The importance of neutrophils in defending Pseudomonas infection find more is reflected by significant
increase in infection rate in neutropenic patients . Winterbourn and colleagues modeled the reactions of oxidant production in neutrophil phagosomes. They calculated that superoxide is produced at a rate of ~312 mM/min and HOCl 134 mM per minute . In this current study, the maximal concentration of H2O2 used was 5 mM and HOCl 0.07 mM. A recent report documented that bleaching of GFP expressed in SA is seen at concentrations of 0.05-0.1 mM HOCl which correlated well with killing of SA by this oxidant , suggesting that similar concentrations of HOCl were likely achieved in vivo. The mathematical model proposed by Winterbourn and colleagues predicts that such levels can be reached within seconds after activation of the NADPH oxidase . Thus, we believe that the selected concentrations of H2O2 and HOCl in our studies are well within the scope of the achievable oxidant levels in neutrophils. Precise
mechanisms of oxidant-mediated bacterial killing are not fully defined. Early studies using EC as a model organism indicated a correlation between EC Quisinostat mw envelope permeabilization and bacterial inactivation by HOCl; however, only low-molecular weight compounds became freely permeable while the cell maintained its selleck chemicals llc barrier function to proteins . Albrich et al. (1986) tested the small-molecule permeability theory in EC by measuring the transport of H+ ion and glycerol and reported that the intercellular movements of these molecules were only marginally affected . Their conclusion was that HOCI inactivation of bacteria does not occur by loss of membrane structural
integrity, which contradicts the previous report. In the current study, we demonstrated that membrane integrity is affected by H2O2 and HOCl, but the effect is organism-specific (Figures 2 and 3). Statistically, permeability of BC and EC caused by H2O2 and HOCl did correlate with loss of viability while permeability of KP with only H2O2 exposure correlated with loss of viability. It is notable that permeability IKBKE and CFU viability were statistically independent of each other for PsA and SA, the two most prevalent CF pathogens, in both H2O2 and HOCl exposures. EC and PsA have been shown to recover from reduced adenylate energy charge, when subsequently supplied with nutrients which facilitate ATP hydrolase activity of the F1F0 complex of the bacterial ATP synthase . After treatment with bactericidal doses of HOCl, however, adenylate energy charge is unrecoverable and further ATP production is abolished . These findings suggest that a potent oxidant-induced killing mechanism may cause destruction of ATP production by specific oxidation of the F1F0 ATP synthase .