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 ␣-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.

HER2-targeting recombinant protein with truncated Pseudomonas Exotoxin: A translocation domain efficiently kills breast cancer cells

“…The E helix was found to be moderately important for PE cytotoxicity, and it has been shown that deletion of this helix results in decreased PEA toxicity. 23 Helix F apparently inhibits translocation, because its deletion results in higher translocation activity than full-length PEA domain II. 13,23 The PEA translocation domain has been successfully engineered as a vehicle to deliver therapeutic molecules into cells.…”

“…23 Helix F apparently inhibits translocation, because its deletion results in higher translocation activity than full-length PEA domain II. 13,23 The PEA translocation domain has been successfully engineered as a vehicle to deliver therapeutic molecules into cells. [15][16][17][18][19] We have previously constructed a series of genes containing different truncated PEA translocation domains (PEA 253-412aa, PEA 253-364aa and PEA253-358aa) fused with the anti-HER2 single-chain antibody (e23sFv) at the 5'-end, and genes encoding diverse apoptosis-inducing molecules such as caspase-3, 20 caspase-6, 21 truncated AIF 22 and granzyme B 13 at the 3'-end.…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…Among the important questions that remain to be answered is how the substrates of T2S (and T4P, where they are involved in secretion) are recognized against the background of myriad periplasmic proteins. No linear secretion signal has been identified, and protein folding is required for secretion competency, suggesting that the signal may be a three-dimensional one (145,407). However, comparison of substrates for which structures are available does not suggest an obvious candidate(s), and thus studies aimed at identifying the elusive secretion signal continue.…”

Exchange of Xcp (Gsp) Secretion Machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes : Species Specificity Unrelated to Substrate Recognition

“…A putative secretion motif involved in this process might be present within the threedimensional structure of the exoproteins (42,53,57). Moreover, recognition of the exoproteins by the Gsp secreton appears to be species specific.…”