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Peroxidase-induced degradation of single-walled carbon nanotubes: hypochlorite is a major oxidant capable of in vivo degradation of carbon nanotubes -

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Bacteria colonizing human intestine adhere to the gut mucosa and avoid the innate immune system. We previously demonstrated that Escherichia coli isolates can adsorb mucin from a diluted solution in vitro. Here, we evaluated the effect of mucin adsorption by E. coli cells on neutrophil activation in vitro. Activation was evaluated based on the detection of reactive oxygen species production by a chemiluminescent reaction (ChL), observation of morphological alterations in neutrophils and detection of exocytosis of myeloperoxidase and lactoferrin. We report that mucin adsorbed by cells of SharL1 isolate from Crohn's disease patient's inflamed ileum suppressed the potential for the activation of neutrophils in whole blood. Also, the binding of plasma complement proteins and immunoglobulins to the bacteria was reduced. Desialylated mucin, despite having the same adsorption efficiency to bacteria, had no effect on the blood ChL response. The effect of mucin suggests that it shields epitopes that interact with neutrophils and plasma proteins on the bacterial outer membrane. Potential candidates for these epitopes were identified among the proteins within the bacterial outer membrane fraction by 2D‐PAGE, fluorescent mucin binding on a blot and HPLC‐MS/MS. In vitro, the following proteins demonstrated mucin adsorption: outer membrane porins (OmpA, OmpC, OmpD and OmpF), adhesin OmpX, the membrane assembly factor OmpW, cobalamine transporter, ferrum uptake protein and the elongation factor Ef Tu‐1. In addition to their other functions, these proteins are known to be bacterial surface antigens. Therefore, the shielding of epitopes by mucin may affect the dynamics and intensity of an immune response.

Myeloperoxidase (MPO), found mainly in neutrophils, is released in inflammation. MPO produces reactive halogen species (RHS), which are bactericidal agents. However, RHS overproduction, i.e., halogenative stress, can also damage host biomolecules, and MPO itself may be targeted by RHS. Therefore, we examined the susceptibility of MPO to inactivation by its primary products (HOCl, HOBr, HOSCN) and secondary products such as taurine monochloramine (TauCl) and taurine monobromamine (TauBr). MPO was dose-dependently inhibited up to complete inactivity by treatment with HOCl or HOBr. TauBr diminished the activity but did not eliminate it. TauCl had no effect. MPO became inactivated when producing HOCl or HOBr but not HOSCN. Taurine protected MPO against inactivation when MPO was catalyzing oxidation of Cl to HOCl, whereas taurine failed to prevent inactivation when MPO was working with Br, either alone or in combination with Cl. SCN interfered with HOCl-mediated MPO inhibition. UV-vis spectra showed that heme degradation is involved in HOCl- and HOBr-mediated MPO inactivation. A negative linear correlation between the remaining chlorinating activity of HOCl- or HOBr-modified MPO and Escherichia coli survival upon incubation with MPO/HO/Cl was found. This study elucidated the possibility of MPO downregulation by MPO-derived RHS, which could counteract halogenative stress.

Cells of E. coli isolates from the gut of healthy volunteers (N=5) and patients with Crohn's disease (N=5) and laboratory E. coli strain DH5α bound mucin in vitro in similar amounts ranging from 0.02 to 0.12 mg/mg of bacterial dry weight. Binding was evaluated by the decrease in optical absorption of mucin solution at 214 nm after incubation with bacteria. Detailed analysis of mucin binding by one of isolates showed that during incubation of 0.09 mg/ml bacteria in 0.15 M NaCl containing 0.1 mg/ml mucin at 25C, maximum binding was reached in 30 min, while in the presence of 14 mM α-methyl mannoside, mucin binding decreased by 46% (p<0.05). Confocal microscopy revealed intensive binding of FITC-labeled mucin to the surface of a small number of bacterial cells. Mucin binding did not significantly affect zeta potential of bacteria and their energetic status assessed by ATP content; at the same time, ATP content in the extracellular environment slightly increased.

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