Showing papers by "Myron S. Cohen published in 1992"

Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase.

Abstract: Pseudomonas aeruginosa produces a blue pigment, pyocyanin. Pyocyanin is a redox-active phenazine compound that kills mammalian and bacterial cells through the generation of reactive oxygen intermediates. We examined the mechanisms by which P. aeruginosa resists pyocyanin. [14C]pyocyanin was taken up by both Escherichia coli and P. aeruginosa, though more slowly by the latter. Cyanide-insensitive respiration, used as an indicator of intracellular superoxide and/or hydrogen peroxide production, was 50-fold less in pyocyanin-treated P. aeruginosa than in E. coli. P. aeruginosa showed less cyanide-insensitive respiration than E. coli upon exposure to other redox-active compounds (paraquat, streptonigrin, and plumbagin). Electron paramagnetic resonance spectrometry and spin trapping showed that P. aeruginosa generated less pyocyanin radical and superoxide than E. coli. Cell extracts from E. coli contained an NADPH:pyocyanin oxidoreductase which increased the rate of reduction of pyocyanin by NADPH. Conversely, cell extracts from P. aeruginosa contained no NADPH:pyocyanin oxidoreductase activity and actually decreased the rate of pyocyanin-mediated NADPH oxidation. Antioxidant defenses could also reduce the sensitivity of P. aeruginosa to pyocyanin. Under culture conditions of limited phosphate, both pyocyanin production and catalase activity were enhanced. Superoxide dismutase activity was also increased under low-phosphate conditions. When cells were grown in a high-phosphate succinate medium, P. aeruginosa formed a previously described iron-superoxide dismutase as well as a manganese-cofactored superoxide dismutase. These results demonstrate that P. aeruginosa resists pyocyanin because of limited redox cycling of this compound and that under conditions favoring pyocyanin production, catalase and superoxide dismutase activities increase.

Interaction of lactoferrin and lipopolysaccharide (LPS): effects on the antioxidant property of lactoferrin and the ability of LPS to prime human neutrophils for enhanced superoxide formation.

Abstract: Lactoferrin is a 77-kDa iron-binding protein to which a wide variety of divergent biologic functions have been ascribed. It has recently been reported that lactoferrin interacts with bacterial lipopolysaccharide (LPS) in such a fashion as to affect the binding of lactoferrin to myeloid cells. Two other potential interactions of LPS and lactoferrin were explored. Lactoferrin prevents hydroxyl radical formation by binding iron, even at low pH. Lactoferrin inhibited iron-catalyzed formation of hydroxyl radical in the presence of LPS at pH 7.4 and 4.5. Low concentrations of LPS can be used to "prime" neutrophils toward enhanced function, such as formation of stimulated superoxide anion. Lactoferrin inhibited LPS priming of neutrophils if LPS contamination of the protein (provided by commercial suppliers) was first reduced. Inhibition of LPS priming was observed whether apolactoferrin or iron-saturated lactoferrin was used. Similar inhibition of LPS priming was observed when neutrophils were incubated with other serum proteins (e.g., albumin, apotransferrin, or iron-saturated transferrin). These results show that LPS should not be expected to affect the free radical biology of lactoferrin, which is a crucial physiologic function of this protein. However, lactoferrin inhibits LPS priming, and this effect requires consideration in experimental models of inflammation.

Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils.

Abstract: We studied the effects of oxidant stress on the catalase activity and hydrogen peroxide sensitivity of Neisseria gonorrhoeae. N. gonorrhoeae is an obligate pathogen of man that evokes a remarkable but ineffective neutrophil response. Gonococci make no superoxide dismutase but express high catalase activity. Gonococcal catalase activity increased threefold when organisms were subjected to 1.0 mM hydrogen peroxide. This increase in catalase activity was marked by a parallel increase in protein concentration recognized by a rabbit polyclonal antibody raised against the purified gonococcal enzyme. Catalase was primarily localized to the gonococcal cytoplasm in the presence or absence of stress; only a single isoenzyme of catalase could be identified. Exposure of gonococci to neutrophil-derived oxidants was accomplished by stimulating neutrophils with phorbol myristate acetate or by using gonococcal Opa variants that interacted with neutrophils with different degrees of efficiency. Gonococci exposed to neutrophils demonstrated a twofold increase in catalase activity in spite of some reduction in viability. Exposure of gonococci to 1.0 mM hydrogen peroxide made the organisms significantly more resistant to higher concentrations of hydrogen peroxide and to neutrophils than control organisms. These results suggest that catalase is an important defense for N. gonorrhoeae during attack by human neutrophils. The rapid response of this enzyme to hydrogen peroxide should be taken into consideration in studies designed to evaluate the interaction between neutrophils and gonococci.