Influence of deletions within domain II of exotoxin A on its extracellularsecretion from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium that secretes many prot eins into the extracellular medium via the Xcp machinery. This pathway, con served in gram-negative bacteria, is called the type II pathway. The exopro teins contain information in their amino acid sequence to allow targeting t o their secretion machinery. This information may be present within a confo rmational motif. The nature of this signal has been examined for P. aerugin osa exotoxin A (PE). Previous studies failed to identify a common minimal m otif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one fo und additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six alpha-helices (A to F). Th e secretion behavior of PE derivatives, individually deleted for each helix , was analyzed. Helix E deletion has a drastic effect on secretion of PE, w hich accumulates within the periplasm. The conformational rearrangement ind uced in this variant is predicted from the three-dimensional PE structure, and the molecular modification is confirmed by gel filtration experiments. Helix E is in the core of the molecule and creates close contact,vith other domains (I and III). Deletion of the surface-exposed helix F has no effect on secretion, indicating that no secretion information is contained in thi s helix. Finally, we concluded that disruption of a structured domain II yi elds an extended form of the molecule and prevents formation of the conform ational secretion motif.