Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates.

Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.

noxR3, a novel gene from Mycobacterium tuberculosis, protects Salmonella typhimurium from nitrosative and oxidative stress.

Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) produced by activated macrophages participate in host defense against the facultative intracellular pathogens Mycobacterium tuberculosis and Salmonella typhimurium. To survive within macrophages, such pathogens may have evolved ROI and RNI resistance mechanisms. ROI resistance pathways have been intensively studied. Much less is known about the mechanisms of resistance to RNI. To identify possible RNI resistance genes in M. tuberculosis, a mycobacterial library was expressed in S. typhimurium and subjected to selection by exposure to the NO donor S-nitrosoglutathione (GSNO) in concentrations sufficient to kill the vast majority of nontransformed salmonellae. Among the rare surviving recombinants was a clone expressing noxR3, a novel and previously anonymous M. tuberculosis gene predicted to encode a small, basic protein. Expression of noxR3 protected S. typhimurium not only from GSNO and acidified nitrite but also from H2O2. noxR3 is the third gene cloned from M. tuberculosis that has been shown to protect heterologous cells from both RNI and ROI. This suggests diversity in the repertoire of mechanisms that help pathogens resist the oxidative and nitrosative defenses of the host.

Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival.

Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems.

Safe administration of iron dextran to a patient who reacted to the test dose.

Parenteral iron therapy is infrequently required but generally well tolerated. We present a case in which intravenous iron dextran was successfully given to a patient who had an anaphylactoid reaction to the test dose. After pretreatment with methylprednisolone, diphenhydramine, ephedrine, and dextran 1, 2 g of iron dextran were safely given over several days; pretreatment was administered only on day 1. In the rare cases in which an anaphylactic agent must be given to a patient with a history of a life-threatening reaction to the agent, pretreatment along with slow escalation of dose may allow safe administration of the offending drug.

Identification of nitric oxide (NO) in human blood.

The amount of nitric oxide (NO) in the blood of residents of urban and suburban areas was measured under steady-state conditions by isotopic dilution with N15O, followed by field-ionization mass spectrometry. Approximately 20 nmoles of NO per ml of blood was characteristic of both smokers and nonsmokers, except for one of the eight subjects who had a significantly lower level. Monkeys (Macaca speciosa) had values comparable to those of seven human subjects, and rats had values like that of the unique human subject. Whether the origin of the NO was endogenous or exogenous was not determined.

Determination of the concentration of explosives in air by isotope dilution analysis.

The concentrations in air of dinitrotoluene (DNT), trinitrotoluene (TNT), nitroglycerin (NG), ethylene glycol dinitrate (EGDN), and pentaerythritol tetranitrate (PETN) were measured at 25 degrees C under equilibrium conditions, and that of cyclomethylene trinitramine (RDX) was measured at elevated temperature by means of an isotope dilution technique. Isotopically multi-labeled compounds were synthesized and used as diluents. Field ionization mass spectrometry was used to measure the abundance ratios of the unlabeled materials. The concentrations in air at 25 degrees C of DNT, TNT, NG, EDGN, PETN, and RDX are 184, 4, 31, 37,000, 7, and 0.8 ppb v/v, respectively. The data obtained may be used for the assessment of the required sensitivity of air-monitoring detection systems.