Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS

Abstract

Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain. We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity. Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP). In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment. Using [ 32 P]NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS. Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro.To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of [ 32 P]NAD, the substrate for ADP-ribosylation. After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated. The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.

Identification of a Stomatin Orthologue in Vacuoles Induced in Human Erythrocytes by Malaria Parasites

“…Selective Permeabilization of the Infected Red Cell Membrane Using Tetanolysin-Tetanolysin is a bacterial toxin that can be used to selectively permeabilize cellular plasma membranes (48). Infected red cells (0 -8-h post-invasion) were placed on poly-L-lysine-coated coverslips as described above.…”

Ezrin/Radixin/Moesin Proteins Are High Affinity Targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS

“…ExoS ADP-ribosylates a 70-kDa Protein Early during Infection-A tetanolysin permeability assay was used to identify cellular proteins ADP-ribosylated by type III-delivered ExoS at early time points (21). At 3 h post-infection, several predominant radiolabeled bands were observed, notably at 70, 50, 33, and 26 kDa (Fig.…”

“…Construction of pUCP-ExoS, pUCP-ExoS-R146K, pUCP-ExoS-E381D, and pUCP-ExoS-R146K/E381D have been described (21,34). Plasmids encoding the ORFs of the ERM proteins were purchased from the ATCC (6925743, moesin; 5265085, erzin; 7019745, radixin) and amplified by PCR using the following primer set: moesin forward, 5Ј-GATCGATCCTCGAGGCCATATGCCCAAAACGATCAGT-3Ј, and reverse, 5Ј-GATCGATCGAATTCTTAGGTCGACGCCATAGACTCAAAT-TCGTCAATG-3Ј; ezrin forward, 5Ј-GATCGATCCTCGAGGCGCATAT-GCCGAAACCAATCAATGTCC-3Ј, and reverse, 5Ј-GATCGATCGAAT-TCTTAGGTCGACGCCAGGGCCTCGAACTCGTCGA-3Ј; radixin forward, 5Ј-GATCGATCCTCGAGGCGCATATGCCGAAACCAATCAACG-TA-3Ј, and reverse, 5Ј-GATCGATCGGATCCTTAGGTCGACGCCATT-GCTTCAAACTCATCGATA-3Ј.…”

“…Proteins ADP-ribosylated by type III-delivered ExoS in cultured HeLa cells were determined by a "tetanolysin permeability assay" that has been previously described with some modifications (21). Confluent HeLa cells (85-mm dishes) were infected with P. aeruginosa expressing the indicated form of ExoS at an m.o.i.…”

“…ExoT ADP-ribosylates CrkI/II (12) and acts as an antiinternalization factor for P. aeruginosa (13). ExoS ADP-ribosylates a diverse group of proteins (being poly-substrate specific), including vimentin (14), IgG (15), Ras, Ras-like GTPases (16 -20) and is auto-ADP-ribosylated (21). ADP-ribosylation inhibits the interaction of Ras and Rap with their cognate guanine exchange factors (22,23).…”