Nitrosative stress in enterobacteria - the S-nitrosoproteome and an assessment of cellular protective functions in vitro and in vivo

Molecules that are important in biology include not only familiar organic macromolecules (nucleic acids, proteins, fats, carbohydrates) but also small inorganic molecules or ions. One of the most important of these, and the most topical, is nitrogen monoxide (nitric oxide, NO). This gas is produced by animals, plants and microbes and is also generated chemically in many environments. NO is potentially toxic to microbes, which must defend themselves from its chemical reactivity. The current interest in NO by research scientists arises from the fact that it is an important signaling molecule in animals and plants, involved in controlling blood vessel relaxation, nerve function and immunity. In addition, NO reacts with macromolecules, modifying their function with important biological implications. These processes (called nitrosation, nitrosylation, nitration), in which the NO group is covalently bound to the macromolecule, are highly selective and so we cannot predict which proteins will be modified. This research project sets out to measure the extent of these reactions inside intact bacteria. We wish also to test the idea that the defences used by bacteria against NO and related chemicals lead to a reduction in protein modification by NO. Finally, we wish to see whether bacteria that have been engulfed by white blood cells are prone to these modifications and whether the bacterial defence mechanisms limit these reactions. Overall, these experiments will help us to understand which molecules within bacteria are susceptible to NO attack and whether Man's antimicrobial strategies might benefit from this knowledge.

Nitric oxide (NO) is one of the most important small molecules in biology. NO is a key signalling molecule in plants and animals, a powerful weapon in the anti-microbial armoury of mammalian and plant cells, and is responsible in animals and plants for controlling numerous functions by targeting protein thiols, metal centres, and other biomolecules. In particular, the covalent attachment of NO groups to protein sulfhydryls and transition metals is a precisely regulated post-translational modification. Bacteria respond to NO by sensing NO and activating transcription of genes encoding protective enzymes, notably, in enterobacteria, Hmp (flavohaemoglobin) and NorVW (flavorubredoxin). However, the effectiveness of these defence systems in preventing or limiting cellular damage and the extent to which pathogens are damaged by NO in vivo are not understood. The purpose of this project is (a) identification of the protein and haem targets of the different nitrosative stress agents to which bacteria are exposed (including targets important in signalling, not only defence), and (b) an assessment of the effectiveness of the various NO detoxification and NO-responsive mechanisms in preventing this damage. The answers generated by this concerted transcriptomic, proteomic and biochemical study will provide new insights into a key bacterial process that is important in bacterial survival in natural environments as diverse as the phagolysosome of a macrophage, the gastrointestinal and urinary tracts, and soil and water where reactive nitrogen species exist. This proposal represents the first attempt to define the degree and specificity of protection afforded by Hmp, NorVW and other proteins that may be involved in the bacterial nitrosative stress response. Joint with BB/E015247/1