediate that oxidizes NADH, converting ˙NO to N2O (82). Special interest attaches to the flavohemoglobin: like AhpC, it is widely expressed and enzymatically catabolizes preformed RNI; and like SOD, it may help prevent formation of peroxynitrite by drawing off a precursor.

Glucose 6-phosphate dehydrogenase, encoded by zwf, initiates the hexose monophosphate shunt to generate NADPH. S. typhimurium disrupted in zwf were hypersusceptible to S-nitrosoglutathione. The mutant bacteria regained partial virulence in mice whose NOS2 was inhibited (83). Dependence of salmonella on zwf for resistance to S-nitrosoglutathione may reflect the role of NADPH in the redox cycling of glutathione, thioredoxin, AhpC, and/or flavohemoglobin.

Low molecular weight thiols, such as glutathione and homocysteine, react with ˙NO. Mutation of gshB (26), which controls glutathione synthesis, and of metL (58), which controls the biosynthesis of homocysteine, each rendered S. typhimurium hypersusceptible to RNI. Virulence of the metL-disrupted organisms was reduced in mice and restored by inhibition of NOS (58). It is usually assumed that low molecular weight thiols protect cells from RNI by scavenging them. It is possible that low molecular weight thiols function additionally or alternatively to help reverse the oxidation or S-nitrosylation of other targets by RNI. Both mechanisms (scavenging and repairing) must be reconciled with the fact that the resulting S-nitrosothiols, such as S-nitrosoglutathione, can themselves be bacteriostatic (72) or bactericidal (57, 76). Presumably, formation of S-nitrosothiols in the course of scavenging RNI or repairing their lesions is only protective if the rate of formation of S-nitrosothiols does not exceed the pathogens' capacity to catabolize S-nitrosothiols. The rate-limiting step in the latter process remains to be identified (84). Thioredoxin can accelerate the decomposition of S-nitrosothiols (85), but whether this reaction protects bacteria from RNI is untested. Glutathione peroxidase can catabolize OONOin vitro (86), and many small biological molecules can react with OONO or its toxic products, including glutathione, cysteine, methionine, and tyrosine, but their contributions to microbial defense against OONO are undefined.

(iii) Mechanisms likely to involve repair of RNI-dependent lesions. RNI acting in the service of host defense have DNA as their ultimate target. OONO, ˙NO2, and higher oxides of nitrogen are mutagenic (87, 88 and 89). The DNA repair proteins RecB and RecC are important for survival of S. typhimurium in mice and macrophages (90). A recBC mutant of S. typhimurium was attenuated in both wild-type and NOS2-deficient mice, but regained partial virulence in phox-deficient mice and complete virulence in mice deficient in both NOS2 and phox (33). Thus, it appears that RNI help control S. typhimurium in part by promoting DNA damage, although to a lesser extent than ROI. Repair of oxidized proteins in RNI-stressed bacteria also has an enzymatic basis (G. St. John, J. Ruan, N. Brot, H. Weissbach, and C.N., unpublished work).

(iv) Mechanisms unknown. NOXR1 and NOXR3, each cloned from M. tuberculosis, confer enhanced resistance to RNI upon S. typhimurium, E. coli, and Mycobacterium smegmatis, but their mechanism of action is unknown (62, 63). Transformed M. smegmatis expressing NOXR1 were markedly more resistant than vector-transformed controls to being killed by macrophages, whether the macrophages were wild-type, NOS2-deficient, or phox-deficient. This was consistent with the evidence that NOXR1 afforded resistance to both RNI and ROI (62).

Perspective

Thirteen years ago conventional wisdom held that mammals could not produce RNI because RNI would be toxic. Now it is accepted that RNI production is widespread among eukaryotes in both nontoxic (signaling) and toxic formats. The toxicity is harnessed for host defense at a cost to the host. This state of affairs parallels the past history and current view of mammalian production of ROI. As for ROI, there are enzymes with the capacity to protect both pathogens and host cells from RNI. The genes encoding some RNI resistance enzymes are widely distributed, and a given cell may express several. The distribution and diversity of RNI resistance genes suggest that RNI exert evolutionary pressure and that different RNI pose distinct biochemical challenges. Agents that inhibit pathogens' RNI resistance mechanisms may improve immunity in those diseases for which RNI represent an important but imperfect element of host control.

Critical comments by R. Bryk, A. Ding, S. Ehrt, M. Fuortes, K. Hisert, D. Marciano, J. McKinney, and G. St. John are much appreciated. We thank C. Bogdan for sharing preprints. The National Heart, Lung, and Blood Institute supported this work.

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