The role of PtdIns3P in the killing of bacteria and fungi by the neutrophil NADPH oxidase

Neutrophils are white blood cells in our body which are responsible for finding, eating and killing nasty microbes. An important part of the killing process is the delivery of large quantities of toxic oxygen molecules into a specialist chamber inside the neutrophil called a phagosome, which contains the microbe. These toxic chemicals are made by an enzyme called the NADPH oxidase and we know it must be important because of the discovery of a rare disease called CGD (chronic granulomatous disease), where patients carrying mutations in one of the six proteins which make up this enzyme suffer life-threatening bacterial and fungal infections. The way in which the NADPH oxidase is turned on is complicated, because it has to switch on in response to eating a wide variety of different microbes but at the same time it mustn?t cause too much damage to surrounding body tissues. An understanding of how the NADPH oxidase works is thus likely to improve our understanding of what goes wrong when some infections and inflammations are not dealt with properly. The applicants have recently discovered that enzymes called PI3Ks may play a key role in regulating the NADPH oxidase by synthesising a messenger molecule around the phagosome called PtdIns3P, which they have shown binds tightly to one of the six NADPH oxidase components. They propose to use chemicals recently developed by the pharmaceutical industry to specifically block PI3Ks to assess their role in the synthesis of PtdIns3P and NADPH oxidase function. Most of these experiments will be done on neutrophils purified from human blood taken from local volunteers. The applicants have also created mutant mice in which the gene encoding the expression of the oxidase component which binds PtdIns3P has been altered to prevent this interaction. These mice are healthy and fertile when housed under pathogen-free conditions and they now propose to evaluate the effect of this mutation on the ability of these mice to combat bacterial and fungal infections; most of the experiments will be done on neutrophils purified from the bone marrow of these animals. This group has a strong track record in publishing their work in the public domain and are regularly asked to present their work at international conferences. They have also developed good links with the pharmaceutical industry and hence useful results from the work should be quickly disseminated.

The NADPH oxidase of neutrophils plays a key role in our defence against microbial pathogens. This is clearly evident from the life-threatening bacterial and fungal infections which present in cases of CGD, a disease caused by mutations in several of the components of this enzyme complex. The NADPH oxidase is responsible for directing the delivery of toxic reactive oxygen species into phagosomes where they play an important role in the killing of engulfed pathogens. The signalling pathways which lead to the appropriate assembly and activation of the oxidase are necessarily complex, both because of the wide variety of receptors which are required to trigger this event and the need for tight control to limit damage to host tissues. This proposal seeks to understand the role of phosphoinositide 3-kinase (PI3K) signalling pathways in NADPH oxidase activation and killing of specific bacteria and fungi.

There are seven different catalytic subunits of PI3Ks which fall into three functional classes; of relevance here are the class I and class III enzymes which synthesise the phospholipid messengers PtdIns(3,4,5)P3 and PtdIns3P, respectively. A major objective of this proposal is to use newly developed PI3K isoform-selective inhibitors and PI3K ?knock-out? mice to define the involvement of specific PI3Ks in human and mouse neutrophil phagocytosis, NADPH oxidase activation and killing of specific bacteria and fungi. The majority of these analyses will be carried out in vitro on neutrophils purified from human blood and mouse bone marrow. A second objective will be to measure PtdIns3P synthesis and subcellular localisation during selected examples of phagocytosis by confocal fluorescence imaging of neutrophils expressing GFP-tagged, PtdIns3P-binding domains. A third objective will be to measure the effects of a mutation in one of the components of the oxidase, p40phox, which prevents binding to PtdIns3P; this will be done by utilising p40phox R58A ?knock-in? mice recently created in the applicants? laboratory and will involve characterising NADPH oxidase responses in vitro and killing of bacteria and fungi both in vitro and in vivo.

It is anticipated that the proposed work will define some important paradigms for how PI3Ks are used to regulate NADPH oxidase activity during infection and inflammation and the applicants are well placed to disseminate this information through publication in peer reviewed journals, presentations at scientific conferences and through good collaborations with the pharmaceutical industry.