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Infection and Immunity, April 2002, p. 2230-2232, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2230-2232.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Membrane Localization Contributes to the In Vivo ADP-Ribosylation of Ras by Pseudomonas aeruginosa ExoS

Matthew J. Riese and Joseph T. Barbieri*

Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received 3 October 2001/ Returned for modification 20 November 2001/ Accepted 5 January 2002


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ABSTRACT
 
Type III-delivered exoenzyme S (ExoS) preferentially ADP-ribosylated membrane-associated His6HRas, relative to its cytosolic derivative His6HRas{Delta}CAAX. This indicates that the subcellular protein distribution contributes to in vivo ADP-ribosylation by ExoS.


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TEXT
 
Exoenzyme S (ExoS) is a type III bifunctional cytotoxin produced by Pseudomonas aeruginosa (2). The N terminus of ExoS encodes a Rho-GAP domain, while the C terminus of ExoS comprises a 14-3-3-dependent ADP-ribosyltransferase (7). Introduction of the ADP-ribosyltransferase domain of ExoS into eukaryotic cells results in cell death (9). ExoS exhibits broad specificity in vitro, but preferentially ADP-ribosylates several groups of proteins, including Ras GTPases, at limiting dilutions (4). Ras GTPases are also ADP-ribosylated following type III delivery of ExoS by P. aeruginosa (8). Recently Ral, a membrane-bound Ras-like GTPase has been reported to be ADP-ribosylated by type III-delivered ExoS (5). ADP-ribosylation of Ras and a related protein, Rap, at Arg41 inhibits interaction with their respective guanine nucleotide exchange factors, resulting in their inactivation (6, 11). While elegant assessments of individual in vivo targets of ExoS have been performed (5, 8), a global evaluation of ADP-ribosylated proteins has not been reported until recently, when a direct measurement of the ADP-ribosylation of target proteins was analyzed following the type III delivery of ExoS by P. aeruginosa (11a). Infection of epithelial cells with ExoS-producing P. aeruginosa resulted in the ADP-ribosylation of ExoS itself and a group of 25-kDa proteins, which included Ras.

Although the fate of type III proteins upon intracellular delivery has received limited investigation, there is growing evidence that several type III proteins localize to distinct subcellular sites (10, 12), which may influence the modulation of host cell physiology. In this study, we address the observation that the majority of proteins that are ADP-ribosylated by type III-delivered ExoS are membrane associated (11a) and report that the membrane localization of Ras, one of the in vivo target proteins of ExoS, contributes to its efficient ADP-ribosylation.

ADP-ribosylation of His6HRas in CHO cells infected with P. aeruginosa. CHO cells or pCMV-His6HRas-transfected CHO cells were infected with an ExoS-expressing strain of P. aeruginosa, PA103 {Delta}exoU exoT::Tc (pUCPExoS), for 3 or 3.5 h in serum-free media at a ratio of 8 to 1 (bacteria/CHO cells). After removal of the medium, CHO cells were washed with phosphate-buffered saline and treated with tetanolysin to generate pores within the plasma membrane for intracellular diffusion of [32P]-NAD (Riese et al., submitted). Briefly, tetanolysin (0.4 µg/ml) was applied to CHO cells in cold HGI buffer (1) (20 mM PIPES, 2 mM Na-ATP, 4.8 mM magnesium acetate, 0.15 M K-glutamate, 2 mM EGTA, KOH adjusted to pH 7.0) at 4°C for 10 min to permit binding to cell membranes. Cells were washed with cold HGI buffer and then incubated in HGI buffer containing [32P]-NAD (1 µCi/ml) at 37°C for 25 min. At this time, cells were harvested in HB2 buffer (0.25 M sucrose, 3 mM imidazole adjusted to pH 7.4, 0.5 mM EDTA, 2 µg of phenylmethylsulfonyl fluoride per ml) and disrupted, and the nuclei and unbroken cells were removed by low-speed centrifugation. This postnuclear supernatant was centrifuged at 68,000 x g for 30 min to produce membrane (pellet) and cytosol (soluble) fractions. Membranes were suspended in HB2 plus 1% Triton X-100.

Infection of CHO cells with ExoS-producing P. aeruginosa and subsequent incubation with tetanolysin and [32P]-NAD resulted in preferential incorporation of radiolabel into 25- and 50-kDa proteins (Fig. 1A, CHO + ttl). In addition to these radiolabeled proteins, infection of His6HRas-transfected CHO cells with ExoS-producing P. aeruginosa yielded a unique tetanolysin-dependent, radiolabeled protein, which migrated with an apparent molecular mass greater than the 25-kDa radiolabeled protein (Fig. 1A, arrow, CHO-His6Ras, + ttl). This unique radiolabeled protein was identified as His6Ras based upon its immunoprecipitation with {alpha}-His6 antisera (Fig. 1B) and reactivity to His probe by Western blot procedures (Fig. 1). To address the possibility that the incorporation of radiolabel could be due to secreted ExoS or bacterially associated ExoS, but not translocated ExoS, pUCPExoSHA was transformed into P. aeruginosa deficient in type III translocation (PA103{Delta}pcrV) and the ability of this type III-deficient strain to deliver ExoS into CHO cells was determined. CHO cells infected with PA103{Delta}pcrV(pUCPExoSHA) did not incorporate radiolabel into Ras, using the tetanolysin assay system (data not shown). This showed that type III translocation was required for the observed incorporation of radiolabel into Ras and that bacterially delivered ExoS ADP-ribosylated His6HRas via a tetanolysin-dependent mechanism. In addition, subcellular fractionation preserved the ADP-ribosylation patterns observed in CHO cells harvested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled (data not shown), which indicated that little detectable modification had occurred after harvesting.



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  		FIG. 1. ExoS ADP-ribosylates His6-HRas in tetanolysin-treated CHO cells. CHO cells or pCMV-His6-HRas (3.0 µg)-transfected CHO cells (85-mm-diameter dishes) were infected for 3 h with ExoS-producing P. aeruginosa. Cells were then incubated for 10 min at 4°C, alone or with 2.4 µg of tetanolysin in 6 ml of HGI buffer, and washed with ice-cold HGI buffer. Next, 6 ml of HGI buffer containing 6 µCi of 20 nM 32P-NAD was added and cells were placed at 37°C in 5% CO2. Cells were harvested and broken, and the postnuclear supernatant was fractionated into cytosol and membrane components. (A) Membrane fractions were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF), and exposed to X-ray film (upper panel; the arrow indicates the unique radiolabeled 28-kDa protein), or blotted with His6-Probe (lower panel). (B) Membrane fractions were subjected to immunoprecipitation, using {alpha}-His6 antibody. The immunoprecipitate was subjected to SDS-PAGE, transferred to PVDF, and analyzed by autoradiography (upper panel; the arrow indicates the unique radiolabeled 28-kDa protein) or His6-Probe blotting (lower panel).

Membrane localization enhances the efficiency of the ADP-ribosylation of Ras by ExoS. The contribution of intracellular localization for efficient ADP-ribosylation by ExoS was addressed by measuring the efficiency of in vivo ADP-ribosylation of Ras, a membrane associated protein, and Ras{Delta}CAAX, a C-terminal deletion protein of Ras that localizes in the cytosol (3). Subcellular fractionation showed that like endogenous Ras, 93% of the His6-HRas was in the membrane fraction, while 83% of His6-HRas{Delta}CAAX was present in the cytosol (Fig. 2A). Cotransfected green fluorescent protein (GFP) was present in the cytosol, which indicated that the fractionation produced little contamination of cell membranes in the cytosol (Fig. 2A). Treatment of transfected cells with tetanolysin did not alter the distribution of His6-HRas or His6-HRas{Delta}CAAX (data not shown).



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  		FIG. 2. Expression of Ras and Ras{Delta}CAAX in CHO cells. (A) CHO cells were transfected with 0.5 µg of pEGFP and 1 µg of pCMV-His6HRas or 0.5 µg of pEGFP and 3 µg of pCMV-His6HRas{Delta}CAAX for 18 h. Cells were fractionated into membrane (M) and cytosolic (C) fractions, which were subjected to SDS-PAGE and transferred to PVDF. PVDF filters were subjected to ECL, using {alpha}-Ras immunoglobulin G (IgG) (upper panel) or {alpha}-GFP IgG (lower panel) as primary antibodies. Exposures of developed X-ray film are shown (upper panel). His6HRas (upper arrow) migrates more slowly than endogenous Ras (lower arrow). Pos contls, reactivity of authentic His6HRas (1.5 ng) and GFP (1.1 ng). (B to C) CHO cells in 12 wells were transfected with the indicated amounts in micrograms (Amt) of pHis6-HRas or pHis6-HRas{Delta}CAAX for 18 h. Cells were infected with a control strain of P. aeruginosa that did not express ExoS (panel B, lane pUCP) or an ExoS-producing P. aeruginosa (panel B, lane pUCPExoS, and panel C, all lanes) for 3.5 h, treated with tetanolysin and then 32P-NAD (see Fig. 1). Cell lysates were prepared and subjected to SDS-PAGE and autoradiography (upper panel; the arrow indicates ADP-ribosylated His6Ras) or immunoblotting with {alpha}-Ras IgG as the primary antibody (lower panel). (D) CHO cells were transfected with 1.5 µg of pHis6-HRas or 3 µg of pHis6-HRas{Delta}CAAX and harvested. Ten microliters of postnuclear supernatant was incubated with the indicated nanomolar amount of ExoS (232 to 453), 0.2 µM fatty acid synthase, and 0.1 mM NAD in a 20-µl reaction volume of 50 mM Tris-HCl, pH 8.0. After 15 min at room temperature, reactions were stopped by the addition of sample buffer and subjected to SDS-PAGE. Proteins were transferred to PVDF and subjected to enhanced chemiluminescence with {alpha}-Ras. Data from panels C and D are quantified in Table 1.

His6-HRas- or His6-HRas{Delta}CAAX-transfected CHO cells were infected with ExoS-producing P. aeruginosa. Western blot analysis showed that only a small amount of His6-HRas and endogenous Ras was shifted during infection, making quantitative analysis of protein modification difficult. This also indicated that at this time point of analysis, substantial in vivo ADP-ribosylation of Ras or Ras{Delta}CAAX had not yet occurred. Thus, in vivo ADP-ribosylation of the two forms of Ras was determined by direct ADP-ribosylation. After 3.5-h infections, cells were treated with tetanolysin and [32P]-NAD. When normalized for protein expression, His6-HRas was observed to be radiolabeled 20-fold more efficiently than His6-HRas-{Delta}CAAX (Fig. 2C and Table 1). Controls showed that, in vitro, both His6-HRas and His6-HRas-{Delta}CAAX were ADP-ribosylated by ExoS at similar rates (Fig. 2D and Table 1), which indicated that ExoS did not have an intrinsic preference for either form of Ras. Together, these data showed that membrane localization enhanced the efficiency for ADP-ribosylation of Ras by type III-delivered ExoS.


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  		TABLE 1. ADP-ribosylation of His6Ras and His6Ras-{Delta}CAAX ADP-ribosylation by ExoSa

ExoS is enriched in the membrane-associated fraction. Distribution of ExoS in infected cells was determined by physical and enzymatic measurements. Western blot analysis of subcellular fractions of GFP-transfected CHO cells infected with ExoS-producing P. aeruginosa showed that 86% of ExoS was associated with the membrane fraction, while 14% was in the cytosol (standard deviation [SD], ±6% from two independent determinations). Localization of functional ExoS was determined on cell fractions from CHO cells infected with ExoS-producing P. aeruginosa, using soybean trypsin inhibitor as a target and the cell lysates as a source of ExoS as previously described (10). From this assay, membranes possessed 90.2%, while the cytosol possessed 9.8% (SD, ±1.8% from two independent determinations) of the ADP-ribosyltransferase activity in the postnuclear supernatant.

Conclusion. Type III-delivered ExoS was found to be localized to the membranes of infected CHO cells by functional and physical analysis. Type III-delivered ExoS preferentially ADP-ribosylated membrane-associated His6HRas, relative to its cytosolic derivative His6HRas{Delta}CAAX. This indicates that target protein localization contributes to the in vivo ADP-ribosylation of proteins by ExoS.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the NIH AI-31062 to J.T.B.


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Phone: (414) 456-8412. Fax: (414) 456-6535. E-mail: toxin{at}mcw.edu. Back

Editor: A. D. O'Brien


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Infection and Immunity, April 2002, p. 2230-2232, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2230-2232.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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