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Bacterial type III secretion system drives the translocation of virulence factors into the cystosol of host target cells. In phagocytes and in Epstein-Barr virus immortalized B lymphocytes, NADPH oxidase generates O(-2) through an electron transfer chain the activity of which depends on the assembly of three, p67(phox), p47(phox) and p40(phox) cytosolic activating factors with Rac 1/2 and a membrane redox component, cytochrome b(558). In p67(phox) deficient chronic granulomatous disease (CGD) patients, p67-phox is missing and NADPH oxidase activity is abolished. ExoS is a virulence factor of Pseudomonas aeruginosa which is secreted via the type III secretion system: it was fused with p67(phox). Pseudomonas aeruginosa synthesized and translocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymphocytes via the type III secretion system. Purified ExoS-p67(phox) hybrid protein was as efficient as normal recombinant p67(phox) in cell-free reconstitution of NADPH oxidase activity. Therefore, ExoS-p67(phox) was transferred via the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally reconstituted NADPH oxidase activity. In the complementation process, ExoS acted as a molecular courier for protein delivery: the reconstitution of an active NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy., (Copyright 2000 Academic Press.)

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.