Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells

Abstract

SummaryExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1±234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232±453) encodes an ADP-ribosyltransferase. Utilizing a series of Nterminal deletion peptides of ExoS and an epitopetagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90±234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.

Intracellular Membrane Localization of Pseudomonas ExoS and Yersinia YopE in Mammalian Cells

“…YopE was expressed as a doublet with an apparent molecular mass of ϳ28 kDa, while YopE-(76 -219) migrated as a single protein with an apparent molecular mass of ϳ20 kDa. The expression properties of ExoS-(1-234) and ExoS-(78 -234) were similar to that previously reported (7). Both full-length and N-terminal forms of ExoS and YopE stimulated the rounding of both CHO cells (Fig.…”

“…Type-III-delivered ExoS localizes to intracellular membranes within eukaryotic cells (7). Fusion of the first 107 amino acids of ExoS to the green fluorescent protein (GFP) directed this reporter protein to the perinuclear region of mammalian cells.…”

Cell line differences in bacterially translocated ExoS ADP-ribosyltransferase substrate specificity

“…An alternative, more likely explanation for altered patterns of ExoS ADPRT substrate modification, based on currently available data, is that differences in the targeting of ExoS within the cell affect its ADPRT substrate specificity. Consistent with this hypothesis, both an N-terminal processing site and a membrane localization domain have been identified within the first 78 aa of ExoS (Pederson et al, 2000). These sites appear to influence the subcellular localization of ExoS and may also prove to influence the substrate targeting specificity of ExoS ADPRT activity.…”

Intracellular Localization of Type III-delivered Pseudomonas ExoS with Endosome Vesicles

“…Bacterial Strains and Construction of Vectors-P. aeruginosa strain PA103 (⌬exoU, exoT::Tc) with a pUCP derivative to express the indicated form of ExoS was cultured as described previously (10,11,28). ExoS-KDD and ExoS⌬MLD-KDD were generated by QuikChange using ExoS R146K, E381D (29) as template.…”