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GASTROENTEROLOGY 2004;126:488–498

igh-Molecular-Weight Polyethylene Glycol Prevents Lethalepsis Due to Intestinal Pseudomonas aeruginosa

ICHENG WU,* OLGA ZABORINA,* ALEX ZABORIN,* EUGENE B. CHANG,‡ MARK MUSCH,‡

HRISTOPHER HOLBROOK,* JAMES SHAPIRO,§ JERROLD R. TURNER,� GUOHUI WU,¶,#

A YEE C. LEE,¶,# and JOHN C. ALVERDY*epartments of *Surgery, ‡Medicine, §Biochemistry and Molecular Biology, �Pathology, and ¶Chemistry, and the #Institute for Biophysicalynamics, the James Franck Institute, University of Chicago, Chicago, Illinois

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ackground & Aims: During stress, erosion of protectiventestinal mucus occurs in association with adherence tond disruption of the intestinal epithelial barrier by in-ading opportunistic microbial pathogens. The aims ofhis study were to test the ability of a high-molecular-eight polyethylene glycol compound, polyethylene gly-ol 15–20, to protect the intestinal epithelium againsticrobial invasion during stress. Methods: The ability ofolyethylene glycol 15–20 to protect the intestinal epi-helium against the opportunistic pathogen Pseudomo-as aeruginosa was tested in cultured Caco-2 cells.acterial virulence gene expression, bacterial adher-nce, and transepithelial electrical resistance were ex-mined in response to apical inoculation of P. aerugi-osa onto Caco-2 cells. Complementary in vivo studiesere performed in a murine model of lethal sepsis dueo intestinal P. aeruginosa in which surgical stress (30%epatectomy) was combined with direct inoculation of. aeruginosa into the cecum. Results: High-molecular-eight polyethylene glycol (polyethylene glycol 15–20)onferred complete protection against the barrier-dys-egulating effects of P. aeruginosa in Caco-2 cells. Intes-inal application of polyethylene glycol 15–20 intressed mice protected against the lethal effects ofntestinal P. aeruginosa. Mechanisms of this effecteem to involve the ability of polyethylene glycol 15–20o distance P. aeruginosa from the intestinal epitheliumnd render it completely insensate to key environmentaltimuli that activate its virulence. Conclusions: High-olecular-weight polyethylene glycol has the potential

o function as a surrogate mucin within the intestinalract of a stressed host by inhibiting key interactivevents between colonizing microbes and their epithelialell targets.

he ability of the intestinal epithelium to resist theadhesion of invading microbes is generated, in large

art, by the presence of variously charged mucins com-rising the so-called unstirred water layer of the intesti-al mucosa. Recent studies on the biochemistry and

hysiology of intestinal mucins suggest that these com-ounds protect the intestinal epithelium against micro-ial invasion by affecting key physicochemical surfaceroperties of both the bacteria and epithelial cells suchhat a net repulsive force is generated.1 Under homeo-tatic conditions, many antagonistic and opposing forcesct to distance bacteria from the epithelial surface: thesenclude van der Waals forces, surface electric charge, andydrophobicity. To a large degree, various charged mu-ins within the unstirred water layer of the intestinalpithelium seem to play a key role in the governance ofuch forces.2 For example, the removal of intestinal mu-us with specific detergents results in significant adher-nce of indigenous bacteria to the intestinal epitheliumn association with a significant defect in intestinal per-eability.3 Similarly, the introduction of a systemic

tress, such as the injection of lipopolysaccharide3 orestraint stress,4 results in depletion of the mucus layeroupled with marked alterations in intestinal permeabil-ty and barrier function to invading indigenous bacteria.inally, an additional role for the protective effect ofucus against bacterial invasion of the intestinal epithe-

ium is suggested by the observation that preincubationf pathogenic strains of bacteria with intestinal mucusignificantly alters their surface characteristics and re-uces their ability to adhere to cultured intestinal epi-helial cells.5,6 Taken together, these studies stronglyuggest that stress-induced depletion of intestinal mucuslone could promote bacterial-mediated disorders of thentestinal tract by eroding an important component of

Abbreviations used in this paper: AFM, atomic force microscopy; cfu,olony-forming units; DIC, differential interference contrast; EGFP, en-anced green fluorescent protein; HSL, homoserine lactone; LDH,actate dehydrogenase; PA-I, Pseudomonas aeruginosa lectin/adhesin; TEER, transepithelial electrical resistance.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2003.11.011

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February 2004 POLYETHYLENE GLYCOL PREVENTS LETHAL SEPSIS 489

he innate intestinal defense system that has a uniqueunction within the host/pathogen interface.

Polyethylene glycol compounds (PEGs) may be ideallyuited to act as surrogate mucin compounds within thentestinal tract of a stressed host on the basis of theirydrophobic/hydrophilic properties. Depending on theirolecular weight, linear conformation, and concentra-

ion, PEGs can anchor to innate or living surfaces andxert major changes in surface electric charge, hydropho-icity, and van der Waals forces to approaching proteins.he precise mechanism for these effects is yet to be fullynderstood; however, PEG compounds display uniqueehavior on biological surfaces as the ability of theseolymers to adopt higher-order intrachain structureshanges their conformational freedom and, hence, theirepellant effect to approaching proteins.7 The completelynert and nontoxic nature of these compounds makeshem highly attractive as therapeutic agents to chemi-ally shield the intestinal epithelium during clinicalisorders in which epithelial barrier function to highlynvasive pathogens is compromised.

In this study, we sought to determine whether intralu-inal application of a high-molecular-weight PEG com-

ound (PEG 15–20 daltons) could functionally reestab-ish the mucus barrier of the intestinal epithelium androtect against a highly toxic and lethal opportunisticathogen expressing a high adherence phenotype underonditions of catabolic stress. Our laboratory has devel-ped a novel model of lethal gut-derived sepsis by ex-osing normal mice to catabolic stress and short-termtarvation (24–48 hours of H2O only) in combinationith the direct introduction of Pseudomonas aeruginosa

nto the cecum.8 This model results in clinically severeepsis and a 100% mortality rate at 48 hours. Lethalityue to intestinal P. aeruginosa in this model is dependentn the in vivo expression of a key virulence determinantn P. aeruginosa, the P. aeruginosa lectin/adhesin I (PA-I),hich facilitates adhesion of the organism to the intes-

inal epithelium. We have also shown that the PA-Iectin plays a major role in the pathogenesis of intestinal. aeruginosa by creating a defect in the intestinal epi-helial barrier to 2 lethal cytotoxins of this organism:xotoxin A and elastase.9 The critical role of PA-I ex-ression in lethal gut-derived sepsis due to intestinal P.eruginosa is suggested by our previous work showinghat null mutant strains of P. aeruginosa lacking PA-I areompletely apathogenic in this model. We proposed thathis model would be ideally suited to test the ability ofEG to prevent gut-derived sepsis caused by P. aerugi-osa as both a specific bacterial virulence marker and anpithelial barrier function that could be tracked through

he course of infection. Therefore, the aims of this studyere to determine the efficacy and mechanism of actionf high-molecular-weight PEG (PEG 15–20) as aethod to prevent lethal gut-derived sepsis in mice due

o intestinal P. aeruginosa.

Materials and MethodsBacterial Strains, Epithelial Cell Lines, andPolyethylene Glycol Solutions

P. aeruginosa strain 27853 (PA27853; American Typeulture Collection, Manassas, VA) is a nonmucoid clinical

train originally isolated from a blood culture. All experimentsere performed with Caco-2/C2bbe, or C2, cells (passage1–68) and were obtained as a generous gift of Dr. Markooseker (Yale University, New Haven, CT). C2 cells areaco-2 subclones that are highly differentiated intestinal epi-

helial cells.10 Two PEGs of different molecular weights wereurchased from Sigma (St. Louis, MO): PEG 3350 (catalog no.4338) and PEG 15,000–20,000 (catalog no. P2263). Theyre designated as PEG 3.35 and PEG 15–20, respectively.

PA27853/Enhanced Green FluorescentProtein and PA27853/PLL–EnhancedGreen Fluorescent Protein Constructs

To develop stable PA-I reporter strains in PA27853,trains were constructed to PA27853/PLL-enhanced green flu-rescent protein (EGFP) by using the E. coli shuttle vector asutlined11 in Figure 1. The final constructs were termedA27853/PLL-EGFP to denote them as constructs using theriginal strain PA27853 with the Promoter of the PA-I Lectinlus the PA-I Lectin gene itself fused to EGFP protein.

Mouse Model of Gut-Derived Sepsis

Female BALB/c mice were anesthetized and subjectedo hepatectomy as reported.7 All mouse experiments werepproved by the Animal Care and Use Committee of theniversity of Chicago. A 30% bloodless excision of the liver

long the floppy left lobe was performed by using a surgicallectrocautery. Control mice underwent manipulation of theiver without hepatectomy. In all mice, the cecal lumen wasccessed by direct needle puncture into the base. One milliliterf 0.9% NaCl, 5% PEG 3.35, or 2.5%–5% PEG 15–20 wasnjected retrograde into the small bowel. Next, 200 �L of 107

olony-forming units (cfu) per milliliter of PA27853 dilutedn 0.9% NaCl, 10% PEG 3.35, or 2.5%–10% PEG 15–20 wasnjected into the cecum through the same needle. The punc-ure site was tied off with a silk suture and swabbed withlcohol. Mice were returned to their cages, given H2O only,nd followed up for mortality for 48 hours. Reiterative exper-ments were performed after infection whereby mice (n � 5)ere gavaged with water, 5% PEG 3.35, or 5% PEG 15–20

750 �L per os via feeding tube) at 4 and 8 hours afterepatectomy and cecal injection of PA27853. Mice were al-

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490 WU ET AL. GASTROENTEROLOGY Vol. 126, No. 2

owed free access to water only and were then observed forigns of sepsis and mortality for 48 hours. Quantitative cul-ures of feces, vigorously washed cecal mucosa, liver, and bloodere performed on Pseudomonas isolation agar as previouslyescribed.8 Mice who seemed moribund were killed beforeatural death by exsanguination before the tissue harvest.

PA-I Expression In Vivo

Reporter strain PA27853/PLL–EGFP was used in theouse model of gut-derived sepsis to follow the expression ofA-I in vivo. Two hundred microliters of 106 cfu per milliliter

igure 1. Construction of PA-I reporter plasmid pUCP24/PLL–EGFP.A) The entire PA-I gene and upstream regulator region in P. aerugi-osa strain PA27853. A lux box–type element of quorum-sensingignaling system and RpoS (�S) consensus are shown. (B) Fusiononstruct: the pll region (440 base pairs [bp] upstream plus 366 bpf the entire PA-I [minus stop codon]) was amplified from PA27853enomic DNA. The 440 bp upstream included the C-terminal ofRNALeu followed by the regulator region of PA-I. The restriction sitespnI and XbaI were inserted by using the following respective primers:

orward, 5�-GGTACCCCGGTTCGACCCCGGCTCCGG-3�; and reverse, 5�-CTAGAGGACTGATCCTTTCCAATAT-3�. The Egfp gene coded for greenuorescent protein was amplified with the pBI-EGFP plasmid (Clon-ech, Palo Alto, CA) as a template. XbaI and PstI restriction sites werentroduced by using the following respective primers: forward, 5�-CTAGAACTAGTGGATCCCCGCGGATG-3�; and reverse, 5�-GCAGACTAG-TCGACAAGCTTGATATC-3�. pll was fused with egfp via the XbaI re-triction site and ligated into pUCP24 digested with KpnI and PstI toreate pUCP24/PLL–EGFP. Reporter plasmid pUCP24/PLL–EGFP waslectroporated into PA27853 electrocompetent cells to create re-orter strain PA27853/PLL–EGFP. PA27853/PLL–EGFP constructsere selected on the basis of gentamicin (100 �g/mL) resistance anduorescence induced by the cognate quorum-sensing signaling mole-ule for PA-I, C4-homoserine lactone (C4-HSL; 0.1 mmol/L). (C)A27853/PLL–EGFP grown in the presence of C4-HSL showed signif-

cant fluorescence. (D) C4-HSL concentration-dependent induction ofA-I expression estimated by Northern blot and immunoblot.

f PA27853/PLL–EGFP was introduced into the cecum ofham-operated and 30% hepatectomy mice with or without0% PEGs. After 24 hours, the mice were killed, and cecalontents (feces) were collected and mixed into a crude liquid/articulate fraction in phosphate-buffered saline (PBS). Theiquid portion of the mixture was harvested and spun at 5000gor 10 minutes. The cecum was wrapped with OCT mediumIMEB, Sakura, Japan), frozen in liquid nitrogen, and cryosec-ioned at 5-�m intervals. The processed fecal samples and cecalucosal sections were viewed under a Zeiss Axioplan fluores-

ence microscope (Thornwood, NY) by using differential in-erference contrast (DIC) and GFP filters. To quantitate fluo-escence of the processed fecal samples, samples wereesuspended in sonication buffer (50 mmol/L NaH2PO4, 10mol/L Tris HCl, pH 8.0, and 200 mmol/L NaCl) and

isrupted with a Sonic Dismembrator (Fisher, Chicago, IL).ebris was removed by centrifugation while the fluorescence of

he cytosolic fraction was measured with a Versafluor fluorom-ter (Bio-Rad, Hercules, CA). Protein concentration was rou-inely measured with the micro-bicinchoninic acid methodPierce, Rockford, IL). Fluorescence was expressed as relativeuorescence units per microgram of protein.

Bacterial Adherence Assay

The number of bacteria that were associated with thepithelial monolayers was determined by using a previouslyublished method.12 Briefly, overnight cultures of P. aeruginosaere inoculated at 1:1000 dilution with or without 10% PEGsnto confluent Caco-2 cells and cocultured for 4 hours (37°C;% CO2). Media were completely removed from the Caco-2ell culture wells, and P. aeruginosa colony counts were deter-ined by plating 10-fold dilutions on Pseudomonas isolation

gar. Next, Caco-2 cells were washed with PBS twice andcraped into centrifuge tubes followed by extensive washingith PBS, spun down at 900g for 10 minutes, and homoge-ized in 0.5 mL of PBS. The number of adherent P. aeruginosaas determined by quantitative culture on Pseudomonas isola-

ion agar. Colony counts were normalized to the protein con-entration of homogenized Caco-2 cells. The total amount of. aeruginosa was determined by collecting and combiningoth media and scrapped Caco-2 cells after co-incubation withA27853.

Analysis of PA-I Expression In Vitro

Northern hybridization for PA-I messenger RNAmRNA) was measured as previously described.9 PA-I proteinas measured by Western blotting as previously described.9

eporter strain PA27853/PLL–EGFP was used to quantitateA-I expression by measuring the expression of the reporterene EGFP. After they were grown in the presence of C4-omoserine lactone (HSL) and PEGs, PA27853/PLL–EGFPacteria were washed with PBS and disrupted by sonication,nd fluorescence of the cytosol fraction was measured as de-cribed previously.

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Influence of Polyethylene Glycol on GrowthCharacteristics and Membrane Integrity ofPseudomonas aeruginosa

Growth curves for PA27853 were generated by plating0-fold dilutions of samples taken at different points of growthn the absence and presence of 10% PEGs. The activity of theousekeeping enzyme lactate dehydrogenase (LDH) was mea-ured with a coupled diaphorase enzymatic assay by using aubstrate mix from CytoTox 96 (Promega, Madison, WI).

Atomic Force Microscopy

PA27853 was cultured in tryptic soy broth with orithout 10% PEGs for 4 hours. Cells were extensively washedith PBS, and 1 drop of the bacterial suspension was dried on

he top of mica in blowing air for 10 minutes and imagedmmediately. Imaging of the dried bacteria with tapping modetomic force microscopy (AFM) was performed in air with aultimode Nanoscope IIIA Scanning Probe Microscope

MMAFM; Digital Instruments, Woodbury, NY). Monolay-red Caco-2 cells were treated with 10% PEGs for 4 hours andashed with PBS extensively. Imaging of the cells was per-

ormed in PBS by AFM without using an O-ring.

Electron Microscopy

PA27853 was grown overnight in tryptic soy brothith or without C4-HSL 1 mmol/L and 10% PEGs. Cells were

igure 2. Mortality rates in mice at 48 hours subjected to either shaA27853 into the cecum. Mice underwent a 30% bloodless left lobef PA27853 (n � 7 per group). Control mice underwent sham laparoA) Protective effect of PEG 15–20 on mortality in mice after hepatectB) Time course of mortality in mice undergoing hepatectomy and treaffect of PEG 15–20. (D) Quantitation of bacterial cultures in cecal cofter 30% surgical hepatectomy and direct cecal injection of PA278tatistically significant increase in bacterial counts in the cecum, liver,†P � 0.05) in liver and blood bacterial counts was observed in miceith mice undergoing hepatectomy without PEG treatment (Hep). (Edministered as an oral solution 4 and 8 hours after infection protecte.01; Fisher exact test).

iluted in NaCl 0.5 mol/L at 1:100 and stained with uranylcetate before examination with a Philips CM 120 electronicroscope (Tallahassee, FL).

The Effect of Polyethylene Glycol Solutionson the Dispersion/Clumping Pattern ofPseudomonas aeruginosa

PA27853/EGFP was used in the following experi-ent. One hundred microliters of PA27853/EGFP, withGFP expressed under the Plac by 0.5 mmol/L isopropylthio-alactoside, was mixed with 1 mL of Caco-2 cell medium withr without 10% PEGs and poured into a 0.15-mm-thick dTC3ish (Bioptech, Bulter, PA) with or without monolayeredaco-2 cells. The dispersion/clumping pattern of live bacterialells was monitored with an Axiovert 100 TV fluorescence-nverted microscope (Zeiss) with DIC and GFP filters.13 The-dimensional imaging software (Slidebook) from Intelligentmaging Innovations (Denver, CO) was used to image theispersion pattern in the z plane by using the GFP filter.

ResultsPEG 15–20 protects against lethal gut-derived

epsis due to P. aeruginosa in mice after 30% hepatec-omy. Direct cecal injection of PA27853 into mice whonderwent a 30% surgical hepatectomy resulted in a

arotomy or 30% surgical hepatectomy followed by direct injection ofectomy (Hep) that was immediately followed by direct cecal injectionfollowed by injection of equal amounts of PA27853 into the cecum.nd direct cecal injection of PA27853 (P � 0.001; Fisher exact test).ith PEG 3.35 or PEG 15–20. (C) Concentration-dependent protectivets (feces), washed cecal mucosa (cecum), liver, and blood 24 hoursll data are mean � SEM. Sum-rank analysis of variance showed a

blood of mice after hepatectomy (*P � 0.001). A significant decreasehepatectomy treated with PEG 15–20 (Hep � PEG 15–20) comparedeffect of PEG rescue therapy on mortality after hepatectomy. PEGinst mortality after hepatectomy and cecal injection of PA27853 (P �

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tate of clinical sepsis with no survivors at 48 hoursFigure 2A). Mice undergoing sham laparotomy withoutepatectomy (controls), similarly injected with P. aerugi-osa, survived completely without any clinical signs ofepsis (Figure 2A). To determine the ability of PEGolutions to prevent mortality in this model, PA27853ells were suspended in one of two 10% (wt/vol) solu-ions of PEG (PEG 3.35 or PEG 15–20). PEG 3.35 washosen because it represents the molecular weight ofEGs that are available for clinical use as intestinalleansing agents (GoLYTELY; Braintree Inc., Braintree,A).14 We compared PEG 3.35 with high-molecular-eight PEG solutions (15–20 kilodaltons) because sim-

lar molecular weight PEGs have been reported to have arotective effect on the intestine by an unknown mech-nism before transplantation.15 Two hundred microlitersf the bacterial/PEG suspension was introduced into theecum by direct puncture. PEG 15–20 was completelyrotective against mortality in mice after hepatectomy,hereas PEG 3.35 delayed the onset of mortality, it hado protective effect at the 48-hour time point (Figure 2And B). Mice treated with PEG 3.35 seemed septicruffled fur, chromodacryorrhea, and loose stools) beforeeath, whereas mice treated with PEG 15–20 seemedompletely healthy at 48 hours. Mice treated with theEG solutions did not develop diarrhea. Mice undergo-

ng hepatectomy that drank only water expelled 7–11ecal pellets in 24 hours. As mice became septic, theirtools became loose yet formed in pellets, and they didot develop diarrhea. PEG-treated mice had a smallncrease in stool frequency (compared with hepatectomyithout PEG) to 14–21 pellets per 24 hours for PEG.35 and 18–25 pellets per 24 hours for PEG 15–20.Dose–response experiments showed a 5% solution to

e the minimal concentration of PEG 15–20 that wasrotective in this model (Figure 2C). Hepatectomy in-reased the adherence of PA27853 to the cecal mucosand its translocation to the liver and blood. Only PEG5–20 decreased the translocation of PA27853 to theiver and blood, although neither PEG 3.35 nor PEG5–20 decreased adherence of PA27853 to the cecum asssessed by quantitative culture alone (Figure 2D). Be-ause these studies were performed by administeringEG directly into the cecum and small bowel simulta-eously with the infectious inoculum, we sought toetermine whether PEG could also protect mice when itas administered orally after the initial infection, asight occur clinically. Results from the rescue experi-ents, in which mice were gavaged with water, 5% PEG

.35, or 5% PEG 15–20 at 4 and 8 hours after hepatec-omy and cecal injection of PA27853, showed that 5 of

mice survived after oral gavage with PEG 15–20 andeemed completely healthy at the 48-hour time point.nly 1 of 5 mice survived in the PEG 3.35–treatedroup and 0 of 5 in the water-only–treated group (FigureE). All mice in these 2 groups seemed septic. Cultureesults of tissues were similar to the previous set ofxperiments and showed that orally gavaged PEG 15–20ttenuated the magnitude of P. aeruginosa disseminationo the liver and blood (data not shown).

PEG 15–20 inhibits virulence expression of P. aerugi-osa in response to the quorum-sensing signaling mole-ule C4-HSL. A diverse array of virulence factors in P.eruginosa is released in response to molecules of theuorum-sensing signaling system (for review, see Fuquand Greenberg16). In particular, PA-I expression in P.eruginosa is regulated by the transcriptional regulatorhamnolipids regulatory gene (rhlR) and its cognate ac-ivator C4-HSL.17 We have previously shown that expo-

igure 3. The inhibitory effect of PEGs on C4-HSL–induced PA-I ex-ression. (A) Immunoblot. (i) Exposure of PA27853 to 1 mmol/L ofhe quorum-sensing signaling molecule C4-HSL resulted in a statisti-ally significant increase (*P � 0.00; 1-way analysis of variance) inA-I protein expression that was significantly inhibited (†‡P � 0.01s. HSL) in the presence of both 10% PEG 3.35 and 10% PEG 15–20,lthough to a greater degree with PEG 15–20. (ii) The minimum

nhibitory concentration of PEG 15–20 on C4-HSL–induced PA-I ex-ression was 5% (*P � 0.001; analysis of variance). (B) Northernlot. Exposure of PA27853 to 0.1 mmol/L of C4-HSL resulted in atatistically significant increase (*P � 0.001; analysis of variance) inA-I mRNA expression that was inhibited with 10% PEG 15–20 (†P �.01 vs. HSL). (C) Effect of PEGs on the expression of the reportertrain PL-EGFP/27853. Corroborative experiments showed that PL-GFP/27853 was also insensitive to C4-HSL–induced fluorescence inhe presence of PEG 15–20 in overnight experiments. All data areean � SEM.

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ure of PA27853 to C4-HSL results in a significantncrease in PA-I expression.18 Inhibition of both PA-Irotein and PA-I mRNA was observed in PA27853xposed to C4-HSL in the presence of 10% PEG 15–20Figure 3Ai and B). A significant inhibitory effect waslso seen with PEG 3.35, although this seemed less at theevel of mRNA. The minimum concentration of PEG5–20 that inhibited C4-HSL–induced expression ofA-I protein was 5% (Figure 3Aii). The ability of PEG5–20 to also render PA27853/PLL–EGFP unresponsiveo C4-HSL was seen in overnight experiments (FigureC).

PEG 15–20 inhibits the virulence expression of P.eruginosa in response to cultured intestinal epithelialells and protects epithelial cells against P. aeruginosa–nduced barrier disruption. Exposure of PA27853 toaco-2 cells resulted in expression of PA-I mRNAithin 4 hours of incubation (Figure 4A). This effect was

ignificantly inhibited in the presence of PEG 15–20Figure 4A). As previously reported, PA27853 resultedn a profound decrease in the transepithelial electricalesistance (TEER) of confluent Caco-2 cells.7,8 This effectas significantly attenuated in the presence of PEG5–20, but not PEG 3.35 (Figure 4B).

PEG solutions do not affect the growth pattern of P.eruginosa. Growth curves for PA27853 grown overnightn the presence of PEGs showed that bacterial quantityas unaffected by the presence of either PEG solution

Figure 5). The activity of a housekeeping enzyme in-olved in energy metabolism, LDH, was measured at thearious time points from the exponential to the station-

igure 4. The effect of PEGs on PA-I expression induced by co-ncubation with Caco-2 cells and their effect on P. aeruginosa–in-uced disruption of Caco-2 cell barrier function (TEER). (A) Caco-2ells caused an increase in PA-I mRNA (*P � 0.001; analysis ofariance) after a 4-hour exposure to bacterial cells of PA27853. PEG5–20 resulted in a significant attenuation (†P � 0.001; 1-way anal-sis of variance) in Caco-2 cell–induced PA-I mRNA. (B) Apical inocu-ation of P. aeruginosa (PA27853 ) onto Caco-2 cells caused a signif-cant (*P � 0.001) decrease in TEER. This effect was significantlyttenuated (†P � 0.01 vs. PA27853) in the presence of PEG 15–20.ll data are mean � SEM of triplicate cultures (n � 7).

ry phase of growth. No change in LDH activity inytosolic extracts of PA27853 grown in the presence ofEGs was observed (data not shown).Electron microscopy and AFM showed a distinct coat-

ng effect of PEG solutions on P. aeruginosa bacterialells. Overnight exposure of PA27853 to C4-HSL 1mol/L changed the appearance of bacterial cells from

hat of a typical convoluted and rugged surface (FigureA; control) to a cellular appearance showing a flaturface without visible surface convolutions (Figure 6A;SL). Bacteria exposed to overnight PEG 15–20 re-

ained a morphological picture similar to that of strainsot exposed to C4-HSL (Figure 6A; PEG 15–20). Aisible electron halo was seen (arrow) around the surfacef the bacterium in the presence of PEG 15–20. AFM ofacterial cell surfaces showed topographic changes atanometer scales in the presence of the 2 polymers. PEG.35 created a smooth, uniform surface on bacterial cells,hereas the PEG 15–20 polymer appeared rugged on the

urface of the bacterial cell (Figure 6Biii). Measurementsf the height deflection of the polymer/bacterial surfacey AFM showed an increase in the height of the bacte-ial/polymer surface with PEG 15–20 compared withEG 3.35 (Figure 6C).PEG 15–20 affects the clumping behavior of P. aerugi-

osa and its spatial relationship to the apical surface ofultured human intestinal epithelial cells. In medialone, bacteria are seen uniformly dispersed as planktonic. aeruginosa cells on DIC and GFP images (Figure 7And B). However, in the presence of Caco-2 cells, bacte-ial cells in media alone were adherent to the epithelialonolayers (Figure 7C). In the presence of 10% PEG

.35, however, bacteria formed large mushroom-shapedlumps (Figure 7A) and adhered to the bottom of theell (Figure 7B). In the presence of Caco-2 cells, bacteria

igure 5. The effect of PEG solutions on growth of PA27853.A27853 growth curves appeared identical in the presence of both0% PEG solutions when compared with PEG-free tryptic soy brothedium. All data are mean � SEM.

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xposed to 10% PEG 3.35 were clumped and suspendedn the order of 8 �m above the plane of the epithelialells (Figure 7C). A similar, yet distinct, pattern was seenith PEG 15–20. For the first 0.5–1 hours of incubation,acterial cells formed spider-shaped microclumps thatere close to the bottom of the well (Figure 7A and B).ithin several hours, spider leg–shaped microclumps

ccupied the entire space/volume of the medium (nothown). When co-incubated with Caco-2 cells, P. aerugi-osa cells were elevated high above the plane of thepithelium (30–40 �m) in the presence of 10% PEG5–20 (Figure 7C).

Experiments in which bacterial adhesion to Caco-2ells was assessed as a ratio of adherent to nonadherentA27853 cells showed that PEG 15–20 resulted in areater concentration of bacteria remaining in the cultureedia. No difference in bacteria adherent to Caco-2 cellsas observed in these experiments (Table 1).To determine whether the expected up-regulation of

A-I in P. aeruginosa introduced into the cecum of micendergoing hepatectomy could be suppressed in theresence of PEG solutions, the reporter strain PA27853/LL–EGFP was injected into the cecum of mice afterurgical hepatectomy and retrieved 24 hours later byarvesting and processing cecal contents (feces) and cecalucosal tissues. Hepatectomy resulted in an increase in

igure 6. (A) Electron microscopy of individual bacteria cells exposedf bacterial cells to C4-HSL showed a loss of transparent surface convells. Electron microscopy of bacterial cells exposed to 10% PEG 1onvoluted surface, similar to control cells. In addition, PEG 15–20 creo determine the nature of this halo effect, atomic force microscopicroscopy (AFM). Besides recording topographic images, AFM generaeflection is recorded as a function of its vertical displacement in nand in PEG 15–20 (iii) show distinct topographic images and height fl.01; analysis of variance) off the bacterial/polymer surface. Data a

A-I expression in both luminal bacteria (Figure 8A and) and mucosally associated bacteria (Figure 8C). PEG5–20 inhibited fluorescence in PA27853/PLL–EGFP inoth luminal and mucosally associated bacteria (FigureA–C).

DiscussionIn this study, the ability of PEG 15–20 to protect

he intestinal epithelium against an opportunistic colo-izing pathogen was assessed. Specifically, the ability ofEG to inactivate the lethal effects of intestinal P. aerugi-osa, one of the more common causes of fatal gut-derivedepsis in an immunocompromised host, was tested.19–21

ata from this show that there are striking effects of

-HSL in the presence and absence of 10% PEGs. Overnight exposurens typically seen in normal gram-negative bacteria as seen in control

, but not 10% PEG 3.35, showed retention of this transparent andan electron halo effect around the perimeter of bacterial cells (arrow).s used to examine bacterial cells. (C) Mechanism of atomic forcerce–distance measurements at a given x, y location as the cantileverters. (B) AFM images of PA27853 without PEG (i), in PEG 3.35 (ii ),

ations. (D) PEG 15–20 shows the highest height of deflection (*P �an � SEM for multiple observations.

able 1. Bacterial Counts (mean � SD) in Media and inCaco-2 Cell Preparations After Apical Exposure ofCell Cultures to 107 cfu/mL of P. aeruginosa27853 for 4 Hours

Group(n � 5)

Total(log cfu/mLa)

Media(log cfu/mL)

Caco-2 cells(log cfu/mLa)

ontrol 6.87 � 0.16 5.43 � 0.17 6.15 � 0.74EG 3.35 6.94 � 0.21 5.75 � 0.17 6.46 � 0.60EG 15-20 7.05 � 0.12 6.56 � 0.30b 6.76 � 0.70

Values adjusted to 100 mg protein.P � 0.05 compared with controls.

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EG 15–20 that parallel the function of intestinal mu-us. Because intestinal mucus exerts its effects on botholonizing bacteria and the intestinal epithelium, both ofhese compartments were examined in some detail. Sim-lar to the reported effects of intestinal mucus on bacte-ial growth, we did not observe PEGs to have any effectn bacterial growth patterns.22 However, like intestinalucins, PEG compounds affected the clumping pattern

f P. aeruginosa similarly to that observed for Escherichiaoli exposed to mouse cecal mucus.20 Mucus-inducedacterial clumping has been shown to be a major mech-nism by which intestinal colonization by luminal patho-ens is prevented.20 In fact, mutanized bacterial strainshat can resist mucus-induced clumping have beenhown to colonize and invade the mouse intestinal epi-helium more efficiently than their parent strains.20

A major mechanism by which PEG 15–20 was pro-ective in these studies seemed to be by simply distanc-ng P. aeruginosa from the intestinal epithelium. A lack ofacterial contact to the epithelial Caco-2 monolayers inhe presence of PEG 15–20 was suggested from experi-ents that showed preservation of the TEER of theaco-2 monolayers exposed to PA27853. That PEG5–20 prevented a contact-induced decrease in TEERfter apical exposure of Caco-2 cells to P. aeruginosa was

igure 7. The effect of PEG solutions on bacterial clumping patterns ashows overhead images of bacteria grown in Caco-2 cell high-glucosshow z-plane reconstructions of multiple stacked images of PA278

olumn A show that PEG 3.35 induces large clump formation in bacterluorescent images in column B show that in media alone without Cachereas in the presence of PEG 3.35 or PEG 15–20, bacteria form cresence of the Caco-2 cells changes the clumping pattern and spatells (column B). In column C, when bacteria are in media alone, theell surface. In the presence of PEG 3.35, bacteria are again seen cpproximately 8 �m. When bacteria are exposed to PEG 15–20 in thistributed about the well and remain suspended as much as 32 �m

rst suggested by the finding that counts of nonadherentacteria remaining in the Caco-2 cell free culture mediaere 10-fold higher in the presence of PEG 15–20,

lthough bacterial adherence assessed by culture ofashed cells was not increased, perhaps because of an

nability to wash PEG off of the bacterial/PEG/epithelialell layer. Nonetheless, fluorescent bacterial images ofA27853/EGFP did show that bacteria are significantlyistanced away from Caco-2 cell monolayers exposed toEG 15–20 to a greater degree than that seen with PEG.35, raising the possibility that a critical spatial inter-ace exists between pathogen and host beyond whichacterial pathogenicity does not occur. The importancef assessing these spatial differences beyond bacterialulture may be essential to understanding such events.thers have shown that polymers such as PEGs can exertrepellant or distancing effect to approaching variously

harged proteins by undergoing a change in their con-ormational entropy in response to contact onto a bio-ogical surface.23 The anchoring of PEG 15–20 to bothaco-2 and PA27853 cell surfaces could impose molec-lar constraints on PEG compounds and result in a lossf elasticity or conformational freedom of the polymer.24

lternatively, coating of both epithelial and bacterialells with PEG 15–20 could lead to an alteration in the

atial orientation of bacteria in relation to Caco-2 cell surface. Columnuglas minimum Eagels media without epithelial cells. Columns B andGFP in the absence (B) and presence (C) of Caco-2 cells. Images inereas PEG 15–20 induces a distinct microclumping pattern (arrows).ells, bacteria remain uniformly dispersed throughout the culture well,and fall to the bottom of the culture well. Column C shows that the

ientation of bacteria compared with bacterial images without Caco-2clumps and fall to the bottom of the wells attaching to the Caco-2

ed, but they remain suspended above the epithelial cell surface atsence of Caco-2 cells, they seem more dispersed and more evenlyve the epithelial cell surface.

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urface electrical charge or hydrophobic characteristics ofoth biological bodies such that a net repulsive force isenerated in response to like charges. Evidence for thisotion can be seen in Figure 7, in which PEG solutionslone do not suspend bacteria above the bottom of theulture wells, whereas in the presence of Caco-2 cells,acteria are seen suspended above the bottom of theulture wells. Collectively, these findings suggest atrong interactive component to the repulsive force ofEGs that seems to be activated when present on Caco-2ells.

To better understand such forces, both electron mi-roscopy and AFM were used to characterize the surfaceorphology of bacterial cells in the presence of the 2EG molecules. AFM force measurements make it pos-ible to probe the physical properties of the surface of liveells with nanometer resolution25 (Figure 6). AFM scan-ing of bacterial cells showed a significant increase in the

igure 8. The effect of PEG 15–20 on PA-I expression in vivo. Reporteresence and absence of PEG 3.35 and PEG 15–20 after hepatectoarvested, processed, and imaged with fluorescence microscopy. Fluof cytosolic fractions of bacterial cells. (A) Overlays of images of bacEG 15–20 in the mouse cecum protected against PA-I expression instatistically significant increase (*P � 0.05; analysis of variance) af

he presence of PEG 15–20 (†P � 0.05; analysis of variance). (C) Oveepatectomy had visibly fluorescent bacteria on and within the mucosaere pretreated with PEG 15–20. Pretreatment with PEG 3.35 didepatectomy.

eight diameter of bacterial cells in the presence of PEG5–20, an effect that appeared as an electron halo aroundhe bacteria by electron microscopy. Although thesendings are preliminary, a molecular weight–dependentffect on bacterial cell surface properties seems to be aistinct feature of PEG 15–20. The extent to which thesehanges direct the behavior and response of bacteriaithin the intestinal epithelial environment will requireore detailed study.A second major mechanism by which PEG might have

een protective in this model was by inhibition of bac-erial virulence gene expression. In this study, PEG5–20 rendered P. aeruginosa insensate to both C4-HSLnd epithelial cells, as judged by a lack of PA-I expres-ion. Although the PA-I lectin is among the manyirulence determinants necessary to initiate epithelialnfection, its co-regulation with other virulence genes inesponse to quorum-sensing signaling molecules makes

in PA27853/PLL–EGFP was introduced into the mouse cecum in theMice were killed after 24 hours, and feces and cecal tissues werence was quantified in processed fecal samples (n � 3) by fluorometryharvested from the cecum from the various groups. The presence of. (B) Quantitation of PA-I expression in feces by fluorometry showingepatectomy. All data are mean � SEM. This effect was attenuated inof images of cecal tissues from the various groups. Mice undergoing

orescent bacteria were not seen on or within cecal tissues when miceprevent PA27853/PLL–EGFP from fluorescing in mice undergoing

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t an appropriate “read-out” for virulence gene activationn P. aeruginosa. The finding that PEGs can desensitize P.eruginosa to its cognate quorum-sensing molecules isignificant, given that quorum sensing controls hundredsf genes that encode for the toxicity of this organismgainst the host.17,26 Mutant strains of P. aeruginosa thatannot communicate via the quorum-sensing signalingystem are seriously impaired in their ability to causenfection in animal models.27 Yet only PEG 15–20 sup-ressed PA-I expression in vivo and protected againstortality in mice subjected to catabolic stress. Data from

igure 8 show that there are yet-to-be-described factorseleased into the intestinal lumen after catabolic stresshat activate PA-I expression in P. aeruginosa. The extento which PEG 3.35 and PEG 15–20 can shield P.eruginosa from these inducing molecules and suppressA-I expression may not be able to be assessed by in vitroxperiments alone. For example, PEG 3.35 inhibitedA-I expression in response to C4-HSL but did notrotect against Caco-2 cell–induced PA-I expression,nlike PEG 15–20, which protected against both effects.owever, PEG 3.35 maintained a significant distancing

ffect on P. aeruginosa from the epithelial surface (Figure), although this effect was not to the same degree as thateen with PEG 15–20. Because it is well established thatost-cell contact alone is capable of inducing bacterialirulence gene expression,28,29 the greater ability of PEG5–20 to distance P. aeruginosa from the epithelium, asbserved in the in vitro experiments, may have played aignificant role in suppressing PA-I expression in vivo, aseen in the experiments in Figure 8. Therefore, althoughEG 3.35 did have some effects in vitro, its inability touppress PA-I expression in vivo may be a critical factorn its inability to protect mice. Finally, major differencesn the rate by which PEG compounds are degraded inivo because of the presence of the commensal flora couldxplain some of the discordances between in vitro and inivo experiments.30 Studies are under way to clarify thisssue.

The use of PEG 15–20 as an intestinally appliedompound to prevent gut-derived sepsis in immunocom-romised individuals harboring opportunistic pathogensay have clinical merit. In this mouse model, PEG

5–20 was protective against fatal gut-derived sepsis dueo P. aeruginosa when directly introduced into the cecumt the time of infection or when administered as an oralolution after infection. Although other biological effectsf PEG 15–20 on the intestinal epithelium—such as itsbility to stimulate endogenous mucus or other intestinalefense proteins—may exist, its ability to chemicallyhield the intestinal epithelium may offer a novel ap-

roach to bacterial-mediated disorders of the intestinalract. In addition, whether PEG 15–20 affects probioticacteria will require further study. It will be importanto specifically address the role of endogenous mucus onhe protective effect of PEG, because many of the ob-erved effects closely mimic those of intestinal mucins.

e have administered PEG 15–20 orally to mice for upo 4 weeks without any noticeable change in weight orverall health (data not shown). In addition, PEG 15–20oes not change the Na/H exchange characteristics ofultured Caco-2 cells (data not shown). The lack of anffect of PEG 15–20 on P. aeruginosa growth and viabil-ty may offer a more ecologically neutral approach toontaining virulent opportunistic pathogens in immuno-ompromised hosts over the current approach of multiplend prolonged antibiotic use.31,32 Given the completelynert and nontoxic nature of PEGs, the use of PEGompounds to treat and prevent bacterial-mediated dis-rders of the intestinal epithelium deserves further in-estigation.

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Received July 18, 2003. Accepted October 23, 2003.Address requests for reprints to: John Alverdy, M.D., F.A.C.S., De-artment of Surgery, Center for Surgical Infection Research, Universityf Chicago, Pritzker School of Medicine, 5841 S. Maryland, MC 6090,hicago, Illinois 60637. e-mail: [email protected];ax: (773) 834-0201.Supported by National Institutes of Health Grants RO1 GM62344-01

to J.C.A.) and DK619131 (to J.R.T.), Digestive Diseases Research Coreenter Grant DK 47722 (to E.B.C.), the David and Lucile Pachardoundation Grant 99-1465 (to K.Y.C.L.), and the University of Chicagoaterials Research Science and Engineering Centers Program of theational Science Foundation under Award DMR 9808595 (to K.Y.C.L.).The authors thank Dr. Klaus Winzer and Dr. Paul Williams, Universityf Nottingham, for helpful technical advice; Raphael Lee, M.D., Sc.D.,irector of the Center for Molecular Repair, University of Chicago, andilliam Cromie, M.D., for helpful conversations; and Shirley Bond,ancer Center Digital Light Microscopy Facility at the University ofhicago, for technical assistance. The Escherichia coli/Pseudomonaseruginosa shuttle vector pUCP24 was a gift of Dr. H. P. Schweizer,olorado State University, Fort Collins, Colorado. G.W. is grateful forhe support of the Burroughs Wellcome Cross Disciplinary Trainingrogram.
L.W. and O.Z. contributed equally to this work.

High-molecular-weight polyethylene glycol prevents lethal sepsis due to intestinal Pseudomonas...

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