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INFECTION AND IMMUNITY, Mar. 2007, p. 1089–1098 Vol. 75, No. 30019-9567/07/$08.00�0 doi:10.1128/IAI.01184-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A2 Activityof Pseudomonas aeruginosa Cytotoxin ExoU�†

Vincent T. Lee,1* Stefan Pukatzki,1 Hiromi Sato,4 Eriya Kikawada,2 Anastasia A. Kazimirova,1Jin Huang,1 Xiaohua Li,3 Jonathan P. Arm,2 Dara W. Frank,4 and Stephen Lory1

Department of Microbiology and Molecular Genetics, Harvard Medical School,1 and Dana Farber Cancer Institute,2 Boston,Massachusetts 02115; Broad Institute, Cambridge, Massachusetts 021423; and Department of Microbiology and

Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 532264

Received 28 July 2006/Returned for modification 11 October 2006/Accepted 9 December 2006

A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directlyinto the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections expressa potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly intocontacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via itsphospholipase A2 (PLA2) activity. A high-throughput cell-based assay was developed to screen libraries ofcompounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compoundswere identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition,these compounds specifically inhibited the PLA2 activity of ExoU in vitro, whereas eukaryotic secreted PLA2and cytosolic PLA2 were not inhibited. This novel inhibitor of ExoU-specific PLA2 activity, named pseudolipasin A,may provide a new lead for virulence factor-based therapeutic design.

Pseudomonas aeruginosa is the leading cause of hospital-acquired infections by gram-negative bacteria and is responsi-ble for chronic infections of individuals with cystic fibrosis (15).As P. aeruginosa and other bacterial pathogens become mul-tidrug resistant (22), there is a continual need for the identi-fication of compounds directed at novel targets that could bedeveloped into therapeutic agents. The ability of P. aeruginosato cause a number of distinct infections has been attributed toits large genome, which encodes a variety of virulence factors(35). Prominent among these is the type III secretion system(TTSS), which allows the bacteria to deliver proteins directlyinto the host cell cytoplasm (39). ExoU is one of the toxiceffector proteins delivered by the type III secretion apparatus,and its expression is associated with strains that cause acuteinfections (11, 12). ExoU is a member of the patatin family ofphospholipase A2 (PLA2), and this activity is required forcytotoxicity toward eukaryotic cells (33, 34). In contrast toeukaryotic phospholipases, which remodel cellular membranesand synthesize proinflammatory secondary messengers such asarachidonic acid and leukotrienes (2, 9), ExoU PLA2 activityprimarily causes disruption of the host cytoplasmic membrane,resulting in cell lysis (24, 32, 33).

We sought to identify compounds that inhibit type III secre-tion-mediated cytotoxicity by protecting tissue culture cellsfrom infection by P. aeruginosa strains that elaborate ExoU asa sole cytotoxic effector. From a synthetic small-molecule li-brary, we have identified compounds that protected Chinesehamster ovary (CHO) cells from the cytotoxic activity of P.

aeruginosa expressing ExoU. One of the most potent com-pounds is pseudolipasin A (Pseudomonas phospholipase inhib-itor A). Pseudolipasin A does not interfere with type III se-cretion in general, suggesting that the protection observedoccurs downstream of the delivery of ExoU. Pseudolipasin Anot only protected CHO cells from intoxication but also res-cued the amoeba Dictyostelium discoideum from ExoU-medi-ated killing by P. aeruginosa. Furthermore, the cytotoxicity ofcytoplasmically expressed ExoU in Saccharomyces cerevisiaewas inhibited by pseudolipasin A. Pseudolipasin A inhibitedthe PLA2 activity of ExoU in vitro but did not affect any of theother eukaryotic PLA2 enzymes tested, including the cytosolicphospholipase that promotes the generation of arachidonicacid and subsequent generation of a proinflammatory immuneresponse. This screen of a small-molecule chemical libraryidentified a specific inhibitor of the enzymatic activity of a toxinthat could be a prototype of therapeutic agents targeting bac-terial virulence factors.

MATERIALS AND METHODS

Strains and media. P. aeruginosa strain PAK-VL1 (PAK �exoS �mexABatt::exoU spcU/pMMB-exsA) harbors a deletion of the exoS gene, a deletion in themexAB genes encoding the major drug efflux pump, a chromosomal insertion ofexoU-spcU from PA103 at the ctx site, and the type III transcriptional activatorexsA under the control of isopropyl-�-D-thiogalactopyranoside (IPTG) on thepMMB67 plasmid (37). Strains PAK, PA103, and PA14 have been describedelsewhere (4, 18, 27). P. aeruginosa strains were routinely grown in Luria-Bertanibroth (LB) and induced for type III secretion by chelating Ca2� with 5 mMEGTA. Plasmid pMMB-exsA was maintained with 50 mg/ml carbenicillin, andexsA expression was induced with 1 mM IPTG. Assays for secretion and injectionutilized pVL710 and pVL712. pVL710 is a pMMB-Ap-based plasmid that con-tains an exoU promoter driving the expression of �-lactamase. pVL712 containsthe promoter of exoU driving the expression of an ExoU(S142A)–�-lactamasefusion protein. S. cerevisiae BY4742 was grown on yeast-peptone-dextrose me-dium. Plasmid pDH105 was maintained in BY4742 by growth on minimal syn-thetic defined (SD) base medium supplemented with �Leu dropout supplement(SD �Leu; Clontech, Mountain View, CA). D. discoideum strain AX3 waspropagated in HL5 medium (per liter, 10 g of dextrose, 5 g of yeast extract, 5 g

* Corresponding author. Present address: Department of Cell Biol-ogy and Molecular Genetics, University of Maryland, College Park,MD 20742. Phone: (301) 405-9397. Fax: (301) 314-9489. E-mail: [email protected].

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 18 December 2006.

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of Thiotone peptone, 0.67 g of Na2HPO4 � 7H2O, 0.34 g of KH2PO4, and 0.05 gof streptomycin-sulfate).

Chemical library and screening facility. The known PLA2 inhibitors methylarachidonyl fluorophosphonate (MAFP) and bromoenol lactone (BEL) werepurchased from Cayman Chemical (Ann Arbor, MI). The Microformat E col-lection from ChemBridge (San Diego, CA) consists of 50,000 compounds andwas screened in duplicate in a high-throughput screening assay. In this assay, thefinal concentration of compounds after pin transfer from a stock solution of 5mg/ml is 12.5 �g/ml (approximately 25 to 50 �M depending on the molecularweight of each compound). The screening was performed at the Institute ofChemistry and Cell Biology at Harvard Medical School.

Inhibition of CHO cell killing by P. aeruginosa expressing ExoU. CHO cellswere grown in F-12 medium supplemented with 10% fetal bovine serum and 2mM glutamine. A total of 1 � 104 CHO cells per well were seeded in 384-wellplates. Compounds were pin transferred into 384-well plates, and the A405 wasread to exclude compounds with high absorbance at the output wavelength. CHOcells were infected at a multiplicity of infection (MOI) of 10 with PAK-VL1grown to a mid-log-phase culture in 1 mM IPTG to induce expression of the typeIII secretion system. Antibiotics were added 2 h postinfection to restrict thecytotoxicity mediated by ExoU as well as to eliminate bacteria that would inter-fere with the readout. After an additional 5 h of incubation, a tetrazolium-basedreagent, WST (Roche Applied Science, Indianapolis, IN), was added to the wellsto detect the total reducing potential, which is an indicator of the number of livecells as measured by absorbance reading at 405 nm. Compounds that providedprotection were retested in 96-well plates. In addition, compounds that protectedcells from ExoU-mediated lysis were also tested by measuring the release ofcellular lactate dehydrogenase (LDH; Roche Applied Science, Indianapolis,IN) (17).

High-throughput secretion assay for the fusion of ExoU to the mature portionof �-lactamase lacking the signal peptide (BlaM). P. aeruginosa PAK orPAK�pscC containing either pVL710 and pVL712 was grown in LB containing50 �g/ml carbenicillin. The bacteria were subcultured to an optical density at 600nm (OD600) of 0.005 in LB supplemented with 5 mM EGTA to induce the TTSS,and 25 �l was dispensed into 384-well plates and incubated for 3 h at 37°C. A25-�l volume of LB supplemented with 5 mM EGTA and 50 �g/ml nitocefin(EMD Biosciences, Madison, WI) was added to each well, and plates were readat 490 nm and 650 nm over a 60-min period.

High-throughput injection assay for ExoU-BlaM fusion. Confluent monolay-ers of CHO cells were grown in black, clear-bottom, 384-well plates. Cells wereinfected for 2 h at an MOI of 10 with P. aeruginosa PAK or PAK�pscC contain-ing either pVL710 or pVL712. CCF4-AM (Invitrogen, San Diego, CA) wasadded according to the manufacturer’s instructions for 30 min. The plates wereread in a TECAN fluorescence plate reader using excitation at 360 nm andemission at 450 nm for coumarin and excitation at 360 nm and emission at 535nm for fluorescence resonance energy transfer of coumarin to fluorescein iso-thiocyanate.

Protection of Dictyostelium discoideum from P. aeruginosa PA103-mediatedkilling. P. aeruginosa PA103-mediated killing of D. discoideum strain AX3 onSM/50 agar plates (per liter, 0.1 g of MgSO4, 1.9 g of KH2PO4, 1.0 g of K2HPO4,0.2 g of glucose, 0.2 g of Bacto peptone, 0.02 g of Bacto yeast extract, and 20 gof Bacto agar; Difco, Franklin Lakes, NJ) was carried out as described previously(25). Inhibitors were added to the agar medium at the indicated concentrationsand poured into 6-well plates.

Protection of Saccharomyces cerevisiae from ExoU-mediated killing. For het-erologous expression of exoU in S. cerevisiae, plasmid pDH105-ExoU, whichallows copper-inducible-expression of exoU, was constructed. The exoU gene wasamplified from plasmid pET24aExoU.9 (1) by PCR with primers 5�-GAGCTCATGCATATCCAATCGTTGG-3� and 5�-GCTAGCTCATGTGAACTCCTTATTC-3�, and the resulting product was introduced into the TA cloning vectorpCRII-TOPO (Invitrogen, San Diego, CA). ExoU was excised as a SacI/NheIfragment, blunt ended, and ligated into XbaI-cut and blunt-ended plasmidpDH105 (a gift from Daniel B. Hall, Harvard Medical School).

S. cerevisiae strain BY4742 harboring plasmid pDH105-exoU was grown over-night in SD �Leu to maintain selection of the plasmid. Inhibitors were dilutedto various concentrations in SD �Leu and placed in wells of a 96-well plate asindicated. The yeast culture was diluted to an OD600 of 0.05 and aliquoted intowells of 96-well plates containing inhibitors, and exoU expression was inducedwith 0.5 mM CuSO4. Plates were incubated in a humidified chamber at 30°C for24 h and resuspended by pipetting prior to spectrophotometer reading at 600 nm.Protection on SD �Leu agar was performed with 0.75 mM CuSO4 at theindicated concentrations of inhibitors.

In vitro PLA2 assays. Recombinant N-terminal six-His-tagged ExoU and cat-alytically inactive ExoU(S142A) were purified from Escherichia coli BL21 har-

boring the pET19 plasmid, containing the genes encoding these proteins (34).After purification over Ni2�-nitrilotriacetic acid resin, the proteins were concen-trated into a buffer containing 20 mM HEPES (pH 7.4)–50 mM NaCl and storedas aliquots at �80°C. The CHO cell extract was purified as described previously(38), resuspended in 20 mM HEPES (pH 7.4)–50 mM NaCl, and frozen inaliquots at �80°C. Inhibitors were resuspended in dimethyl sulfoxide (DMSO) at1 mg/ml. A cytosolic PLA2 (cPLA2) assay was purchased from Cayman Chemical(26), and the cPLA2 activity of ExoU was assayed according to the manufactur-er’s protocol. Inhibition of ExoU activity was performed with reaction mixturescontaining 5 �g of ExoU, 5 �g of CHO extract, 5 �g of each inhibitor in DMSO,and the arachidonic thioester phosphatidylcholine (TEPC) substrate. PLA2

cleavage results in the generation of free thiol groups that are detected byaddition of 5,5�-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent; DTNB) andcontinuous reading of the plate at 414 nm. The percentage of hydrolysis wascalculated from the 60-min time point.

Alternatively, ExoU PLA2 activity was monitored by the release of oleic acidfrom 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine (POPC) (Amersham Bio-sciences, Piscataway, NJ) as described previously (34). Briefly, 1 �g of recombi-nant ExoU was mixed with 10 �g of yeast extract and added to 14C-labeledPOPC-1-palmitoyl-2-oleoyl-phosphatidyl serine (POPS) vesicles for 6 h at 30°Cwith and without inhibitors at the indicated concentrations.

Secreted PLA2 (sPLA2) was assayed using the sPLA2 detection kit fromCayman Chemical using diheptanoyl thioester phosphatidylcholine as the sub-strate. Bee venom PLA2 was obtained from Cayman Biochemical. Human groupIID, IIE, V, X, and XII PLA2 were generously provided by BioVendor Labora-tory Medicine (Candler, NC).

Inhibition of ExoU-mediated lysis of peripheral blood PMN. Peripheral bloodpolymorphonuclear neutrophils (PMN) were isolated from freshly isolated bloodby centrifugation over RPM medium (ICN, Irvine, CA) and washed once withmodified Eagle’s medium. A total of 5 � 105 PMN were infected with strainPA103 opsonized with serum at an MOI of 10 and treated with 1% DMSO withor without 10 �g/ml of inhibitor. After 1 h of incubation at 37°C, the supernatantwas collected and assayed for LDH (Roche Applied Sciences).

Production of LTC4 by BMMC. Experiments were performed as describedpreviously (21). Cultured bone marrow-derived mast cells (BMMC) were sensi-tized with monoclonal immunoglobulin E against trinitrophenyl (TNP) for 60min. After being washed with WEHI-3 medium, cells were resuspended to 2 �107/ml and mixed with pseudolipasin A or MAFP at the indicated concentrationfor 10 min. The cells were stimulated at 37°C with 100 ng/ml TNP-conjugatedbovine serum albumin. After 10 min, the reaction was stopped by centrifugationof the cells at 120 � g for 5 min at 4°C, and the supernatants were assayed forleukotriene C4 (LTC4).

RESULTS

Screening of a compound library for inhibitors of P. aerugi-nosa cytotoxic activity. To identify inhibitors that prevent in-toxication of mammalian cells by P. aeruginosa expressingExoU, we developed an assay that measures the total reducingpotential of cells after P. aeruginosa infection as an indicationof their viability. For this assay, we engineered strain PAK-VL1 (PAK �mexAB �exoS att::exoU spcU/pMMB-exsA whichcarries a deletion of the genes encoding the MexAB effluxpump such that the bacteria would be sensitized to small mol-ecules acting on intracellular targets. exoS, the gene native tostrain PAK that also encodes a type III secreted cytotoxin, isdeleted, and the exoU operon from PA103 is placed in thectx site on the chromosome (17). PAK-VL1 injection ofExoU via the TTSS resulted in a rapid cytotoxic effect onCHO cells (17, 36).

The high-throughput assay of ExoU-mediated cytotoxicityallowed rapid screening of 50,000 compounds in ChemBridgeMicroformat Library E. Cells infected with P. aeruginosa PAK-VL1 (Fig. 1A, 1st and 2nd columns) are intoxicated and unableto reduce WST, whereas cells protected by gentamicin (Fig.1A, 23rd and 24th columns) are viable and reduce WST to thesame extent as uninfected controls. Active compounds that can

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protect CHO cells from infection allow reduction of WST andresult in a change in absorbance at 405 nm (Fig. 1A). Theinitial screen yielded 88 compounds that were able to protectCHO cells from the cytotoxic activity of PAK-VL1. One ofthese hits was the antibiotic ciprofloxacin, which has knownbactericidal activity against P. aeruginosa, thus providing evi-dence that the screen was sensitive enough to detect bacterialgrowth inhibitors that could protect CHO cells. Upon subse-quent retesting of active compounds, a subset demonstratedthe ability to protect CHO cells from the cytotoxic action of P.aeruginosa (see Table S1 in the supplemental material). Themost potent compound was 9H-fluorene-4-carboxylic acidamide, which we named pseudolipasin A (Pseudomonas phos-pholipase inhibitor A) (Fig. 1B). Using an LDH release assayto determine the amount of cell lysis, we determined thatpseudolipasin A has a 50% inhibitory concentration (IC50) of1 to 7.5 �M against a variety of P. aeruginosa strains expressingExoU, including PAK-VL1, PA103, and PA14 (Fig. 2). P.aeruginosa strains lacking the MexAB efflux pump are moresensitive to pseudolipasin A, whereas the up-regulation of theTTSS by overexpression of ExsA resulted in strains that wereless sensitive to pseudolipasin A. The cytotoxicity of P. aerugi-nosa strains that lack ExoU, such as the parental PAK strain,was not affected by pseudolipasin A (Fig. 2). The structure and

purity of pseudolipasin A were confirmed by liquid chroma-tography-mass spectrometry and 1H nuclear magnetic reso-nance (see Fig. S1 in the supplemental material). To testwhether pseudolipasin A is toxic to eukaryotic cells, CHO cellswere propagated in 20 �g/ml of pseudolipasin A; they grew atthe same rate as cells treated with the DMSO carrier over thecourse of 7 days. Additionally, no morphological changes wereobserved in the treated cells compared to cells grown in me-dium only (data not shown). These data suggest that pseudo-lipasin A is not toxic to mammalian cells.

Active compounds could act at several stages of intoxication,including interference with the assembly of the type III secre-tion machine, the delivery of ExoU into the host cell cyto-plasm, the interaction of ExoU with its host activator, and thePLA2 activity of ExoU (34). Moreover, compounds that eitheraffect the synthesis of ExoU, block general protein synthesis, orkill P. aeruginosa by any mechanism would also lead to therescue of CHO cells. However, none of the compounds otherthan ciprofloxacin possessed antibacterial activity at 10-�g/mlconcentrations. The other possibilities were investigated, andthe results are described below.

Pseudolipasin A does not inhibit type III secretion or typeIII injection into mammalian cells. We conducted several ad-ditional assays for type III-dependent secretion and injectionby utilizing a �-lactamase reporter that fused BlaM to the Cterminus of ExoU (Fig. 3A). As a result, type III secretion canbe detected by �-lactamase cleavage of nitrocefin in the cal-cium-depleted TTSS-induced culture (23). Secretion of theExoU-BlaM fusion (from plasmid pVL712) can be readilymeasured using the nitrocefin assay (Fig. 3B). Neither BlaMalone (expressed from pVL710), which lacks the type III se-cretion signal, nor a type III-defective strain (�pscC) can se-crete BlaM via the TTSS. These data demonstrate that the

FIG. 1. Identification of pseudolipasin A. (A) A total of 50,000compounds were screened in a 384-well plate format for compoundsthat protect CHO cells from cytotoxicity mediated by P. aeruginosatype III secretion pathway-delivered ExoU. Each screening plate in-cluded negative controls in which CHO cells were mock treated withDMSO only (1st and 2nd columns) and positive controls in which theantibiotic gentamicin was added to the wells prior to the addition of P.aeruginosa PAK-VL1 (23rd and 24th columns). Yellow wells indicateviable CHO cells that were protected from P. aeruginosa killing. Redcircles indicate compounds that were able to protect CHO cells fromP. aeruginosa infection, with a Z score for the plate of �3. (B) Com-pound structure of pseudolipasin A.

FIG. 2. Pseudolipasin A protects CHO cells from lysis mediated byExoU delivered by various strains of P. aeruginosa. Protection of CHOcells is calculated as the inverse of the percentage of LDH release frominfected cells. The following strains of P. aeruginosa were tested: PA14(light blue), PAK �exoS att::exoU (red), PAK �mexAB �exoS att::exoU(yellow), PAK �exoS att::exoU pMMB67EH-exsA (green), PAK�mexAB �exoS att::exoU pMMB67EH-exsA (dark blue), and PA103(orange). Strains containing pMMB67EH-exsA were induced with 1mM IPTG.

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export of the BlaM reporter depends on fusion to a type IIIsecretion signal and a functional TTSS (Fig. 3B). Addition ofpseudolipasin A to PAK/pVL712 had no effect on low-calcium-induced type III secretion (Fig. 3B). As a control, pseudolipasin Awas added to nitrocefin, which resulted in no measurable chem-ical cleavage of nitrocefin (Fig. 3B).

Type III-dependent injection of the hybrid protein can bedetected by �-lactamase cleavage of CCF4 in the cytosol ofintoxicated cells (5, 40). Only wild-type P. aeruginosa with afunctional TTSS expressing the ExoU-BlaM fusion protein wasable to deliver BlaM into the host cells as detected by CCF4cleavage (Fig. 3C). Addition of pseudolipasin A had no effecton the delivery of ExoU–�-lactamase into the cytosol of CHO

cells (Fig. 3C). These results suggest that pseudolipasin A actsat a step downstream from TTSS-mediated delivery of ExoU.

Protection of Dictyostelium discoideum from P. aeruginosaexpressing ExoU. In order to assess whether pseudolipasin Ainterference with P. aeruginosa-mediated killing is specific forCHO cells, we examined other eukaryotic models that aresensitive to type III-mediated killing. The amoeba D. dis-coideum has previously been shown to be susceptible to killing byP. aeruginosa PA103 in a type III secretion-dependent manner,and the PLA2 activity of ExoU was shown to be responsible forthis toxic activity (25). In this assay, amoebae fed on a lawn ofbacteria will form plaques unless the D. discoideum is killed.The presence of pseudolipasin A in the agar protected D.discoideum from P. aeruginosa PA103, allowing the formationof visible plaques (Fig. 4A). Pseudolipasin A protected D.discoideum with an IC50 of 0.2 �M. Addition of MAFP, asubstrate analog inhibitor for cytosolic PLA2 that also inhibitsExoU PLA2 activity (24, 34), did not protect D. discoideumfrom PA103 (Fig. 4B). Although MAFP did not permit thegrowth of D. discoideum on E. coli, MAFP did not affectgrowth in liquid medium (data not shown). These data suggestthat a PLA2 is required for the amoeba to utilize bacteria as afood source and that MAFP, rather than inhibiting an un-known essential target, has an activity that is highly specificagainst D. discoideum PLA2. Thus, pseudolipasin A acts toprotect the eukaryotic cell from ExoU-dependent killing in asystem in which the known PLA2 inhibitor MAFP cannot,suggesting that pseudolipasin A has a greater specificity forExoU. These results also demonstrate that protection bypseudolipasin A occurs in a number of eukaryotic cells.

Protection of Saccharomyces cerevisiae from intracytoplasmi-cally expressed ExoU. Since pseudolipasin A appears to actdownstream of type III injection of ExoU, we asked whetherthe activity of this compound requires a functional TTSS. Weused an S. cerevisiae expression system in which exoU transcrip-tion was controlled by the copper-inducible promoter-1 (cup1).Addition of Cu2� to the culture medium leads to expression ofexoU and subsequent cell death, thus providing a method fordetecting protection against ExoU activity in the absence ofbacterial delivery. The PLA2 inhibitor MAFP was able to pro-tect yeast induced with copper from ExoU-mediated killing atan IC50 of 80 nM (Fig. 5). Pseudolipasin A also protects yeastfrom killing after ExoU induction with an IC50 of 7 �M forliquid medium and 1.5 �M for solid medium (Fig. 5A and B).Thus, pseudolipasin A acts directly on ExoU within the eu-karyotic cell, independently of the bacterial type III secretionmechanism.

Inhibition of the PLA2 activity of ExoU in vitro. Inhibition ofExoU PLA2 activity by pseudolipasin A was examined usingtwo different enzymatic assays. The first assay is based on thecleavage of arachidonyl-TEPC, resulting in the generation of afree thiol at the sn-2 position that in turn reduces DTNB,yielding a colorimetric readout (29). Since the PLA2 activity ofExoU requires activation by an unknown eukaryotic cellularfactor, each reaction was supplemented with a CHO or yeastcell extract. When arachidonyl-TEPC was used as the sub-strate, purified ExoU showed CHO extract-dependent PLA2

activity. A mutant variant of ExoU [ExoU(S142A)] with asubstitution in the phospholipase catalytic dyad was completelyinactive in this assay (data not shown). When this assay was

FIG. 3. Secretion of ExoU-BlaM via the P. aeruginosa type IIIsecretion system is not affected by pseudolipasin A. (A) Gene con-structs of �-lactamase fusions BlaM (pVL710) and ExoU-BlaM(pVL712). (B) Type III secretion of BlaM (pVL710) or ExoU-BlaM(pVL712) was assayed for P. aeruginosa strain PAK and a type IIIsecretion system-defective (�pscC) mutant in low-calcium medium.Secretion of ExoU-BlaM is measured by an increase in the cleavage ofnitrocefin detected as the absorbance at 490 nm. (C) Type III injectionwas assayed by infecting CHO cells with P. aeruginosa strain PAK or atype III secretion system-defective (�pscC) mutant harboring a plas-mid encoding BlaM (pVL710) or ExoU-BlaM (pVL712). Subsequentaddition of CCF4 allows detection of BlaM as a change in the ratio ofrelative fluorescence intensities emitted at 465 nm and 535 nm.



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used to test inhibition of the PLA2 activity of ExoU by pseudo-lipasin A and MAFP, pseudolipasin A had an IC50 of 7 �M(Fig. 6A), whereas the PLA2 inhibitor MAFP irreversibly in-hibited ExoU with an IC50 of 20 nM (Fig. 6A).

The second assay is based on detection of cleavage productsof 14C-labeled phosphatidylcholine by thin-layer chromatogra-phy (34). ExoU activity was also assessed by measuring the

release of 14C-labeled fatty acid from palmitoyl oleoyl phos-phatidylcholine as described previously (34). MAFP inhibitedthis reaction by 75%, while BEL inhibited this reaction by 60%(Fig. 6B). Pseudolipasin A inhibited 80% of the reaction atconcentrations similar to those of MAFP and BEL (Fig. 6B).These results indicate that pseudolipasin A inhibits the PLA2

activity of ExoU in vitro.Chemically related pseudolipasin A analogs and inhibitory

activity. Compounds with structures similar to that of pseudo-lipasin A were identified (Table 1) and tested for their abilitiesto inhibit ExoU PLA2 activity in vitro. Modification of theprimary amine in pseudolipasin A resulted in inactive com-pounds. Furthermore, hydrophilic modification or addition ofa fluorene ring also produced inactive compounds. The inabil-ity of structural analogs to act on the same process supports thefinding that pseudolipasin A, identified in this screen, has adefined chemical specificity toward ExoU-mediated cytotoxic-ity. Interestingly, the primary screen and analysis of structur-

FIG. 4. Pseudolipasin A protects D. discoideum from P. aeruginosaPA103 killing. (A) D. discoideum plated on agar with P. aeruginosaPA103 results in no plaque formation (in wells marked “0,” whichcontain no pseudolipasin A). D. discoideum forms plaques (indicatedby arrowheads) in the presence of the indicated micromolar concen-trations of pseudolipasin A. (B) D. discoideum fails to form plaques onagar containing the indicated nanomolar concentrations of MAFP.

FIG. 5. Pseudolipasin A protects against expression of ExoU in S.cerevisiae. (A) S. cerevisiae strain BY4742 harboring plasmid pDH105-exoU was induced for ExoU expression with SD �Leu medium con-taining 0.5 mM CuSO4 in a 96-well plate with either pseudolipasin Aor MAFP at the indicated concentrations. Growth of yeast was mon-itored by the OD600. (B) BY4742 harboring plasmid pDH105-exoU wasinduced for ExoU expression on SD �Leu agar containing 0.7 mMCuSO4 with the inhibitor pseudolipasin A (added at the indicatedmicromolar concentrations) or MAFP (added at 135 nM) or withDMSO (added at 1% as a solvent control). The sizes of coloniesindicate the survival of S. cerevisiae.



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ally related compounds revealed another family of compoundsthat show a possible structural relationship to pseudolipasin A[2-(4-cyclohexyl-phenylamino)-4,6-dimethyl-nicotinamide and2-(3-chloro-phenylamino)-4,6-dimethyl-nicotinamide] (see Ta-bles S1 and S2 in the supplemental material). Phenylamino-4,6-dimethyl-nicotinamide derivatives were capable of protect-ing CHO cells from killing by ExoU-expressing P. aeruginosa,but they were not as potent as pseudolipasin A. Both phe-nylamino-4,6-dimethyl-nicotinamide and pseudolipasin A havea free acetamide moiety and two 6-member rings with littlehydrophilicity. Given the conservation of some of the struc-tural features of these compounds, it is conceivable that theyinhibit ExoU by similar mechanisms.

Pseudolipasin A is an ExoU-specific inhibitor of PLA2. Anumber of eukaryotic PLA2 have been implicated in immunefunction by eliciting a proinflammatory response via the gen-eration of secondary molecules such as leukotrienes (LTC4)and arachidonic acid (2, 9). In addition, sPLA2 have demon-

strated antibacterial activity (8, 13, 16, 30). Pseudolipasin Awas tested against a panel of sPLA2 to determine its range ofbiological inhibition. The activity of sPLA2 was measured byuse of the substrate diheptanoyl-TEPC (28). The cleavage ofdiheptanoyl-TEPC results in generation of free thiol at thesn-2 position, which can be measured by the reduction ofDTNB. Pseudolipasin A did not inhibit bee venom PLA2

(group IV) or human group IID, IIE, V, X, or XII PLA2 at81 �M concentrations (see Table S3 in the supplementalmaterial).

cPLA2 share several conserved sequence features withExoU, including the same residues in the catalytic dyad (34).The activity of cPLA2was measured indirectly in murine bonemarrow-derived mast cells as the generation of the secondarymolecule LTC4 from arachidonic acid released by cPLA2. Theaddition of pseudolipasin A up to 50 �M had no effect onLTC4 production, whereas the addition of 1 �M MAFP re-sulted in a decrease in LTC4 production (Fig. 7A). Theseresults suggest that pseudolipasin A is a specific inhibitor forExoU PLA2 activity.

A consequence of the proinflammatory response is the re-cruitment of neutrophils to the site of PLA2 activation, and thisrecruitment is an immediate host response to P. aeruginosainfections (20, 31). The compounds identified from our screenwere tested for their abilities to provide protection of primaryperipheral blood neutrophils from lysis mediated by ExoU-expressing P. aeruginosa. Peripheral blood neutrophils wereinfected with strain PAK-VL1 opsonized with serum at anMOI of 10. In the absence of compounds, the neutrophils werelysed rapidly (Fig. 7B). In the presence of the known inhibitorMAFP, 60% of the neutrophils were protected from lysis (Fig.7B). Pseudolipasin A provided 40% protection at approxi-mately twice the concentration of MAFP (Fig. 7B). Theseresults indicate the possibility of these compounds and theirderivatives may act as therapeutic agents for in vivo infectionmodels.

DISCUSSION

In this study, we describe a cell-based assay for the detectionof inhibitors of mammalian cell killing by the cytotoxins deliv-ered by bacterial type III secretion systems. Since the assay isbased on cell viability in the presence of cytotoxin-producingbacteria, cytotoxic compounds were excluded. An additionalattractive feature of this screening format was the ability toidentify compounds that target individual components of acomplex pathway, beginning with the synthesis of the effectorsby the bacteria, their delivery, and their intracellular activity.The screen yielded several active compounds, some of whichwere structurally related. We then used additional assays todefine the specificity of the target of the compounds. A subsetof the compounds protected CHO cells against the lethal ac-tion of P. aeruginosa expressing ExoU, but not against anisogenic strain expressing ExoS, an unrelated cytotoxin alsodelivered by the type III secretion system (Fig. 2). We there-fore concluded that this group of molecules targeted ExoUfollowing its entry into CHO cells by interfering with its acti-vation or PLA2 enzymatic activity. The ability of these com-pounds to protect S. cerevisiae from killing by plasmid-encoded

FIG. 6. In vitro inhibition of ExoU PLA2 activity. (A) Inhibition ofExoU PLA2 activity in vitro by pseudolipasin A. Serial dilutions ofpseudolipasin A were added to a reaction mixture containing six-His-tagged ExoU (inset), CHO cellular extract, arachidonic TEPC sub-strate, and Ellman’s reagent (DTNB). ExoU cleavage of arachidonyl-TEPC results in generation of free thiol, which is detected by DTNBas an increase in the absorbance at 414 nm and reported as a percentageof ExoU PLA2 activity. (B) Inhibition of PLA2 cleavage of 14C-radio-labeled POPC-POPS mixed micelles. ExoU activity was determined byincreased mobility of cleaved 14C-labeled oleic acid by thin-layer chro-matography and reported as a percentage of ExoU PLA2 activity.



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TABLE 1. Activities of compounds similar to pseudolipasin A

Source Compound Structure % PLA2 activity a

at 200 �M% Yeast growthb

at 20 �M

ChemBridge 5633885 1.8 90.7

Maybridge BTB10979SC 81.8 0.2



ChemBridge 5635520 56.5 1.1

ChemBridge 5305847 57.0 8.9


Sigma S349925 62.9 0.7

Maybridge CC28302CB 103.6 0.6

Continued on following page



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ExoU confirmed this hypothesis (Fig. 5). Pseudolipasin A actsby inhibiting ExoU-catalyzed PLA2 activity in vitro.

Depending on the assay system used, the potency of pseudo-lipasin A compared to that of known inhibitors of PLA2 varied,and their activities in in vitro and in vivo assays did not alwayscorrelate well. A number of factors may contribute to theseobserved differences in pseudolipasin A activities. One of therate-limiting steps in the activity of an inhibitor active in thecytoplasm is efficient uptake and accumulation of sufficientinhibitory levels. Therefore, any differences in the permeabilityof PLA2 inhibitors in various cell types would be reflected intheir relative potencies, which would be significantly differentfrom those observed in in vitro enzymatic assays. Alternatively,different compounds could have different fates, depending ontheir interactions with various cellular constituents, leading tointerference with their inhibitory activities. Finally, we cannotexclude the possibility that the target of pseudolipasin A is theunknown cellular activator of ExoU, while the known PLA2

inhibitors very likely interfere directly with the enzymatic ac-tivity. Therefore, target specificity would also account for dif-ferent efficiencies of PLA2 inhibitors in various assays.

Identification of ExoU PLA2 inhibitors provides leads tothree areas of research: identification of novel functions forPLA2 enzymes, dissection of the enzymatic activity of ExoU,and development of novel therapeutics. An interesting obser-vation is that the reaction intermediate PLA2 inhibitor MAFPprevented D. discoideum from forming plaques on bacteria butdoes not affect the growth of the amoebae in liquid medium(V. T. Lee, unpublished data). Since MAFP is a potent inhib-

itor of ExoU-mediated cytotoxicity, the inability of D. dis-coideum to form plaques indicates that MAFP inhibits a PLA2, anarachidonate binding protein, or another off-target functionrequired for the phagocytosis of bacteria. A survey of phos-pholipase genes in the Saccharomyces genome database (6)and the D. discoideum database (dictyBase) (7) revealed onlyone PLA2 in yeast, which has been shown to be involved inmeiotic but not in haploid growth, whereas in D. discoideum noPLA2 could be identified. Data from this study confirm thatPLA2 activity is dispensable for S. cerevisiae growth (19). Thus,the MAFP-mediated inhibition of D. discoideum growth on abacterial lawn may be due to other arachidonate-utilizing pro-teins that could be inactivated by MAFP. A search of thedictyBase for arachidonate revealed a putative arachidonate12-lipoxygenase (lipA) that is transcriptionally induced in theearly response to Legionella infection (10). Further character-ization of lipA will reveal whether it is essential for phagocyticutilization of bacteria as a food source. Future experimentsdetermining the inhibitory target of MAFP that prevents thegrowth of D. discoideum on bacteria could reveal a novel func-tion of PLA2 in the phagocytic process.

Pseudolipasin A could be a useful tool for probing the mech-anism of the intracellular PLA2 activity of ExoU. Studies ofother PLA2 suggest that there are two sequential steps foractivity, binding to the aqueous-lipid interface to gain access tothe substrate followed by enzymatic cleavage of lipids along theinterface, which could also apply to ExoU (3). In the scenarioof type III secretion-delivered ExoU, the incoming toxin mayact on the lipids located around the injection needle, thus

TABLE 1—Continued

Source Compound Structure % PLA2 activity a

at 200 �M% Yeast growthb

at 20 �M

ChemBridge 5135308 41.0 �0.7

ChemBridge 5162800 66.2 1.6

ChemBridge 5768352 36.9 0.6

ChemBridge 6687996 52.7 3.8

a Calculated as a percentage relative to ExoU activity in the absence of inhibitors.b Calculated as a percentage relative to yeast growth in the absence of Cu2� induction of exoU expression.



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bypassing the requirement for interfacial binding. However,ExoU expressed directly with cells also results in cytotoxicity,suggesting that a cytosolic protein or a protein in the innerleaflet of the plasma membrane can recruit ExoU. Alterna-tively, the host activator could be required for activating thePLA2 activity of ExoU. One possible mechanism to account forthe inhibition of the PLA2 activity of ExoU and the lack of anydiscernible effect on eukaryotic PLA2 is that pseudolipasin A,in preventing either the recruitment or the activation of PLA2,interferes with the interaction of ExoU with this unknown hostfactor. A prediction from this proposed mechanism is thatpseudolipasin A is a noncompetitive allosteric inhibitor thatdoes not occupy the active site. Testing of this hypothesisawaits the identification of the host activator. An in vitro assaywith purified components will allow elucidation of the mecha-nism of PLA2 inhibition by pseudolipasin A and other ExoUinhibitors identified in this screen. In addition, the inhibitorsshould distinguish the function of the host factor in interfacialbinding or activation of PLA2 activity. Thus, pseudolipasin Arepresents a novel class of inhibitor that is specific for thephospholipase A2 activity of a bacterial virulence factor.

In analogy with recent work targeting virulence mechanismsin pathogenic bacteria (14), pseudolipasin A represents an-

other member of the emerging group of anti-infectives that donot kill or inhibit the growth of the microorganisms but insteadattenuate their virulence. P. aeruginosa has been shown torequire ExoU in a number of infection models, and expressionof ExoU correlates with severe outcomes of certain humaninfections. Pseudolipasin A may be a lead compound for atherapeutic agent, due to its specificity for the PLA2 activity ofthe ExoU toxin and its lack of identified effects on a variety ofeukaryotic cells. Increasing the potency of analogous com-pounds could provide suitable candidates for testing in animalmodels.

ACKNOWLEDGMENTS

V.T.L. was supported by a NIAID postdoctoral research fellowship.S.L., A.A.K., and J.H. were supported by NIH grant R21-HL079393.J.P.A. and E.K. were supported by NIH grant R01-HL070946. D.W.F.was supported by NIH grant R01-AI49577.

We thank Stephen Juris and Roger S. Smith for critical reading ofthe manuscript.

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