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Microbial Pathogenesis 44 (2008) 61–70

www.elsevier.com/locate/micpath

Transcriptional responses of murine macrophages to the adenylatecyclase toxin of Bordetella pertussis

Gordon Y.C. Cheunga,b,�, Paul Dickinsonb, Garwin Singb, Marie Craigonb, Peter Ghazalb,Roger Partona, John G. Cootea

aDivision of Infection and Immunity, Institute of Biomedical and Life Sciences, Glasgow Biomedical Research Centre,

University of Glasgow, 120 University Place, Glasgow G12 8TA, UKbScottish Centre for Genomic Technology and Informatics, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent,

Edinburgh EH16 4SB, UK

Received 25 May 2007; accepted 1 August 2007

Available online 14 August 2007

Abstract

Three different recombinant forms of CyaA were used to investigate transcriptional responses of murine bone marrow-derived

macrophages (BMMs) using Affymetrix Mouse Genome GeneChipss. These forms were enzymically active, invasive CyaA, non-

enzymically active, invasive CyaA (CyaA*) and non-enzymically active, non-invasive CyaA (proCyaA*). BMMs, treated with 20 ng/ml

of CyaA for 24 h, showed over 1000 significant changes in gene transcription compared with control cells. CyaA caused an increase in

transcription of many inflammatory genes and genes associated with various signalling cascades such as those involved in cyclic

AMP-dependent protein kinase A signalling. Most strikingly, CyaA caused down-regulation of numerous genes involved in cell

proliferation. CyaA* at 20 ng/ml significantly up-regulated the transcription of only twelve genes after 24 h whereas proCyaA* at this

concentration significantly increased the transcription of only two genes.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Bordetella pertussis; Adenylate cyclase toxin; CyaA; Macrophage; Microarray; Transcriptional responses

1. Introduction

Bordetella pertussis, the aetiological agent of whoopingcough, secretes an adenylate cyclase toxin (CyaA) thatbelongs to the Repeats in ToXin (RTX) family [1]. CyaA, a177 kDa protein, is synthesised as a protoxin (proCyaA)that is post-translationally acylated by a separateprotein, CyaC. CyaA has two functional domains: theC-terminal domain (of about 1300 amino acids) which hasmembrane-targeting and pore-forming activity; and the400 amino acid N-terminal domain which has adenylatecyclase (AC) enzymic activity. Binding of Ca2+ ions tothe C-terminal glycine/aspartate repeats is a prerequisite

e front matter r 2007 Elsevier Ltd. All rights reserved.

icpath.2007.08.007

ing author. NIH/NIAID, Laboratory of Bacterial Diseases

h Drive, Building 33, Room 1W20, Bethesda, MD 20892-

.: +1 301 443 5325; fax: +1 301 480 9997.

ess: cheunggo@niaid.nih.gov (G.Y.C. Cheung).

for toxin activity. Interaction with, and invasion of,mammalian target cells, such as monocytes and neutro-phils, that express the CD11b/CD18 (CR3) receptor, isfacilitated by acylation of CyaA [2]. Upon entry intothe cell, the N-terminal AC enzymic moiety is activatedby host calmodulin to produce supraphysiologicallevels of cyclic AMP (cAMP) (a process referred to asintoxication) [2].CyaA has a range of biological effects on phagocytic

cells including impairment of chemotaxis, inhibition ofphagocytosis of bacteria, inhibition of the oxidative burst,induction of apoptosis and, at higher concentrations, celldeath as a result of pore-formation [2]. Modulation of theresponses of immune effector cells by cAMP are incom-pletely defined. Classically, cAMP signalling involvesactivation of protein kinase A (PKA) which phosphor-ylates downstream target proteins, such as the cAMPresponse element-binding (CREB) protein [3]. However,

ARTICLE IN PRESSG.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–7062

PKA-independent actions of cAMP have been reported,such as activation of exchange protein activated by cAMP(Epac-1) which has a role in the suppression of phagocy-tosis [4]. To help define more clearly the signallingmechanisms resulting from interaction of CyaA with targetcells, the gene transcriptional responses of mouse bonemarrow-derived macrophages (BMMs) were studied bymicroarray analysis. Three different forms of highlypurified recombinant CyaA, low in LPS, were used toassess the relative contributions of the enzymic andinvasive activities of the toxin to transcriptional changes.

2. Results and discussion

2.1. Cytotoxicity of recombinant CyaA forms towards

BMMs

At a CyaA concentration of 20 ng/ml, �95% and �80%of BMMs remain viable after incubation for 2 and 24 h,respectively (data not shown). Thus, this concentration wasconsidered to be suitable, as it would be expected topromote target cell responses without extensive loss of cellviability over the time intervals examined. CyaA* andproCyaA* at 20 ng/ml, or urea at the same concentration(400 mM) as in the CyaA preparations, did not affectviability of BMMs after treatment for 24 h (data notshown). BMMs treated with CyaA, but not CyaA* or pro-CyaA*, for 24 h showed a spherical morphology withruffled membranes and the production of long thinprocesses (data not shown), features previously noted afterCyaA treatment of BHK21 cells [5].

Fig. 1. Line diagram showing differences in gene transcription 43-fold after in

three separate incubations, pooled and cRNA hybridised to Affymetrix MG_U

down-regulation of genes is represented by red or blue, respectively, as indica

2.2. Gene responses in BMMs after treatment with CyaA,

CyaA* or proCyaA*

A preliminary experiment in which BMMs were treatedwith 20 ng/ml of CyaA for 2 h or 24 h was done to comparetranscriptional changes at these two time intervals. Usingan Affymetrix MG_U74Av2 microarray (12,488 probesets), it was found that the fold-changes of gene transcrip-tion after 24 h were generally greater than those at 2 h(Fig. 1) (accession number GPX-00031.1) with 40 moregenes up- or down-regulated 43-fold at 24 h compared to2 h, although the majority (84%) of the transcriptionalchanges at the two times were identical. Thus, a period of24 h was chosen for incubation of BMMs with the differentCyaA forms. A comparison was done of BMM generesponses to CyaA, CyaA* or proCyaA* (all at 20 ng/ml in400 mM urea) or with 400 mM urea alone for 24 h. A total ofnine separate incubations were made for each treatmentand the RNA was pooled from three incubations creatingthree separate RNA preparations which were each madeinto cRNA and hybridised to a Affymetrix MOE430_2GeneChips (45,000 probe sets), thus generating a total oftwelve arrays.

2.2.1. Responses to CyaA

With CyaA, 5.8% (2613) of the total number of genes onthe array were up- or down-regulated greater than 2-fold(Po0.05). Of these, 574 and 629 were up- and down-regulated 43-fold (Po0.01), respectively. A selection ofthese genes of known function that showed the greatestfold-changes is shown in Tables 1 and 2.

cubation with CyaA for 2 h (left) or 24 h (right). RNA was prepared from

74Av2 mouse arrays. Each line represents a single gene. Greatest up- or

ted on the right-hand panel.

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Table 1

Selected genes significantly up-regulated in BMMs after treatment for 24 h with CyaA

Affymetrix ID Gene name Fold changea P-value

CD antigens

1450513_at CD33 antigen 3.53 4.33E�06

1437025_at CD28 antigenb 4.08 1.07E�06

1451950_a_at CD80 antigenb 4.76 7.93E�06

1418394_a_at CD97 antigen 5.43 3.69E�07

1449858_at CD86 antigen 13.27 4.29E�07

1428547_at CD73 antigenb 60.13 2.51E�08

1425243_at CD207 antigen (langerin) 130.69 1.69E�07

Cytokines and chemokines

1417266_at Chemokine (C-C motif) ligand 6 3.18 1.96E�06

1449195_s_at Chemokine (C-X-C motif) ligand 16 3.48 3.81E�06

1449984_at Chemokine (C-X-C motif) ligand 2 4.82 4.35E�07

1448995_at Chemokine (C-X-C motif) ligand 4 5.43 2.98E�07

1419209_at Chemokine (C-X-C motif) ligand 1b 5.94 6.43E�07

1419728_at Chemokine (C-X-C motif) ligand 5 26.72 6.34E�08

1449399_a_at Interleukin 1 b 36.76 7.62E�08

1418480_at Chemokine (C-X-C motif) ligand 7 292.04 2.51E�08

Immune response

1426083_a_at B-cell translocation gene 1, anti-proliferative 3.03 1.66E�06

1438934_x_at Semaphorin B, mRNA sequenceb 3.76 1.05E�05

1453055_at Semaphorin transmembrane domain and cytoplasmic

domain 6D

5.35 4.53E�06

Cell surface receptors involved in immunity

1448731_at IL-10 receptor, a 3.18 1.06E�04

1424595_at F11 receptor 2.97 3.12E�05

1417460_at IFN induced transmembrane protein 2 3.32 1.41E�06

1421844_at IL-1 receptor accessory protein 3.58 3.70E�06

1422190_at Complement component 5, receptor 1 3.94 1.95E�06

1419132_at Toll-like receptor 2 3.84 9.21E�07

1422046_at Integrin a M 4.11 5.95E�07

1428018_a_at immunoglobulin superfamily, member 7 4.17 8.93E�07

1455660_at Colony stimulating factor 2 receptor, b 1, low-affinity

(granulocyte-macrophage)

5.10 3.13E�07

1425225_at Fc receptor-like 3 5.28 2.69E�07

1421034_a_at Interleukin 4 receptor, ab 4.99 1.09E�06

1435645_at Monocyte to macrophage differentiation associated 5.66 1.11E�06

1418741_at Integrin b 7 7.31 1.21E�06

1455660_at Colony stimulating factor 2 receptor, b 2, low-affinity

(granulocyte-macrophage)

5.10 3.13E�07

1435560_at Integrin a Lb 11.08 5.75E�07

1440865_at Interferon induced transmembrane protein 6 14.22 1.72E�07

1419609_at Chemokine (C-C motif) receptor 1 18.38 6.41E�08

1424254_at Interferon induced transmembrane protein 1 31.56 6.35E�08

1419532_at Interleukin 1 receptor, type II 36.50 5.02E�08

1447284_at Triggering receptor expressed on myeloid cells

1b(Trem1)

52.35 6.35E�08

1417625_s_at Chemokine orphan receptor 1 57.68 2.51E�08

Cell surface receptors

1420407_at Leukotriene B4 receptor 1 5.74 2.69E�06

1419309_at Glycoprotein 38 7.36 7.01E�06

1419647_a_at Immediate early response 3 11.39 1.18E�06

1420842_at Protein tyrosine phosphatase, receptor type, F 18.38 6.62E�07

1415800_at Gap junction membrane channel protein a 1b 18.51 6.35E�08

Apoptosis

1450997_at Serine/threonine kinase 17bb 4.59 5.97E�07

1456006_at BCL2-like protein 11b 5.10 1.98E�05

1418649_at EGL nine homolog 3 (C. elegans) 5.17 4.02E�06

1418847_at Arginase type IIb 24.76 6.87E�07

G.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–70 63

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Table 1 (continued )

Affymetrix ID Gene name Fold changea P-value

Signalling

1415834_at Dual specificity phosphatase 6 (Dusp6) 3.41 2.08E�04

1438097_at RAB20, member RAS oncogene family 3.97 1.23E�05

1452202_at Phosphodiesterase 2A (Pde2a) 4.08 1.03E�05

1455181_at RAS p21 protein activator 2b (RAS p21) 6.28 3.48E�07

1416701_at Ras homolog gene family, member Eb (Rhoe) 7.06 4.95E�06

1422474_at Phosphodiesterase 4Bb (Pde4b) 14.72 6.73E�08

1449145_a_at Caveolin, caveolae protein 41.36 1.72E�07

Transcription

1417662_at Member of ETS oncogene familyb (Elk3) 3.53 4.60E�06

1427844_a_at CCAAT/enhancer binding protein bb (C/EPB b) 3.97 1.68E�06

1423100_at FBJ osteosarcoma oncogene (c-Fos) 4.38 1.31E�04

1415899_at Jun-B oncogene (JunB) 4.44 5.35E�05

1437247_at Fos-like antigen 2 (Fosl2) 9.65 6.62E�07

1448713_at STAT 4 13.45 3.49E�07

1449037_at cAMP responsive element modulator (CREM) 17.88 2.98E�07

Cell growth/division/differentiation

1420909_at Vascular endothelial growth factor A (VEGFA) 8.63 2.75E�06

1423475_at Cyclin M2 19.03 4.29E�07

1427256_at Chondroitin sulfate proteoglycan (Cspg) 2 200.85 1.25E�08

Mitochondrion

1416381_a_at Peroxiredoxin 5 4.44 6.00E�07

Cellular structure

1416298_at Matrix metalloproteinase 9b 6.06 1.57E�06

1421976_at Matrix metalloproteinase 19b 7.21 4.27E�06

1415943_at Syndecan 1b 13.27 4.26E�07

Transport

1434773_a_at Solute carrier family 2 member 1b 3.18 8.87E�06

1447181_s_at Solute carrier family 7 (cationic amino acid

transporter), member 7

3.10 1.03E�05

1420697_at Solute carrier family 15 member 3 5.58 3.37E�07

1420413_at Solute carrier family 7 member 11 7.06 2.41E�06

1449005_at Solute carrier family 16 member 3 8.22 1.88E�07

1457989_at Solute carrier family 4 member 11 8.28 1.69E�07

Metabolism/biosynthesis

1423414_at Prostaglandin-endoperoxide synthase 1b 5.62 5.22E�07

Adhesion

1460302_at Thrombospondin 1b 382.68 3.59E�08

aMean of fold-change for 3 replicates.bIndicates the gene being recognised by a second probe.

G.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–7064

2.2.1.1. Up-regulated genes

2.2.1.1.1. Immune response genes. A majority of up-regulated genes (Table 1) were associated with inflamma-tion and host immune responses. CyaA caused increasedtranscription of genes for CD80 (B7-1) and CD86 (B7-2)which is in agreement with the findings of Bagley et al. [6]who showed that up-regulation of these genes by CyaA inhuman dendritic cells was dependent on PKA signalling.Co-stimulatory receptors on T cells and their respectiveligands on APCs (CD80 and CD86) are required foroptimal T-cell activation. CD207, the mouse homologue ofhuman langerin [7] was strongly up-regulated and isthought to interact with CD1a to present non-peptideantigens to T cells [8]. There was up-regulation of the

integrin aM gene, which forms one part of the CyaAreceptor [9].Chemokine genes that were up-regulated included Cxcl5,

Cxcl2, Cxcl1 and Cxcl7. In particular, Cxcl7 and Cxcl5both promote adhesion and transmigration of neutrophilsin vitro [10,11]. These data might suggest that macrophagesrecruit leukocytes to the site of infection in response toCyaA. The only interleukin (IL) gene significantly up-regulated was that for IL-1b and its type II receptor(up-regulated 35- and 36-fold, respectively). This isdiscussed further below.

2.2.1.1.2. Apoptosis-related genes. Induction of apop-tosis of macrophages by CyaA requires both AC enzymicactivity and acylation [12]. Three pro-apoptotic genes

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Table 2

Selected genes significantly down-regulated in BMMs after treatment for 24 h with CyaA.

Affymetrix ID Gene name Fold changea P-value

CD antigens

1436346_at CD109 antigen �4.96 9.29E�07

Cytokines and chemokines

1421578_at Ccl 4 �4.29 1.03E�06

1419561_at Ccl 3 �5.43 4.29E�07

Immune response

1421547_at Lymphocyte antigen 78 �4.47 1.73E�05

1427329_a_at Immunoglobulin heavy (of IgM) chain 6 �14.93 1.88E�07

Cell surface receptors involved in immunity

1452431_s_at MHC class II antigen A ab �3.32 6.72E�06

1450648_s_at MHC class II antigen A b 1b �3.36 8.99E�06

1422892_s_at MCH class II antigen E a �3.68 1.27E�05

Cell surface receptors

1424208_at Prostaglandin E receptor �9.65 2.16E�05

1422445_at Integrin a 6 �12.04 1.23E�06

Apoptosis

1424278_a_at Baculoviral IAP repeat-containing 5 (Birc5) �16.00 1.68E�06

Signalling

1434518_at Phosphorylase kinase a �4.96 4.45E�07

1419247_at Regulator of G-protein signalling 2 �7.73 3.37E�07

1451358_a_at Rac GTPase-activating protein 1b �9.78 1.72E�07

1448627_s_at PDZ binding kinase �30.27 3.59E�08

Transcription

1435368_a_at ADP-ribosyltransferase 1 �4.20 4.33E�06

1424629_at Breast cancer 1 �4.76 1.53E�06

1437187_at E2F transcription factor 7 �10.06 5.78E�07

1416258_at Thymidine kinase 1 �21.41 1.17E�07

1415810_at Nuclear protein 95 �34.78 6.35E�08

Cell growth/division/differentiation

1416773_at Wee 1 homolog (S. pombe) �3.34 5.44E�06

1454742_at RasGEF domain family, member 1B �3.86 3.89E�06

1424156_at Retinoblastoma-like 1 (p107) �4.86 8.23E�06

1448635_at Structural maintenance of chromosomes2-like 1 (yeast)b �7.52 1.72E�07

1423877_at Chromatin assembly factor 1, subunit B (p60) �8.94 4.29E�07

1416746_at H2A histone family, member X �9.00 3.19E�07

1416030_a_at Minichromosome maintenance deficient (Mcm) 7 (S.

cerevisiae)

�19.29 1.85E�06

1415945_at Mcm 5 cell division cycle 46 (S. cerevisiae)b �20.68 4.85E�08

1416309_at Nucleolar and spindle associated protein 1 �22.32 1.88E�06

1438852_x_at Mcm 6 (S. pombe)b �32.45 5.39E�07

Cell division cycle (Cdc) genes

1426002_a_at Cdc 7 (S. cerevisiae) �4.44 1.54E�04

1456077_x_at Cdc 25 phosphatase mRNA �5.24 8.91E�06

1416575_at Cdc 45 homolog (S. cerevisiae) �5.82 1.07E�05

1448314_at Cdc 2 homolog A (S. pombe) �5.78 2.96E�07

1417019_a_at Cdc 6 homolog (S. cerevisiae) �6.06 4.71E�06

1439377_x_at Cdc 20 homolog (S. cerevisiae)b �6.63 2.90E�06

1448441_at Cdc 28 protein kinase regulatory subunit 1 �8.22 3.13E�07

1430811_a_at Cdc associated 1 �11.96 1.12E�07

Cyclins

1417420_at Cyclin D1b �4.38 1.03E�06

1416868_at Cyclin-dependent kinase inhibitor 2C �6.63 1.38E�06

1422513_at Cyclin F �7.26 3.23E�06

1450920_at Cyclin B2 �7.46 1.27E�07

1419943_s_at Cyclin B1b �8.17 6.13E�07

1422535_at Cyclin E2 �11.16 3.50E�07

1417910_at Cyclin A2b �19.70 1.41E�06

G.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–70 65

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Table 2 (continued )

Affymetrix ID Gene name Fold changea P-value

Kinesins

1452315_at Kinesin family member 11b �10.85 1.85E�06

1437611_x_at kinesin family member 2Cb�11.16 6.80E�06

1451128_s_at kinesin family member 22b �11.79 2.37E�07

Mitochondrion

1423392_at Chloride intracellular channel 4b �3.36 3.46E�06

1422978_at Cytochrome b-245, b polypeptide �6.36 6.20E�06

Cellular structure

1425476_at Procollagen, type IV, a 5 �4.14 4.30E�06

1451989_a_at Microtubule-associated protein, RP/EB family, member 2 �4.35 7.21E�06

1428976_at Thymopoietinb �6.28 1.68E�06

1434748_at Cytoskeleton associated protein 2 �8.28 8.23E�06

DNA replication

1416641_at Ligase I, DNA, ATP-dependent �5.28 4.68E�07

1417541_at Helicase, lymphoid specific, mRNAb�10.06 2.45E�07

1450862_at RAD54 like (S. cerevisiae) �11.16 1.12E�07

1454694_a_at Topoisomerase (DNA) II a �14.22 1.23E�07

1419397_at Polymerase (DNA directed), a 1 �14.72 6.35E�08

1418281_at RAD51 homolog (S. cerevisiae) �19.03 7.62E�08

1448226_at Ribonucleotide reductase M2b �36.25 9.17E�07

Transport

1416629_at Solute carrier family 1 (neutral amino acid transporter),

member 5

�3.84 1.50E�05

1418326_at Solute carrier family 7 (cationic amino acid transporter,

y+ system), member 5

�14.52 1.09E�06

1437052_s_at Solute carrier family 2 (facilitated glucose transporter),

member 3

�15.24 9.53E�07

Metabolism/biosynthesis

1423569_at L-arginine:glycine amidinotransferase �5.50 3.93E�06

1433966_x_at Asparagine synthetaseb �12.04 3.70E�06

1431056_a_at Lipoprotein lipase �118.60 3.13E�07

aMean of fold-change for 3 replicates.bIndicates the gene being recognised by a second probe.

G.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–7066

(Stk17b, Egln3 and Bcl2-like protein 11 (Bcl2l11)), but noanti-apoptotic genes, were up-regulated (Table 1). Only alow level of apoptosis, as measured by caspase 3/7 activity,was found after exposure of J774.2 mouse macrophage-likecells to 20 ng/ml of CyaA (Cheung et al., unpublisheddata).

2.2.1.1.3. cAMP-influenced genes and pathways. Thegenes coding for the cAMP-responsive element modulator(CREM) and CCAAT/enhancer-binding protein b (C/EPBb) were up-regulated. These regulatory proteins are founddown-stream of the PKA signalling pathway [3,13]. PKA isa well-known cAMP-induced signalling effector molecule.The changes in cell morphology of mouse fibroblast cellsinduced by the oedema toxin of Bacillus anthracis (EdTx),which includes cell rounding and the production of longthin processes, is PKA dependent [14]. Therefore, theCyaA-induced morphological changes in BMMs notedabove were likely to have been a consequence of cAMP-activated PKA signalling. Activated PKA can alsodirectly phosphorylate Rho proteins, interfering with theirability to interact with proteins that are involved in the

maintenance of cellular morphology [15]. Rho proteins areintimately connected with the regulation of the actincytoskeleton [16] and it is interesting to note in this contextthat there was up-regulation of genes from the Rho familyof small GTPases, such as RAB20, RAS p21 and Rhoe.Intracellular cAMP levels have been shown to up-

regulate syndecan-1 [17] and arginase [18] genes and CyaAalso markedly up-regulated these genes (Table 1). Arginasecompetes with nitric oxide synthase (NOS) for arginine as asubstrate, which is imported from the extracellularenvironment into the cells by cationic amino acidtransporters [19]. Increased arginase activity may causedecreased NO production by NOS [20] and so underminethe macrophage response to B. pertussis which involves NOproduction [21]. In addition, there was up-regulation oftwo cationic amino acid transporters (solute carrier family7, members 7 and 11) (Table 1) that import L-arginine [22].Elk3, up-regulated by CyaA (Table 1), acts as a transcrip-tional repressor of NOS in mouse macrophages [23].This may be another mechanism by which CyaA is ableto down-regulate NO production. Recently, increased

ARTICLE IN PRESSG.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–70 67

arginase production in macrophages has been associatedwith apoptosis and a decrease in NO production [24].Excessive cAMP accumulation also induces transcriptionof phosphodiesterase genes which degrade cAMP andcGMP, thereby regulating cAMP/cGMP-mediated cellsignalling. This feature was reflected in up-regulation ofphosphodiesterases 2A and 4B (Table 1).

In eukaryotic cells, three well-characterised subfamiliesof mitogen-activated protein kinases (MAPKs), extracel-lular response kinase (ERK), c-Jun N-terminal Kinase(JNK) and p38 have been described. cAMP-activated PKAhas been shown to activate B-raf, a kinase which serves as acentral intermediate to relay signals from Ras to ERK [25].Phosphorylated MAPKs activate many target proteinsubstrates, such as transcription factors (C/EBPb, JunB,CREM, Elk3, c-Fos and Fosl2). Genes that code fortranscription factors found down-stream of the ERKsignalling pathway (Fos and Fosl2) and the JNK signallingpathways (Jun and JunB) were up-regulated by CyaA(Table 1). These transcription factors combine to form theheterodimeric AP-1 regulatory complex which can mod-ulate cell proliferation and cell death [26]. JunB, forinstance, has anti-proliferative effects [27]. Dual specificityphosphatase 6 (Dusp6), up-regulated by CyaA (Table 1),dephosphorylates the ERK2 protein, inhibiting the activityof ERK2 [28]. Therefore, CyaA may modulate the MAPKsignalling pathways in this way. B. bronchiseptica CyaAhas been reported to prevent p38 phosphorylation [29].

2.2.1.2. Down-regulated genes. A selection of the genessignificantly down-regulated in BMMs by CyaA is listed inTable 2. Only 10% of the down-regulated genes wereassociated with the immune response compared with32.5% of up-regulated genes (accession numberGPX-00050.1). Those down-regulated included the CDantigen gene CD109, chemokine genes including Ccl4

(Mip-1b) and Ccl3 (Mip-1a) and MHC class II genes.

Table 3

Comparison of genes up-regulated in BMMs after treatment for 24 h with Cy

Affymetrix ID Gene name

1419209_at Chemokine (C-X-C motif) ligand 1

1419627_s_at C-type lectin domain family 4, member n

1420591_at G protein-coupled receptor 84

1422041_at Paired immunoglobin-like type 2 receptor beta 1

1422953_at Formyl peptide receptor, related sequence 2

1423954_at Complement component 3

1425951_a_at C-type lectin domain family 4, member n

1427327_at Paired immunoglobin-like type 2 receptor alpha

1448377_at Secretory leukocyte peptidase inhibitor

1450764_at Acyloxyacyl hydrolase

1450826_a_at Serum amyloid A 3

1427381_at Immunoresponsive gene 1

n/a ¼ no alteration in expression.

Text highlighted in bold ¼ fold change o2.aMean of fold-change for 3 replicates.

Mice deficient in Ccl3 are more susceptible to bacterial,viral and parasitic infections [30]. The down-regulation ofthe gene for MHC class II may be due to the fact that itsexpression is cAMP-regulated [31] and would indicate thatCyaA has a role in interference with antigen presentationby BMMs. Down-regulation of the anti-apoptotic gene,Birc5, an inhibitor of caspase-3 and caspase-7 [32] againindicates that CyaA at 20 ng/ml was pro-apoptotic.

2.2.1.2.1. Effects on cell cycle-associated genes. Manyof the down-regulated genes (approximately 40%) wereinvolved in the cell cycle compared with only �3% of up-regulated genes (accession number GPX-00050.1). Cyclins,a family of proteins that bind with specific cell-dependentkinases (Cdks) to phosphorylate different proteins to helpcells progress through the cell cycle [33], were extensivelydown-regulated, including genes for cyclin B1, cyclin B2and their Cdk partner Cdc2A (also known as Cdk1)(Table 2). These proteins combine to make the M-phasepromoting factor, which permits the entrance of cells intomitosis from the G2 phase [34]. CyaA also down-regulatedseveral enzymes that are central to DNA replication. Theseincluded helicase, topoisomerase, DNA polymerase andribonucleotide reductase (Rnr). This down-regulation ofcell cycle-associated genes, together with up-regulation ofthe anti-proliferative transcription factor JunB, indicatesthat CyaA has a strong inhibitory effect on macrophageproliferation.

2.2.2. Responses to CyaA* and proCyaA*

CyaA* and proCyaA* increased expression of only 12and 2 genes greater than 2-fold (Po0.05), respectively(Table 3). Of these, serum amyloid A3 (Saa3) andimmunoresponsive gene 1 (Irg-1) were significantly up-regulated by both CyaA* and proCyaA* (Table 3). Saa3

and Irg-1 can respond to the presence of LPS [35,36] whichraises the question of whether the LPS present in similar,low concentrations in all three of the CyaA preparations

aA*, proCyaA* and CyaA

Fold changea (P-value)

CyaA* proCyaA* CyaA

2.14 (8.34E�06) n/a 5.94 (7.46E�09)

2.50 (1.83E�07) n/a 8.34 (3.22E�10)

2.45 (3.78E�07) n/a 1.01 (0.834184)

2.30 (1.68E�06) n/a 10.70 (2.24E�09)

3.43 (5.23E�08) n/a 25.11 (9.70E�11)

2.06 (6.67E�07) n/a 2.41 (3.12E�07)

2.50 (3.51E�07) n/a 9.19 (4.28E�09)

2.17 (1.42E�06) n/a 6.63 (2.48E�09)

2.36 (1.09E�05) n/a 22.47 (1.33E�09)

2.04 (2.15E�06) n/a 1.03 (0.79045)

5.28 (6.29E�09) 2.77 (4.31E�07) 0.45 (0.000126)

4.23 (2.23E�09) 2.19 (1.56E�06) 4.26 (6.77E�08)

ARTICLE IN PRESSG.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–7068

were high enough to alter gene responses in BMMs.However, only Irg-1 was significantly up-regulated byCyaA while Saa3 was down-regulated (Table 3). LPSinduces production of IL-6 in murine macrophages in vitro

[37], but no IL-6 was secreted and no alterations in IL-6transcription were observed in response to any of the CyaAforms (data not shown). IL-1b mRNA transcription wassignificantly up-regulated only by CyaA (Table 1),although no IL-1b was detected in the culture supernatesfrom CyaA-treated BMMs (data not shown). This could bedue to lack of cleavage of IL-1b precursor by the IL-1bconverting enzyme or caspase-1, to release the 17.5 kDamature form of IL-1b [38]. It seems reasonable to concludethat the gene transcription changes prompted by CyaAwere predominantly due to CyaA alone. It cannot be ruledout, however, that CyaA and a low level of LPS actedsynergistically [39] to modulate some aspects of genetranscription.

The remaining 10 genes up-regulated by CyaA*(Table 3) were presumably due to binding of CyaA* tothe CD18/CD11b receptor. The non-acylated proCyaA*form would be expected to have reduced receptor binding[40] and this is in keeping with the limited effects of thisprotein on transcription (Table 3). It is possible that othertranscriptional events subsequent to CyaA* interactionwith the BMMs are short-lived and thus undetected after24 h. This interpretation is supported by recent work whereit was shown that CyaA* stimulated the respiratory burstof human neutrophils in a modest, but rapid and transientmanner indicating that CyaA* can initiate signallingprocesses following interaction with neutrophils [41]. Allthe genes up-regulated by CyaA*, except for the G protein-coupled receptor 84, acyloxyacyl hydrolase and serumamyloid A 3 genes were also up-regulated by CyaA(Table 3). No genes were significantly down-regulated byCyaA* or proCyaA*.

2.3. Summary of effects of CyaA forms

The transcriptional changes in BMMs induced by CyaAafter 24 h can be attributed largely to elevated cAMP levels.The extent to which these effects are, in turn, dependent onPKA-associated or other cAMP-signalling mechanismsinvolving, for example, Epac-1 remains to be determined.

3. Materials and methods

3.1. Expression and purification of different CyaA forms

Three forms of CyaA, prepared as recombinant proteinsin E. coli, were used. These were acylated and enzymicallyactive CyaA, an acylated, non-enzymically active CyaAform (CyaA*) and a non-acylated form of CyaA*(proCyaA*). CyaA* retains cell-invasiveness but carries aLeu-Gln di-peptide insertion between codons 188 and 189of the N-terminal AC domain and is unable to raiseintracellular cAMP levels. ProCyaA* is non-acylated

(poorly invasive) and has no AC enzymic activity. Thepurification and characterisation of CyaA, CyaA* andproCyaA* have been described elsewhere [42]. Purifiedpreparations were stored at �80 1C in 8M urea, 50mMTris–HCl (pH 8.0) and containedo0.1 endotoxin units/mgprotein, determined using a Kinetic-QCL

TM

(Biowhittaker)Limulus amoebocyte lysate (LAL) assay.

3.2. Isolation of bone marrow-derived macrophages

BMMs were extracted from the femurs of male,10–12 week old, Balb/c mice (Charles River Labs, UK),as described previously [43]. The bone marrow cellprogenitors were resuspended in DMEM/F12 completegrowth medium containing GlutaMAX

TM

and pyridoxine(Gibco) plus 10% (v/v) foetal calf serum (Gibco), 100U/mlpenicillin/streptomycin (Gibco) and 10% (v/v) culturesupernate from murine L929 connective tissue cells, as asource of macrophage colony-stimulating factor, preparedas described previously [44]. For the cytotoxicity assay,50 ml of the cells, at 4� 105 cells/ml, were dispensed intowells of a 96-well tissue culture plate (Costar) andincubated at 37 1C with 5% CO2 until �70% confluency(see below). For RNA extraction of BMMs after CyaAtreatment, 4ml of the bone marrow cell progenitors wereseeded at a density of 1.6� 106 cells/well in a 6-well tissueculture dish (Costar) and incubated at 37 1C with 5% CO2.After 3 days, the medium was replaced with 2ml of freshDMEM/F12 complete medium and incubated until �70%confluency was achieved (see below).

3.2.1. Cytotoxicity assay

The CellTiter 96TM

kit (Promega) was used to determinea concentration of purified CyaA, CyaA* and proCyaA*that did not kill significant numbers of BMMs aftertreatment for 2 h or 24 h. Briefly, BMMs (50 ml) were mixedwith toxin (50 ml) or urea buffer, serially diluted in non-phenol red DMEM/F12 complete growth medium contain-ing GlutaMAX

TM

and pyridoxine (Gibco) plus 10% (v/v)foetal calf serum (Gibco) and 100U/ml penicillin/strepto-mycin (Gibco) in 96-well tissue culture plates (Costar) andincubated for 2 or 24 h, at 37 1C in a humidified atmo-sphere containing 5% CO2. Cell viability was thenmeasured according to the manufacturer’s instructions.Loss of cell viability was calculated using the formula:100�(((sample OD�positive OD)/(negative OD�positiveOD))� 100). Cells incubated in the presence or absence of1% (v/v) Triton-X100 (Sigma) served as positive andnegative controls, respectively.

3.3. CyaA treatment and sample collection

When BMMs had grown to 70% confluency in 6-wellplates (see above), the medium in each well was replacedwith 1ml of DMEM/F12 medium containing onlyantibiotics plus either 20 ng/ml of CyaA, CyaA*, pro-CyaA* or urea buffer alone and incubated for 2 or 24 h at

ARTICLE IN PRESSG.Y.C. Cheung et al. / Microbial Pathogenesis 44 (2008) 61–70 69

37 1C in 5% CO2. Urea buffer was used at the sameconcentration as in the CyaA samples (400 mM urea) andserved as a negative control. After incubation, the super-nates were collected and stored at �20 1C for detection ofcytokines as described elsewhere [42].

3.4. Microarray analysis

RNA was extracted using Trizol, according to themanufacturer’s instructions (Invitrogen). The integrity ofeach RNA preparation was assessed by the BioAnalysersoftware (Agilent technologies) after electrophoresis onRNA 6000 Nano LabChips (Agilent technologies). cDNAsynthesis, in vitro transcription, and labelling and frag-mentation of cRNA were performed as instructed by theGeneChips manufacturer (Affymetrix, Santa Clara, CA).The cRNA was purified using RNeasy (Qiagen). cRNA(5 mg) was first hybridised to a test array (Affymetrix) andthen 15 mg of cRNA was hybridised to MG_U74Av2(12,488 probe sets) or MOE430_2 (45,000 probe sets)GeneChipss. The GeneChipss were washed in a Gene-Chips Fluidics Station 400 (Affymetrix), and hybridisationvisualised with a Gene Array Scanner 2500 or 3000 usingthe Affymetrix software. Microarray suite 5 (MAS5) andAffymetrix GeneChips Operating Software 1.4 (GCOS1.4)were used to control analysis of Affymetrix GeneChips

microarrays from experimental set up to data output.Additionally, much of the analysis of the Affymetrix arrayswas performed using the Bioconductor 1.9 packagerunning under the R2.4 environment. Empirical basedstatistical testing using Benjamini-Hochberg false discoveryrate correction was used to determine significances betweenreplicates and treatments (Po0.05 or Po0.01). In thisreport, genes that showed changes 43-fold were the focusof interest. A further criterion was created to select up-regulated genes which were flagged as ‘‘present’’, asdetermined by MAS5, in all three replicates. GeneSpring7.3.1 (Silicon Genetics, Redwood City, CA) was used toanalyse the microarray data. A complete list of genes in thedatabase showing genes that were up- or down-regulated42-fold (Po0.05) can be viewed from http://www.gti.ed.ac.uk/GPX/, accession numbers GPX-00031.1 orGPX-00050.1.

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