The B Subunit of an AB5 Toxin Produced by Salmonella entericaSerovar Typhi Up-Regulates Chemokines, Cytokines, and AdhesionMolecules in Human Macrophage, Colonic Epithelial, and BrainMicrovascular Endothelial Cell Lines

Hui Wang,a James C. Paton,a Brock P. Herdman,a Trisha J. Rogers,a Travis Beddoe,b Adrienne W. Patona

Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australiaa; Department of Biochemistry andMolecular Biology, Monash University, Clayton, Victoria, Australiab

The principal function of bacterial AB5 toxin B subunits is to interact with glycan receptors on the surfaces of target cells andmediate the internalization of holotoxin. However, B subunit-receptor interactions also have the potential to impact cell signal-ing pathways and, in so doing, contribute to pathogenesis independently of the catalytic (toxic) A subunits. Various Salmonellaenterica serovars, including Salmonella enterica serovar Typhi, encode an AB5 toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis toxin. However, although the A subunit is able to catalyze ADP-ribosyla-tion of host G proteins, a cytotoxic phenotype has yet to be identified for the holotoxin. We therefore examined the capacity ofthe purified B subunit (ArtB) from S. Typhi to elicit cytokine, chemokine, and adhesion molecule responses in human macro-phage (U937), colonic epithelial (HCT-8) cell, and brain microvascular endothelial cell (HBMEC) lines. Secretion of the chemo-kines monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) was increased in all three tested cell lines, with macro-phage inflammatory protein 1� (MIP-1�), MIP-1�, and granulocyte colony-stimulating factor (G-CSF) also significantlyincreased in U937 cells. ArtB also upregulated the cytokines tumor necrosis factor alpha (TNF-�) and IL-6 in HBMECs andHCT-8 cells, but not in U937 cells, while intercellular adhesion molecule 1 (ICAM-1) was upregulated in HCT-8 and U937 cellsand vascular cell adhesion molecule 1 (VCAM-1) was upregulated in HBMECs. Thus, ArtB may contribute to pathogenesis inde-pendently of the A subunit by promoting and maintaining a strong inflammatory response at the site of infection.

Bacterial AB5 toxins are so termed because they comprise acatalytic A subunit noncovalently linked to a pentameric B

subunit. They exert their effects in a two-step process: first, the Bsubunit pentamer binds to specific glycan receptors on the cellsurface, triggering uptake of the holotoxin; this is followed byinhibition or corruption of essential host functions, mediated bythe enzymatic activity of the A subunit. AB5 toxins are criticalweapons in the armory of virulence factors deployed by majorbacterial pathogens, which collectively kill over a million peopleeach year (1). The AB5 toxins characterized to date are classifiedinto four families according to A subunit sequence homology andcatalytic activity, as well as the structural organization of the ho-lotoxin (1). The A subunits of both the cholera toxin (Ctx) family(comprising Ctx and the enterotoxigenic Escherichia coli labileenterotoxin [LT]) and the pertussis toxin (Ptx) family catalyze theADP-ribosylation of Gs� and Gi� proteins in the host cell cytosol,disrupting their signal transduction pathways. The A subunits ofthe Shiga toxin (Stx) family have RNA N-glycosidase activity andinhibit eukaryotic protein synthesis by cleaving a specific adeninebase from 28S rRNA. The fourth and most recently discoveredAB5 toxin family is subtilase cytotoxin (SubAB), produced by asubset of Shiga-toxigenic E. coli (STEC) strains (2). Its A subunit isa highly specific subtilase-like serine protease that cleaves the es-sential endoplasmic reticulum (ER) chaperone BiP/GRP78,thereby inducing a massive ER stress response and triggering cel-lular apoptosis (3–6).

The B subunits of the Ctx and Stx families (CtxB and StxB,respectively) recognize glycans displayed by host glycolipids (gan-gliosides, including GM1, and glycosphingolipids, such as Gb3

and Gb4) (1). The Ptx family is unique among AB5 toxins, as its Bsubunit is not a homopentamer, comprising 4 nonidentical sub-units (S2, S3, S4, and S5) in a 1:1:2:1 stoichiometry. The S2 and S3subunits both bind to sialylated glycoproteins rather than glyco-lipids (1). The B subunit of SubAB (SubB) is a homopentamer,like CtxB and StxB, but it binds to glycoproteins like Ptx. SubB andthe C-terminal portion of Ptx S2 exhibit strong structural homol-ogy, in spite of relatively low amino acid sequence identity (18%over a 95-amino-acid [aa] overlap) (7). Interestingly, SubB dis-plays a high degree of specificity for glycans terminating in N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is not synthe-sized in humans due to a deletion mutation in the cmah gene.Humans should be genetically resistant to the SubAB toxin. How-ever, assimilation of Neu5Gc from dietary sources enables theexpression of high-affinity receptors on the cell surface, therebyconferring susceptibility to the lethal effects of SubAB (7, 8).

In our original description of SubAB (2), we also reported

Received 25 September 2012 Returned for modification 4 November 2012Accepted 10 December 2012

Published ahead of print 17 December 2012

Address correspondence to Adrienne W. Paton, adrienne.paton@adelaide.edu.au.

Editor: L. Pirofski

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01043-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01043-12

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the presence of homologues of SubB encoded on the genomesof both Yersinia pestis (57% amino acid identity) and Salmo-nella enterica serovar Typhi (51% identity). BLAST searches ofcurrent genome sequences revealed SubB homologues in otherpathogenic Yersinia species and S. enterica serovars, includingSalmonella enterica serovar Paratyphi, Salmonella enterica se-rovar Typhimurium, Salmonella enterica serovar Montevideo,and Salmonella enterica serovar Arizonae. All these proteinshave now been classified as superfamily PRK15265 (subtilasecytotoxin B subunit-like proteins). The Salmonella genes areparts of operons that also contain homologues of the Ptx cata-lytic S1 subunit. The putative S. Typhimurium AB5 toxin hasbeen named ArtAB (for ADP-ribosylating toxin) (9), and re-cent studies have confirmed that, like Ptx S1, S. TyphimuriumArtA is an ADP-ribosyltransferase capable of modifying hostcell G proteins (10). However, to date, no A subunit-dependentcytotoxic phenotype has been identified for ArtAB. Interest-ingly, the ArtAB homologue from S. Typhi (also referred to asPltAB, for pertussis-like toxin) has been reported to associatewith cytolethal distending toxin and to deliver it from an in-tracellular compartment to target cells via autocrine and para-crine pathways (11). Although both A and B subunits wererequired for this property, it was not dependent on A subunitADP-ribosyltransferase activity (11).

Although the principal function of AB5 toxin B subunits is tointeract with receptors on the surfaces of target cells and to medi-ate internalization of holotoxin, interactions with surface mole-cules have the potential to impact a variety of signaling pathways.In so doing, the B subunits may directly contribute to pathogen-esis by triggering host responses independently of their respectivecatalytic (“toxic”) A subunits. In the present study, we have exam-ined the capacity of purified S. Typhi ArtB to elicit cytokine,chemokine, and adhesion molecule responses in human macro-phage and colonic epithelial and brain microvascular endothelialcell lines.

MATERIALS AND METHODSPurification of ArtB and ArtAB. The artB open reading frame (ORF) wasfirst amplified by PCR using an S. enterica serovar Typhi T2 chromo-somal-DNA template and primers STyArtBF (5=-GCGCGCGAATTCTTTTAAACGTACAGGAGAGTAG-3=) and STyArtBHis6R (5=-GCAGCAAAGCTTTTAGTGGTGGTGGTGGTGGTGCTTG-3=). The resultant PCRproduct, which incorporates EcoRI and HindIII restriction sites, as well asfusing a region encoding a His6 tag on the 3= terminus of artB, was thencloned into pBAD18 (12), so that the artB ORF is under the control of thevector ara promoter. The complete artBA ORF was also cloned intopBAD18 by fusing the native artA ORF immediately downstream of artB-His6 in the above construct (unlike most other AB5 toxin operons, artBprecedes artA in the S. Typhi chromosome). These constructs were thentransformed into an E. coli BL21(DE3) lpxM mutant (13) that produces apenta-acylated lipopolysaccharide (LPS) that has very low endotoxic ac-tivity. The recombinant bacterium was grown in 500 ml Terrific Broth(14) at 37°C to late logarithmic phase, diluted 50:50 with fresh mediumsupplemented with 0.2% arabinose to induce artB or artBA expression,and then incubated overnight at 26°C. Cells were harvested by centrifu-gation, resuspended in 20 ml loading buffer (50 mM sodium phosphate,300 mM NaCl, 20 mM imidazole, pH 8.0), and lysed in a French pressurecell. Cell debris was removed by centrifugation at 20,000 � g for 30 min at4°C. The supernatant was then loaded onto a 2-ml column of Ni-nitrilo-triacetic acid (NTA) resin, which had been preequilibrated with 20 mlloading buffer. The column was then washed with 40 ml loading buffer,and bound proteins were eluted with a 30-ml gradient of 0 to 500 mM

imidazole in loading buffer; 3-ml fractions were collected and analyzed bySDS-PAGE, followed by staining with Coomassie blue or Western blot-ting with monoclonal anti-His6. ArtB migrates as a single 16-kDa specieson SDS-PAGE, while ArtAB has an additional 26-kDa species correspond-ing to ArtA; densitometric analysis of ArtAB gels was consistent with 1:5stoichiometry. Fractions containing �95% pure ArtB or ArtAB werepooled. Under the above conditions, ArtB and ArtAB retain their nativefolding and conformation.

Cell culture and toxin treatment. Human brain microvascular endo-thelial cells (HBMEC) were grown at 37°C in 5% carbon dioxide in culturemedium (RPMI 1640 medium [GIBCO 11875] and F-12 Nutrient Mix-ture [GIBCO 11765] [1:1], supplemented with 10 mM HEPES, 2 mML-glutamine, 1 mM sodium pyruvate, 10% heat-inactivated fetal calf se-rum [FCS], 50 IU of penicillin, and 50 �g/ml of streptomycin). U937(human macrophages) and HCT-8 (human colonic epithelial cells) weregrown at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 10% heat-inac-tivated FCS, 50 IU of penicillin, and 50 mg/ml streptomycin. For the toxintreatment, cells were seeded into 24-well tissue culture plates; 1 millionU937 cells per well or confluent monolayers of HBMEC (2 � 105/well) orHCT-8 cells (5 � 105/well) were then exposed to ArtB at the indicatedconcentration in 300 �l of culture medium containing 1% FCS for theindicated times.

Examination of apoptosis and necrosis. Apoptosis and necrosis weredetected by differential staining with annexin V (which stains apoptoticand necrotic cells) and propidium iodide (PI) (which stains necrotic cellsonly) using a Vybrant Apoptosis Assay Kit 2 (Invitrogen V13241) accord-ing to the manufacturer’s instructions. Briefly, 106 U937 cells were treatedfor 24 h with 10 �g/ml ArtB or ArtAB and transferred from the wells of thetissue culture tray into fluorescence-activated cell sorter (FACS) tubes(Falcon 352008). The cells were washed three times with 3 ml annexin Vbinding buffer and resuspended in 50 �l of annexin V working reagent,consisting of 1/25 annexin V-Alexa 488 (which emits at 519 nm) and 1�g/ml PI (which emits at 617 nm) in annexin V binding buffer. The cellswere incubated at room temperature in the dark for 15 to 20 min and thenwashed and analyzed immediately on a FACscan flow cytometer.

Measurement of secreted chemokines, cytokines, and adhesionmolecules. Secreted chemokines, cytokines, and adhesion molecules incell culture supernatants were determined using Cytometric Bead ArrayKits (Bender Medsystems) according to the manufacturer’s instructions.The Human Chemo/Cytokine 9plex kit included 6 chemokines— granu-locyte colony-stimulating factor (G-CSF), interleukin 8 (IL-8), monocytechemotactic protein 1 (MCP-1), monokine induced by gamma interferon(MIG), macrophage inflammatory protein1� (MIP-1�), and MIP-1�—and 3 cytokines—IL-6, tumor necrosis factor -alpha (TNF-�), and IL-1�.The Human Adhesion 6plex kit included the adhesion molecules E selec-tin, intercellular adhesion molecule 1 (ICAM-1), ICAM-3, platelet endo-thelial cell adhesion molecule 1 (PECAM-1), P selectin, and vascular celladhesion molecule 1 (VCAM-1). The cytometric bead array techniqueutilizes beads of different sizes coated with specific capturing antibodies,which are labeled with an internal fluorescent dye (which emits at 700 nm)at discrete intensities. Up to 20 bead populations distinguished by internaldye intensity and bead size allow the simultaneous quantification of up to20 analytes in a small-volume sample. During incubation of the beadswith culture supernatant, different analytes were captured by their corre-sponding beads. The bead mixtures were then mixed with phycoerythrin(PE)-conjugated adhesion molecule-detecting antibodies or biotin-con-jugated chemokine/cytokine-detecting antibodies, followed by streptavi-din-PE to form sandwich complexes. Following incubation and washing,fluorescence data were acquired with a BD FACSCanto (serial numberV0130) or BD LSR II with high-throughput sampler (serial numberH1169) plus BD FACSDiva Software (version 5.0.3) and analyzed withWEASLE v2.6 software. The fluorescence intensity of PE (which emits at575 nm) is proportional to the amount of analytes present in the test

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samples. The data are presented as means and standard errors (SE), anddifferences were analyzed using Student’s t test.

Measurement of cell-bound chemokines, cytokines, and adhesionmolecules. Cell-bound chemokines, cytokines, or adhesion moleculeswere directly probed by incubating ArtB-treated cells (fixed with 1% para-formaldehyde and permeabilized with 0.05% Triton X-100 in phosphate-buffered saline [PBS]) with PE-conjugated adhesion molecule-detectingantibodies or biotin-conjugated chemokine/cytokine-detecting antibod-ies, followed by streptavidin-PE. At the end of the experiments, after 3 PBSwashes, the cells were analyzed with a BD FACSCanto, and the data wereacquired with BD FACSDiva software (version 5.0.3) and analyzed withWEASLE v2.6.

Measurement of mRNA levels of chemokines/cytokines and adhe-sion molecules. HCT-8 cells were seeded in 6-well tissue culture plates at3.6 � 106 cells/well and incubated overnight (to confluence). The HCT-8cells were then washed with PBS and incubated for 4 h in the presence of10 �g/ml ArtB or vehicle control in serum- and antibiotic-free medium.U937 cells were plated into 6-well tissue culture trays at 2.65 � 106 cells/well in serum- and antibiotic-free medium and then incubated for 4 h inthe presence of 10 �g/ml ArtB or vehicle control. RNA was then extractedfrom the HCT-8 or U937 cells using RNeasy minicolumns and QIA-shredder columns according to the manufacturer’s instructions (Qiagen,Valencia, CA). RNasin RNase inhibitor (Promega, Madison, WI) wasadded to samples. Contaminating DNA was digested with RNase-freeDNase I (Roche Applied Science, Sydney, Australia) according to themanufacturer’s instructions. The absence of DNA contamination in allRNA preparations was confirmed by reverse transcription (RT)-PCRanalysis using primers specific for the gene encoding the housekeepingenzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as previ-ously described (15). The RNA was then recleaned using RNeasy minicol-umns according to the manufacturer’s instructions (Qiagen). A total of 1�g RNA was synthesized into cDNA using the RT2 first-strand kit accord-ing to the manufacturer’s instructions (SABiosciences). A subset of hu-

man chemokines/cytokines or adhesion molecule mRNAs (as men-tioned above) were quantitated using PCR arrays or real-time RT-PCRand run in a LightCycler 480 (Roche) according to the manufacturer’sinstructions in 96-well plates (PAHS-225F; SABiosciences) or on aRotorgene RG-2000 Cycler (Corbett Research). Each treatment shownis the result of three independent experiments. The results were calcu-lated using the RT2 Profiler PCR array data analysis template v3.3,which uses the comparative cycle threshold (2���CT)) method andStudent’s t test for data analysis (SABiosciences). The data are pre-sented as means and standard deviations (SD).

RESULTSArtB and ArtAB-induced apoptosis and necrosis in U937 cells.As an initial experiment, general cytotoxicity was assessed by de-termining rates of apoptosis and necrosis in U937 cells exposed to10 �g/ml of purified ArtB or ArtAB for 24 h by flow cytometry(Fig. 1). At this time point, approximately 21% of ArtB- and 12%of ArtAB-treated cells were apoptotic (annexin V positive/PI neg-ative), and 14% of ArtB-treated cells and 0.5% of ArtAB-treatedcells were necrotic (annexin V positive/PI positive), compared toapproximately 7.5% apoptotic and 0.1% necrotic for untreatedcontrol cells. The higher potency of ArtB than ArtAB may be at-tributable to the higher actual dose in molar terms and also servesto underscore the phenotypic dominance of the B subunit. Thus,subsequent experiments were focused on the effects of ArtB alone.Decreases in cell size and complexity were also seen in HCT-8 cellstreated with 10 �g/ml ArtB for 24 or 48 h relative to control cells,as judged by FACS analysis (see Fig. S1 in the supplemental mate-rial). In view of this evidence of general cytotoxicity, 10 �g/ml and24 h were set as the maximum dose and time tested in most ex-periments.

FIG 1 Effect of ArtB on apoptosis and necrosis. U937 cells were incubated with ArtB or ArtAB (10 �g/ml) for 24 h. The cells were then labeled live with annexinV and PI and analyzed by FACS. (A) Dot plots of annexin V versus PI relative fluorescence intensities (RFI). Cells in the lower left quadrant (annexin V and PIdouble negative) were classified as living; cells in the lower right (LR) quadrant (only annexin V positive) were classified as apoptotic; cells in the upper right (UR)quadrant (annexin V and PI double positive) were considered necrotic. (B) Bar chart showing percent apoptosis and necrosis derived from 10,000 cell counts; thedata are representative of two independent experiments.

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ArtB-induced changes in chemokine and cytokine secretion.The effect of treatment with 10 �g/ml ArtB for up to 24 h onsecretion of selected chemokines (G-CSF, IL-8, MCP-1, MIG,MIP-1�, and MIP-1�) and cytokines (IL-6, TNF-�, and IL-1�) byHCT-8 and U937 cell and HBMEC lines was then investigatedusing FACS bead array assays. At 1 h for HCT-8 and U937 cells,there was no significant difference in the secretion of the testedchemokines or cytokines relative to vehicle-treated controls, whilefor HBMEC, a slight increase in TNF-� and MCP-1 was detected(see Fig. S2 in the supplemental material). However, at 4 h, for allthree tested cell lines, IL-8 secretion was significantly elevated (seeFig. S2), while the secretion of IL-6 and MCP-1 increased in HB-

MEC and the secretion of MIP-1�, MIP-1�, and G-CSF increasedin U937 cells (see Fig. S2). At 24 h, there was significant elevationof secretion of the chemokines IL-8 and MCP-1 in all three testedcell lines; secretion of the chemokines MIP-1�, MIP-1�, and MIGwas significantly increased in U937 cells only, while secretion ofthe proinflammatory cytokines TNF-� and IL-6 was elevated inHBMEC and HCT-8 cells, but not in U937 cells (Fig. 2).

Since chemokine and cytokine responses were maximal at24 h post-ArtB treatment, dose response at that time point wasinvestigated (Fig. 3). HBMEC were the most responsive cellline, exhibiting highly significant (P � 0.001) elevations inIL-8, MCP-1, and IL-6 secretion at 1.25 �g/ml, the lowest ArtB

FIG 2 ArtB-induced changes in secreted chemokines/cytokines at 24 h. HCT-8 or U937 cells or HBMEC were treated with 10 �g/ml ArtB for 24 h or untreated.Levels of secreted chemokines or cytokines (as indicated) were assayed using a cytometric bead array (see Materials and Methods). The data are presented as RFIfor each chemokine/cytokine (means and standard errors of the mean [SEM] from at least three experiments). ***, P � 0.001; **, P � 0.01; and *, P � 0.05 relativeto control cells (Student’s unpaired two-tailed t test).

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concentration tested. U937 cells exhibited significant elevationof IL-8, MCP-1, and MIP-1� at 10 �g/ml ArtB and above, whileHCT-8 cells exhibited significant elevation of IL-8 and MCP-1secretion at and above 2.5 and 5 �g/ml ArtB, respectively.

ArtB-induced changes in cell-bound chemokine and cyto-kine levels. Cell-bound chemokine and cytokine levels were alsoassayed. Initially, HCT-8 cells were treated with 10 �g/ml ArtB foreither 1, 4, or 24 h, and increases in expression of G-CSF, IL-8,MCP-1, MIG, MIP-1�, MIP-1�, IL-6, and TNF-� were observedat all time points (see Fig. S3A in the supplemental material).Responses at 24 h were dose dependent for all these chemokines/cytokines, with the maximum responses elicited at 10 �g/ml, ex-cept for IL-6 and TNF, which showed further increases at 20�g/ml (Fig. 4A). For U937 cells, treatment with 10 �g/ml ArtBresulted in higher surface expression of IL-8, MCP-1, MIP-1�,MIP-1�, and G-CSF from 1 h and MIG from 4 h (see Fig. S3B inthe supplemental material). Dose-response experiments after 24 htreatment indicated that U937 cells were somewhat less sensitiveto ArtB than HCT-8 cells, with maximal responses occurring at 20�g/ml in all cases (Fig. 4B). Consistent with findings for secretedcytokines, there were no increases in cell-bound IL-6 or TNF-� inArtB-treated U937 cells (results not shown).

Transcriptional responses to ArtB. To determine whether theeffects of ArtB and ArtAB on chemokine/cytokine expression atthe protein level were also reflected at the transcriptional level,mRNA levels were measured by quantitative real-time reversetranscription-PCR arrays 4 h after treatment of either HCT-8 orU937 cells with 10 �g/ml ArtB (Fig. 5A). For HCT-8 cells, IL-8 andTNF-� mRNA levels were upregulated 18.6-fold and 2.4-fold, re-spectively, relative to untreated control cells (P � 0.05 in bothcases). For U937 cells, MCP-1 and IL-8 mRNAs were upregulated6.4-fold and 132-fold, respectively, relative to untreated controlcells (P � 0.05 in both cases). RT-PCR was also used to comparethe effects of ArtB and the ArtAB holotoxin on IL-1�, TNF-�, andIL-8 mRNA levels in U937 cells at 4 h (Fig. 5B). While both elicitedsignificant gene upregulation at 10 �g/ml, ArtB was slightly morepotent than ArtAB, consistent with the earlier findings for cyto-toxicity (Fig. 5B).

ArtB-induced changes in adhesion molecules. FACS bead ar-rays were also used to measure levels of the adhesion moleculesICAM-1, E selectin, ICAM-3, PECAM-1, VCAM-1, and P selectinin the culture supernatants of the various cell types treated with 10�g/ml ArtB for 1, 4, and 24 h. At 1 h for all three cell lines, therewas no significant difference in the secretion of the tested adhesion

FIG 3 ArtB dose-dependent changes in secreted chemokines/cytokines. HCT-8 or U937 cells or HBMEC were treated with the indicated doses of ArtB for 24 h,and secreted chemokines or cytokines were assayed using a cytometric bead array (see Materials and Methods). The data are presented as RFI for eachchemokine/cytokine (means and SEM from at least three experiments). ***, P � 0.001; **, P � 0.01; and *, P � 0.05 relative to control cells (Student’s unpairedtwo-tailed t test).

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molecules relative to vehicle-treated controls (see Fig. S4 in thesupplemental material). However, at 4 h for HCT-8 cells, the se-cretion of all the tested adhesion molecules (except ICAM-1) waselevated, whereas for U937 cells, no significant increases were ob-served, while secretion of both ICAM-1 and VCAM-1 was in-creased at 4 h in HBMEC (Fig. S4). At 24 h, there was significantelevation of the secretion of ICAM-1 in both HCT-8 and U937cells, while the secretion of VCAM-1was elevated in HBMEC only(Fig. 6A).

Dose-response analysis at 24 h (Fig. 6B) showed that HCT-8cells were more sensitive to ArtB than U937 cells in terms ofICAM-1 response, with significant elevation seen at doses as low as2.5 �g/ml in the former. VCAM-1 responses of HBMEC were alsohighly responsive to ArtB and were near maximal at 2.5 �g/ml.

Expression of cell-bound ICAM-1 and VCAM-1 in ArtB-treated HCT-8 and U937 cells was also examined. At 10 �g/ml,ArtB treatment increased cell-associated expression of ICAM-1,but not VCAM-1, in both cell types at 24 h (Fig. 7A), which isconsistent with the levels observed in culture supernatant (seeabove). Dose-response experiments (Fig. 7B) indicated that cell-associated ICAM-1 expression was maximal in HCT-8 cells at 5�g/ml, while expression in U937 cells increased up to 20 �g/ml,the maximum dose tested.

ICAM-1, VCAM-1, E selectin, and P selectin mRNA levelswere also measured in HCT-8 and U937 cells treated with 10�g/ml ArtB for 4 h (Fig. 8). ICAM-1 was the only adhesionmolecule to exhibit significantly increased transcription (1.3-fold and 3.8-fold, respectively; P � 0.05 in both cases). These

FIG 4 ArtB dose-dependent changes in cell-bound chemokines/cytokines in HCT-8 (A) and U937 (B) cells. HCT-8 or U397 cells were treated with the indicateddoses of ArtB for 24 h. Cell-bound chemokines or cytokines were measured by direct probing with biotin-conjugated specific antibodies, followed by strepta-vidin-PE, and analyzed by FACS. The data are the mean chemokine or cytokine RFI, which were acquired from at least 5,000 cells. The results are representativeof two independent experiments.

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findings are consistent with the data obtained at the proteinlevel.

DISCUSSION

In the present study, we have shown broad effects of ArtB on theproduction of chemokines, proinflammatory cytokines, and ad-hesion molecules in distinct cell types. Secretion of the chemo-kines MCP-1 (CCL2) and IL-8 (CXCL8) was increased in all 3tested cell lines, with MIP-1� (CCL3), MIP-1� (CCL4), and G-CSF also significantly increased in U937 cells. ArtB also upregu-lated the cytokines TNF-� and IL-6 in HBMEC and HCT-8 cells,but not in U937 cells, while ICAM-1 was upregulated in HCT-8and U937 cells and VCAM-1 was upregulated in HBMEC. Thesefindings regarding levels of expressed proteins were largely con-sistent with data obtained at the transcriptional level. However,there were some exceptions; TNF-� mRNA was upregulated inHCT-8 cells at 4 h without significant elevation in soluble TNF-�in supernatants at the same time point. This may be related toposttranslational modification necessary to generate solubleTNF-�, which was indeed detected at elevated levels at 24 h. Also,IL-1� transcripts were elevated in U937 cells, yet IL-1� proteinwas not produced at levels above controls at any time point, againsuggesting possible alterations in posttranslational processing.Nevertheless, the overall net impact of the various changes in-duced by ArtB would be to promote and maintain a strong inflam-matory response, with MCP-1 recruiting monocytes, memory Tcells, and macrophages and IL-8 recruiting neutrophils to the site

of infection or tissue damage. Neutrophil proliferation and releasefrom the bone marrow would be promoted by G-CSF and thenactivated by MIP-1� and MIP-1� produced by macrophages. In-flammation would also be promoted by TNF-� and IL-6. Thesecytokine and chemokine responses are likely to have triggered theobserved upregulation of ICAM-1 and VCAM-1, which wouldpromote transmigration of leukocytes into underlying tissues.

Other AB5 toxins, most notably Ctx, LT, and Ptx, have potentimmunomodulatory properties, including strong adjuvant effects(16, 17). Some of these effects are dependent on the ADP-ribosyl-transferase activity of the respective A subunit, but significant im-munomodulation effects, particularly in lymphocytes, can also beelicited either by mutant holotoxins devoid of catalytic activity orby purified B subunit pentamers (16–19). For CtxB and LTB, ef-fects include altered antigen processing and presentation by mac-rophages, induction of cytokine secretion by monocytes, stimula-tion of proliferation of B and CD4 T cells, and induction ofapoptosis in CD8 T cells (17). Such effects were not observedwhen cells were treated with LTB with a G33D mutation thatabrogated the capacity to bind the ganglioside receptor GM1, sug-gesting that B subunit-GM1 interaction was required for the sig-naling events that result in immunomodulation (17). However,LTB derivatives with either H57A or H57S mutations retainedhigh-affinity binding to GM1 and underwent normal uptake andintracellular trafficking, but were also defective in immunomodu-lation (20, 21). Thus, binding to GM1 alone is insufficient, and

FIG 5 ArtB-induced changes in chemokine/cytokine mRNA in HCT-8 and U937 cells. HCT-8 or U937 cells were treated with 10 �g/ml ArtB for 4 h or leftuntreated (A); alternatively, U937 cells were treated with 10 �g/ml ArtB or ArtAB for 4 h (B). Total RNA was then extracted from the cells, and chemokine/cytokine mRNA (as indicated) was quantitated by PCR arrays (A) or real-time RT-PCR (B) (see Materials and Methods). The results are expressed as the foldchange in [mRNA] relative to levels in control cells. The data are shown as the means and SD of three independent experiments; ***, P � 0.001; **, P � 0.01; and*, P � 0.05 relative to control cells (Student’s unpaired two-tailed t test).

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additional interactions between the GM1-bound B pentamer andother surface molecules are required for immunomodulation.

The capacity of AB5 toxin B subunits to elicit signaling re-sponses in epithelial or endothelial cells, however, has received lessattention. Treatment of T84 (intestinal epithelial) cells with Ctx orLT induced expression of several cytokines/chemokines, includ-ing IL-6, IL-10, IL-1�, and IL-1�, as well as low levels of IL-8.However, this was totally dependent on the ADP-ribosyltrans-ferase activity of the respective A subunit, and catalytically inactiveholotoxin mutants or isolated B subunits were inactive (22). LT-induced reduction in trans-epithelial resistance in polarized T84cells also required functional holotoxin (23). In the present study,treatment of human colonic epithelial (HCT-8) cells with ArtBelicited significant IL-6, IL-8, TNF-�, and MCP-1 secretion, aswell as ICAM-1 expression, at 24 h. Interestingly, at 4 h, expres-sion of other adhesion molecules (E selectin, ICAM-3, PECAM-1,VCAM-1, and P selectin) was also significantly elevated. It hasbeen established that both leukocyte chemoattractants (IL-8 andMCP-1) and activators (TNF-� and IL-6), as well as adhesionmolecules, play essential roles in the initial recruitment of leuko-cytes to the site of infection (24). In particular, MCP-1 has beenshown to play a regulatory role during innate immune responsesto Salmonella infection (25). MCP-1�/� mice show decreased sur-vival after administration of purified Salmonella LPS, with accom-panying elevated levels of TNF-� and IL-6 in the serum (25).Similarly, increased mortality was also observed in normal micetreated with anti-MCP-1 antibodies prior to endotoxin exposure,while administration of MCP-1 during endotoxin challenge pro-tected the mice from death (26). Administration of MCP-1 to

Salmonella-infected mice also results in enhanced macrophage ac-tivation and accumulation (27). Adhesion molecules are also crit-ically involved in leukocyte-epithelial/endothelial cell interactions(28–32). The expression of ICAM-1 is strongly upregulated byinflammatory mediators and plays a key role in acquired immu-nity to salmonellosis (33). Thus, ArtB not only has the potential tosignificantly impact innate immune responses at the intestinal ep-ithelium, the initial site of interaction between S. Typhi and thehost, but also may have systemic effects.

HBMEC were particularly sensitive to ArtB, with significantelevations in IL-6, IL-8, and MCP-1 secretion at 1.25 �g/ml, thelowest dose tested, while VCAM-1 expression was significantlyelevated at 2.5 �g/ml. VCAM-1 is an endothelial cell membraneglycoprotein, which can be upregulated by proinflammatory cy-tokines (e.g., TNF-� and IL-1), and has been implicated in leuko-cyte/endothelial cell interactions in inflammation (34). Therefore,ArtB may play a role in promoting endothelial cell damage andleukocyte extravasation and may induce further tissue or organdamage in the later stages of infection. To our knowledge, theeffects of isolated B subunits of Ctx, LT, or Ptx on human endo-thelial cells have not been investigated. However, purified B sub-units of Stx1 and Stx2 have been reported to induce secretion ofvon Willebrand factor by human endothelial cells and to contrib-ute to thrombotic microangiopathy in mice (35). Interestingly,this appeared to involve activation of distinct signaling pathwaysby Stx1B and Stx2B, dependent upon protein kinase C� and pro-tein kinase A, respectively (36). Thus, Stx B subunits may play adirect role in the endothelial injury that is the hallmark of thesystemic complications of STEC disease in humans.

FIG 6 ArtB-induced changes in secreted adhesion molecules. (A) HCT-8 or U937 cells or HBMEC were treated with 10 �g/ml ArtB for 24 h or left untreated.(B) HCT-8 or U937 cells or HBMEC were treated with the indicated dose of ArtB for 24 h. Levels of secreted adhesion molecules (as indicated) were determinedusing a cytometric bead array (see Materials or Methods). The data are presented as RFI for each adhesion molecule (means and SEM from at least threeexperiments). **, P � 0.01, and *, P � 0.05 relative to control cells (Student’s unpaired two-tailed t test).

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At present, the role of ArtAB in the pathogenesis of salmo-nellosis remains obscure, and to date, no ADP-ribosyltrans-ferase-dependent cytotoxic effects have been reported. How-ever, it is known to be expressed in vivo, as antibodies to the Asubunit have been detected in sera from patients with S. Typhibacteremia (37). Spanò et al. (11) have reported that ArtAB(referred to as PltAB in that study) functions as a delivery ve-hicle for the catalytic subunit of cytolethal distending toxin(CdtB), whose cognate binding subunits (CdtA and CdtC) are

missing in S. Typhi. This property required both the A and Bsubunits of ArtAB, but not A subunit catalytic activity. Thismight be explained if the interaction with CdtB occurred viaArtA, or a domain spanning the ArtA-ArtB interface, while theArtB pentamer mediated membrane binding and transloca-tion. The findings of the present study, however, raised thepossibility that ArtB could also function in pathogenesis in itsown right by triggering signaling responses that result in secre-tion of proinflammatory cytokines and chemokines and adhe-

FIG 7 ArtB-induced changes in cell-bound adhesion molecules. (A) HCT-8 or U937 cells were treated with ArtB (10 �g/ml) for 1, 4, or 24 h or left untreated.(B) HCT-8 or U937 cells were treated with the indicated dose of ArtB for 24 h. The cell-bound adhesion molecules ICAM-1 and VCAM-1 were measured by directprobing with PE-conjugated adhesion molecule antibodies and analyzed by FACS. The data are the mean RFI, which were acquired from at least 5,000 cells. Theresults are representative of two independent experiments.

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sion molecule expression, both in the intestinal epithelium andsystemically. This finding may also have significance for infec-tions caused by Yersinia spp., whose genomes encode homo-logues of ArtB but lack a cotranscribed cognate catalytic sub-unit.

ACKNOWLEDGMENTS

This work was supported by Program Grant 565526 and Project Grant1002792 from the National Health and Medical Research Council of Aus-tralia (NHMRC) and Discovery Grants DP1095420 and DP120103178from the Australian Research Council. A.W.P. is an Australian ResearchCouncil DORA Fellow, J.C.P. is an NHMRC Australia Fellow, and T.B. isa Pfizer Australian Research Fellow.

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