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

Abstract

Pseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for P. aeruginosa exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found 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). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within the periplasm. The conformational rearrangement induced 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 with 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 this helix. Finally, we concluded that disruption of a structured domain II yields an extended form of the molecule and prevents formation of the conformational secretion motif.

FIG. 1

Schematic representation of the PE derivatives used in this study. The representation is not drawn to scale, and helices A to F of domain II are indicated. Numbers represent residues. The names of the PE derivatives are indicated on the left. The predicted molecular sizes are indicated on the right.

FIG. 2

(A) Secretion by P. aeruginosa PAK-NT of PE and PE derivatives deleted within domain II. PAK-NT cells containing the pMMB67HE vector (C⁻) or pMMB67HE derivatives carrying genes encoding wild-type PE and various deletions of domain II helices A to F (PE-delA to PE-delF) were separated into cellular (C) and extracellular (SN) fractions. Samples were loaded on SDS–11% PAGE gels followed by immunoblotting using a rabbit antiserum directed against PE. Only that part of the gel where whole-length PE and PE derivatives migrated is shown. (B) Quantitative analysis of PE secretion levels. The Western blots presented in panel A were scanned, and bands were quantified using Image Quant (Molecular Dynamics). The values presented are means of three independent experiments.

FIG. 3

Subcellular localization of PE derivatives in PAK-NT cells containing the pMMB67HE vector (C⁻) or pMMB67HE carrying genes encoding wild-type PE, PE-delC, or PE-delE. Samples were loaded on SDS–11% PAGE followed by immunoblotting using a goat antiserum directed against PE. C, cell fraction (cytoplasm plus membrane); P, periplasmic fraction; SN, extracellular fraction. Molecular size markers are indicated on the right (in kilodaltons). The position of PE and derivatives is indicated by an arrow.

FIG. 4

Secretion by P. aeruginosa PAK-NT of PE and the PE derivatives PE-2T and PE-46. PAK-NT cells containing the pMMB67HE vector (C⁻) or pMMB67HE carrying genes encoding wild-type PE or PE-2T and PE-46 derivatives were separated into cellular (C) and extracellular (SN) fractions. Samples were loaded on SDS–11% PAGE followed by immunoblotting using a rabbit antiserum directed against PE. Molecular size markers are indicated on the right (in kilodaltons).

FIG. 5

Molecular size determination of PE derivatives by gel filtration chromatography. Purified toxins were analyzed on Superdex 200. Elution profiles of PE, PE-delA, and PE-delB (A), PE, PE-delC, and PE-delD (B), PE and PE-delE (C), and PE and PE-delF (D). The positions of the different molecular size markers are noted at the top (in kilodaltons).

FIG. 6

(A) Ribbon representation of the three-dimensional structure of PE, indicating the organization into domains. Domains Ia and Ib are shown in green and light green, respectively. Domain II is in yellow with the exception of helices C, E, and F, which are red, and domain III is blue. Helix E both plays a central role in the structuration of domain II and ensures the interconnection of domain II with domains I and III. Helices C and F are rather short and highly surface exposed, with few contacts to other structural elements and no contacts to domain I or domain III. The figure was produced with the program Turbo-Frodo (38). (B) Close-up view of helix E, highlighting the zones of interactions with other domains. All residues are represented as backbone except those in domain II (residues in red) that are involved in hydrogen bonds formed with residues of domain III (residues in blue) and with residues from domain Ib (residues in light green).