Protein delivery by Pseudomonas type III secretion system: Ex vivo complementation of p67(phox)-deficient chronic granulomatous disease

Bacterial type III secretion system drives the translocation of virulence f actors into the cystosol of host target cells. In phagocytes and in Epstein -Barr virus immortalized B lymphocytes, NADPH oxidase generates O-2(-) thro ugh an electron transfer chain the activity of which depends on the assembl y 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(pho x) deficient chronic granulomatous disease (CGD) patients, p67-phox is miss ing 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 transl ocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymph ocytes via the type III secretion system. Purified ExoS-p67(phox) hybrid pr otein was as efficient as normal recombinant p67(phox) cell-free reconstitu tion of NADPH oxidase activity. Therefore, ExoS-p67(phox) was transferred v ia the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally r econstituted NADPH oxidase activity. In the complementation process,ExoS ac ted as a molecular courier for protein delivery: the reconstitution of an a ctive NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy. (C) 2000 Academic Press.