Long-lived oxidants in innate immunity

Phagocytosis and intracellular killing of invading pathogens by human granulocytes is a major feature of the innate immune system to overcome infection. When bacteria are engulfed into phagocytic vacuoles, they are attacked by a number of oxidative molecules formed by an enzymatic cascade. While highly reactive oxidants are consumed within seconds in vivo, stable N-chloro compounds (chloramines, R-NHCl) are in contact with the target pathogens for extended time periods. Recently, low molecular weight representatives (i.e. N-chloro amino acids) of these long-lived oxidants (LLO) have been shown to possess microbicidal activity. They are considered to contribute to destruction of bacteria during infection and, moreover, they seem to be of advantage as new antimicrobial agents in topical therapy of infections. The first aim of this project is to clarify the interaction of selected LLO with bacteria and fungi. Surface attack on cell walls and membranes and penetration of LLO into the microbial cytosol will be investigated using spectrophotometric and functional tests, 2D electrophoresis and mass spectrometry of possible target proteins as well as radiolabelling of targets and LLO. Pathogens exposed to LLO for a sublethal period of time are known to lose virulence in an in-vivo model as a first step of inactivation. Phagocytosis and intracellular killing of these attenuated micro-organisms will be addressed. The second aim is to investigate the combined effect of LLO and non-oxidative antimicrobial agents of granulocytes against bacteria and fungi. These agents are cationic proteins (defensins, bactericidal-permeability increasing protein, cathepsin G, lysozyme ...) deposited within the leukocyte granules which attack mainly the surface of pathogens and will act at least in part simultaneously with LLO in vivo. Experiments on the simultaneous impact of LLO and the most important non-oxidants on bacterial and fungal viability will be performed. As a third aim, the effect of LLO on the biology and function of cationic proteins will be monitored by protein electrophoresis, mass spectrometry and functional tests. The fourth aim is to investigate the interaction of LLO with the key components of the complement system, an important plasma-protein based defence system linked with cellular effectors like granulocytes. The results of this project will contribute essentially to the understanding of the action and co-operation of granulocyte antimicrobial agents in eradication of pathogens. In addition, valuable knowledge for application of synthetically generated LLO in human medicine will be gained. The project matches the recently established program of central interest ("Schwerpunktprogramm der Medizinischen Fakultät") on "Infection - immunity and pathogens" of the Medical Faculty at the University of Innsbruck.

N-chlorotaurine (NCT), one of the most important long-lived oxidants produced by the human body to overcome infections, has been synthetized and made available as pure substance in our laboratory. Since it exerts broad- spectrum killing activity against pathogens, the results of our science are important for both its natural role in the human body to inactivate bacteria, fungi, and viruses and for its topical application as aqueous solution to treat infections in human medicine. In this project a number of results have been obtained regarding the chemistry and the mechanism of action of NCT. Its chemical properties (such as determination of purity, stability, storage conditions, chemical reactions) have been thoroughly investigated, which is an obligatory basis for clinical application. NCT can be prepared at 99% purity and stored at 2-4C for one year without the occurrence of noxious decay products. For a therapy cycle, storage at room temperature is sufficient. The chemical reactions of NCT in a therapeutically applied concentration are harmful only to the pathogens invading the body but not to human tissue, as proven repeatedly in clinical studies. The first interaction with bacteria and fungi is oxidation of their surface leading to inhibition of growth and loss of virulence which means that they cannot cause severe infections any more. A very short incubation time of 1 min is sufficient for these effects, which may explain the efficacy of NCT applied for instance as eye drops, where relatively short contact times are warranted that do not necessarily lead to complete killing of the pathogens. Clinical studies within the project proved the tolerability and efficacy of NCT in infected leg ulcers, in the infected outer ear canal. There are also strong hints for efficacy in chronic rhinosinusitis and severe viral eye infections, both of which are in part lacking causative therapy up to date.