Abstract
When neutrophils phagocytose bacteria, myeloperoxidase generates the cytotoxic agent HOCl. However, debate exists as to whether HOCl is the major contributor to bacterial killing. Several studies have demonstrated that HOCl reacts with phagocytosed bacteria at concentrations in excess of those required for killing. In contrast, there is conflicting evidence that bacterial chlorination is lower than if sufficient HOCl had reacted to kill bacteria directly, and that the vast majority of HOCl reacts with neutrophil proteins rather than those of the ingested microbe. The aim of this thesis was to investigate whether HOCl may act together with neutrophil proteins to kill S. aureus indirectly.
Neutrophil proteins are extensively chlorinated during phagocytosis, so it was of interest to determine the cellular location of oxidised and chlorinated proteins. Following phagocytosis of magnetic beads, both the neutrophil phagosome and granule fractions had comparable amounts of chlorinated proteins, while the cytoplasm had relatively low levels of chlorinated proteins. Measurement of protein carbonyls demonstrated that all neutrophil fractions were exposed to high levels of oxidative stress.
As myeloperoxidase is a likely target of HOCl, the ability of oxidatively-modified myeloperoxidase to kill S. aureus was investigated. Oxidatively-modified myeloperoxidase was able to kill S. aureus as effectively as the myeloperoxidase/H2O2/chloride system, but did not chlorinate S. aureus as efficiently. Oxidatively-modified myeloperoxidase could still actively generate HOCl, indicating that myeloperoxidase could chlorinate itself in the phagosome to form an antibacterial protein. Little change to the enzyme structure was observed; however oxidatively-modified myeloperoxidase formed two chloramines per amine group, indicating dichloramines may be required for toxicity.
Investigation of model chloramines revealed that dichloramines were much more unstable than their analogous monochloramines. Stability was affected by substituents on the 4-carbon and a carboxyl group facilitated rapid decay. Unstable dichloramines containing a substituent on their 4-carbon were cytotoxic, although their cytotoxicity declined with time. The stable dichloramines of N-4-acetyl lysine and taurine were not bactericidal up to 10 nmoles per 10^5 S. aureus. None of the analogous monochloramines were cytotoxic at this dose. Dichloramines decomposed to yield chlorimines, aldehydes, and the inorganic gases NH2Cl and NHCl2. Stable products formed during the breakdown of dichloramines were not bactericidal.
To detect exposure of bacteria to reactive chlorine species in the phagosome, the carotenoid staphyloxanthin was isolated from S. aureus as a potential endogenous bacterial sensor and identified by mass spectrometry. Staphyloxanthin was found to react with HOCl, NH2Cl, NHCl2, but not H2O2. The rate constant for the reaction between staphyloxanthin and HOCl was ~3×103 M–1s–1. Clinical isolates of S. aureus were more resistant to HOCl if they contained more staphyloxanthin. S. aureus treated with HOCl, NH2Cl, NHCl2, as well as amino acid chloramines, lost viability in conjunction with loss of staphyloxanthin. Stable taurine dichloramine was able to kill bacteria under these conditions. Staphyloxanthin on phagocytosed S. aureus was not detectably oxidised, so it could not be concluded if sufficient HOCl was produced in the phagosome, although this result indicated that NHCl2 was not the sole bactericidal agent in the phagosome.
In summary, neutrophil proteins are extensively chlorinated during phagocytosis and oxidatively-modified myeloperoxidase has antibacterial properties independent of HOCl generation. Formation of unstable dichloramines should lead to the liberation of the cytotoxic species NH2Cl which may play a role in bacterial killing. Stable peptide dichloramines may also contribute to the bactericidal arsenal. Thus, HOCl may not kill microbes directly, but rather act through the generation of reactive chloramine species.