Bordetella pertussis infects the upper airways of humans and disarms host defense by the potent immuno-subversive activities of its pertussis (PT) and adenylate cyclase (CyaA) toxins. CyaA action near-instantly ablates the bactericidal activities of sentinel CR3-expressing myeloid phagocytes by hijacking cellular signaling pathways through the unregulated production of cAMP. Moreover, CyaA-elicited cAMP signaling also inhibits the macrophage colony-stimulating factor (M-CSF)-induced differentiation of incoming inflammatory monocytes into bactericidal macrophages. We show that CyaA/cAMP signaling via protein kinase A (PKA) downregulates the M-CSF-elicited expression of monocyte receptors for transferrin (CD71) and hemoglobin-haptoglobin (CD163), as well as the expression of heme oxygenase-1 (HO-1) involved in iron liberation from internalized heme. The impact of CyaA action on CD71 and CD163 levels in differentiating monocytes is largely alleviated by the histone deacetylase inhibitor trichostatin A (TSA), indicating that CyaA/cAMP signaling triggers epigenetic silencing of genes for micronutrient acquisition receptors. These results suggest a new mechanism by which B. pertussis evades host sentinel phagocytes to achieve proliferation on airway mucosa.IMPORTANCETo establish a productive infection of the nasopharyngeal mucosa and proliferate to sufficiently high numbers that trigger rhinitis and aerosol-mediated transmission, the pertussis agent Bordetella pertussis deploys several immunosuppressive protein toxins that compromise the sentinel functions of mucosa patrolling phagocytes. We show that cAMP signaling elicited by very low concentrations (22 pM) of Bordetella adenylate cyclase toxin downregulates the iron acquisition systems of CD14+ monocytes. The resulting iron deprivation of iron, a key micronutrient, then represents an additional aspect of CyaA toxin action involved in the inhibition of differentiation of monocytes into the enlarged bactericidal macrophage cells. This corroborates the newly discovered paradigm of host defense evasion mechanisms employed by bacterial pathogens, where manipulation of cellular cAMP levels blocks monocyte to macrophage transition and replenishment of exhausted phagocytes, thereby contributing to the formation of a safe niche for pathogen proliferation and dissemination.

Laboratory of Molecular Biology of Bacterial Pathogens Institute of Microbiology of the Czech Academy of Sciences Prague Czechia

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Melvin JA, Scheller EV, Miller JF, Cotter PA. 2014. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol 12:274–288. doi:10.1038/nrmicro3235 PubMed DOI PMC

Mattoo S, Cherry JD. 2005. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18:326–382. doi:10.1128/CMR.18.2.326-382.2005 PubMed DOI PMC

Paddock CD, Sanden GN, Cherry JD, Gal AA, Langston C, Tatti KM, Wu KH, Goldsmith CS, Greer PW, Montague JL, Eliason MT, Holman RC, Guarner J, Shieh WJ, Zaki SR. 2008. Pathology and pathogenesis of fatal Bordetella pertussis infection in infants. Clin Infect Dis 47:328–338. doi:10.1086/589753 PubMed DOI

Scanlon K, Skerry C, Carbonetti N. 2019. Association of pertussis toxin with severe pertussis disease. Toxins 11:373. doi:10.3390/toxins11070373 PubMed DOI PMC

Coutte L, Locht C. 2015. Investigating pertussis toxin and its impact on vaccination. Future Microbiol 10:241–254. doi:10.2217/fmb.14.123 PubMed DOI

Carbonetti NH. 2016. Pertussis leukocytosis: mechanisms, clinical relevance and treatment. Pathog Dis 74:ftw087. doi:10.1093/femspd/ftw087 PubMed DOI PMC

Fedele G, Schiavoni I, Adkins I, Klimova N, Sebo P. 2017. Invasion of dendritic cells, macrophages and neutrophils by the Bordetella adenylate cyclase toxin: a subversive move to fool host immunity. Toxins (Basel) 9:293. doi:10.3390/toxins9100293 PubMed DOI PMC

Ahmad JN, Sebo P. 2021. Bacterial RTX toxins and host immunity. Curr Opin Infect Dis 34:187–196. doi:10.1097/QCO.0000000000000726 PubMed DOI

Guermonprez P, Khelef N, Blouin E, Rieu P, Ricciardi-Castagnoli P, Guiso N, Ladant D, Leclerc C. 2001. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the αMβ2 integrin (Cd11b/Cd18). J Exp Med 193:1035–1044. doi:10.1084/jem.193.9.1035 PubMed DOI PMC

Bumba L, Masin J, Fiser R, Sebo P. 2010. Bordetella adenylate cyclase toxin mobilizes its β2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog 6:e1000901. doi:10.1371/journal.ppat.1000901 PubMed DOI PMC

Guo Q, Shen Y, Lee YS, Gibbs CS, Mrksich M, Tang WJ. 2005. Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin. EMBO J 24:3190–3201. doi:10.1038/sj.emboj.7600800 PubMed DOI PMC

Wolff J, Cook GH, Goldhammer AR, Berkowitz SA. 1980. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci U S A 77:3841–3844. doi:10.1073/pnas.77.7.3841 PubMed DOI PMC

Ahmad JN, Holubova J, Benada O, Kofronova O, Stehlik L, Vasakova M, Sebo P. 2019. Bordetella adenylate cyclase toxin inhibits monocyte-to-macrophage transition and dedifferentiates human alveolar macrophages into monocyte-like cells. mBio 10:e01743-19. doi:10.1128/mBio.01743-19 PubMed DOI PMC

Cerny O, Kamanova J, Masin J, Bibova I, Skopova K, Sebo P. 2015. Bordetella pertussis adenylate cyclase toxin blocks induction of bactericidal nitric oxide in macrophages through cAMP-dependent activation of the SHP-1 phosphatase. J Immunol 194:4901–4913. doi:10.4049/jimmunol.1402941 PubMed DOI

Cerny O, Anderson KE, Stephens LR, Hawkins PT, Sebo P. 2017. cAMP signaling of adenylate cyclase toxin blocks the oxidative burst of neutrophils through Epac-mediated inhibition of phospholipase C activity. J Immunol 198:1285–1296. doi:10.4049/jimmunol.1601309 PubMed DOI

Confer DL, Eaton JW. 1982. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217:948–950. doi:10.1126/science.6287574 PubMed DOI

Kamanova J, Kofronova O, Masin J, Genth H, Vojtova J, Linhartova I, Benada O, Just I, Sebo P. 2008. Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J Immunol 181:5587–5597. doi:10.4049/jimmunol.181.8.5587 PubMed DOI

Pearson RD, Symes P, Conboy M, Weiss AA, Hewlett EL. 1987. Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin. J Immunol 139:2749–2754. PubMed

Ahmad JN, Sebo P. 2020. Adenylate cyclase toxin tinkering with monocyte-macrophage differentiation. Front Immunol 11:2181. doi:10.3389/fimmu.2020.02181 PubMed DOI PMC

Chitu V, Stanley ER. 2006. Colony-stimulating factor-1 in immunity and inflammation. Curr Opin Immunol 18:39–48. doi:10.1016/j.coi.2005.11.006 PubMed DOI

Guilliams M, De Kleer I, Henri S, Post S, Vanhoutte L, De Prijck S, Deswarte K, Malissen B, Hammad H, Lambrecht BN. 2013. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. J Exp Med 210:1977–1992. doi:10.1084/jem.20131199 PubMed DOI PMC

van de Laar L, Saelens W, De Prijck S, Martens L, Scott CL, Van Isterdael G, Hoffmann E, Beyaert R, Saeys Y, Lambrecht BN, Guilliams M. 2016. Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages. Immunity 44:755–768. doi:10.1016/j.immuni.2016.02.017 PubMed DOI

de Oliveira J, Denadai MB, Costa DL. 2022. Crosstalk between heme oxygenase-1 and iron metabolism in macrophages: implications for the modulation of inflammation and immunity. Antioxidants 11:861. doi:10.3390/antiox11050861 PubMed DOI PMC

Hentze MW, Muckenthaler MU, Galy B, Camaschella C. 2010. Two to tango: regulation of mammalian iron metabolism. Cell 142:24–38. doi:10.1016/j.cell.2010.06.028 PubMed DOI

Gazitt Y, Reddy SV, Alcantara O, Yang J, Boldt DH. 2001. A new molecular role for iron in regulation of cell cycling and differentiation of HL-60 human leukemia cells: iron is required for transcription of p21(WAF1/CIP1) in cells induced by phorbol myristate acetate. J Cell Physiol 187:124–135. doi:10.1002/1097-4652(2001)9999:9999<::AID-JCP1061>3.0.CO;2-E PubMed DOI

Kramer JL, Baltathakis I, Alcantara OSF, Boldt DH. 2002. Differentiation of functional dendritic cells and macrophages from human peripheral blood monocyte precursors is dependent on expression of p21 (WAF1/CIP1) and requires iron. Br J Haematol 117:727–734. doi:10.1046/j.1365-2141.2002.03498.x PubMed DOI

Alcantara O, Kalidas M, Baltathakis I, Boldt DH. 2001. Expression of multiple genes regulating cell cycle and apoptosis in differentiating hematopoietic cells is dependent on iron. Exp Hematol 29:1060–1069. doi:10.1016/s0301-472x(01)00683-x PubMed DOI

Andreesen R, Osterholz J, Bodemann H, Bross KJ, Costabel U, Löhr GW. 1984. Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes. Blut 49:195–202. doi:10.1007/BF00319822 PubMed DOI

Haldar M, Kohyama M, So AY-L, Kc W, Wu X, Briseño CG, Satpathy AT, Kretzer NM, Arase H, Rajasekaran NS, Wang L, Egawa T, Igarashi K, Baltimore D, Murphy TL, Murphy KM. 2014. Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages. Cell 156:1223–1234. doi:10.1016/j.cell.2014.01.069 PubMed DOI PMC

Aisen P. 2004. Transferrin receptor 1. Int J Biochem Cell Biol 36:2137–2143. doi:10.1016/j.biocel.2004.02.007 PubMed DOI

Dautry-Varsat A, Ciechanover A, Lodish HF. 1983. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A 80:2258–2262. doi:10.1073/pnas.80.8.2258 PubMed DOI PMC

Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA. 1997. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388:482–488. doi:10.1038/41343 PubMed DOI

Persson CG, Andersson M, Greiff L, Svensson C, Erjefält JS, Sundler F, Wollmer P, Alkner U, Erjefält I, Gustafsson B. 1995. Airway permeability. Clin Exp Allergy 25:807–814. doi:10.1111/j.1365-2222.1995.tb00022.x PubMed DOI PMC

Persson C. 2019. Airways exudation of plasma macromolecules: innate defense, epithelial regeneration, and asthma. J Allergy Clin Immunol 143:1271–1286. doi:10.1016/j.jaci.2018.07.037 PubMed DOI PMC

Persson CG, Erjefält JS, Greiff L, Erjefält I, Korsgren M, Linden M, Sundler F, Andersson M, Svensson C. 1998. Contribution of plasma-derived molecules to mucosal immune defence, disease and repair in the airways. Scand J Immunol 47:302–313. doi:10.1046/j.1365-3083.1998.00317.x PubMed DOI

Persson CG, Erjefält JS, Andersson M, Greiff L, Svensson C. 1996. Extravasation, lamina propria flooding and lumenal entry of bulk plasma exudate in mucosal defence, inflammation and repair. Pulm Pharmacol 9:129–139. doi:10.1006/pulp.1996.0015 PubMed DOI

Erjefält I, Persson CG. 1989. Inflammatory passage of plasma macromolecules into airway wall and lumen. Pulm Pharmacol 2:93–102. doi:10.1016/0952-0600(89)90030-6 PubMed DOI

Persson CG, Erjefält I, Alkner U, Baumgarten C, Greiff L, Gustafsson B, Luts A, Pipkorn U, Sundler F, Svensson C. 1991. Plasma exudation as a first line respiratory mucosal defence. Clin Exp Allergy 21:17–24. doi:10.1111/j.1365-2222.1991.tb00799.x PubMed DOI

Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman HJ, Law SK, Moestrup SK. 2001. Identification of the haemoglobin scavenger receptor. Nature 409:198–201. doi:10.1038/35051594 PubMed DOI

Finn AV, Nakano M, Polavarapu R, Karmali V, Saeed O, Zhao X, Yazdani S, Otsuka F, Davis T, Habib A, Narula J, Kolodgie FD, Virmani R. 2012. Hemoglobin directs macrophage differentiation and prevents foam cell formation in human atherosclerotic plaques. J Am Coll Cardiol 59:166–177. doi:10.1016/j.jacc.2011.10.852 PubMed DOI PMC

Schaer DJ, Schaer CA, Buehler PW, Boykins RA, Schoedon G, Alayash AI, Schaffner A. 2006. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood 107:373–380. doi:10.1182/blood-2005-03-1014 PubMed DOI

Lanceta L, Li C, Choi AM, Eaton JW. 2013. Haem oxygenase-1 overexpression alters intracellular iron distribution. Biochem J 449:189–194. doi:10.1042/BJ20120936 PubMed DOI

Harrison PM, Arosio P. 1996. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1275:161–203. doi:10.1016/0005-2728(96)00022-9 PubMed DOI

Osicka R, Osicková A, Basar T, Guermonprez P, Rojas M, Leclerc C, Sebo P. 2000. Delivery of CD8+ T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. Infect Immun 68:247–256. doi:10.1128/IAI.68.1.247-256.2000 PubMed DOI PMC

Franken KL, Hiemstra HS, van Meijgaarden KE, Subronto Y, den Hartigh J, Ottenhoff TH, Drijfhout JW. 2000. Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent. Protein Expr Purif 18:95–99. doi:10.1006/prep.1999.1162 PubMed DOI

Riedelberger M, Kuchler K. 2020. Analyzing the quenchable iron pool in murine macrophages by flow cytometry. Bio Protoc 10:e3552. doi:10.21769/BioProtoc.3552 PubMed DOI PMC

Christensen AE, Selheim F, de Rooij J, Dremier S, Schwede F, Dao KK, Martinez A, Maenhaut C, Bos JL, Genieser H-G, Døskeland SO. 2003. cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension. J Biol Chem 278:35394–35402. doi:10.1074/jbc.M302179200 PubMed DOI

Pizza M, Covacci A, Bartoloni A, Perugini M, Nencioni L, De Magistris MT, Villa L, Nucci D, Manetti R, Bugnoli M, Giovannoni F, Olivieri R, Barbieri JT, Sato H, Rappuoli R. 1989. Mutants of pertussis toxin suitable for vaccine development. Science 246:497–500. doi:10.1126/science.2683073 PubMed DOI

Komohara Y, Ohnishi K, Kuratsu J, Takeya M. 2008. Possible involvement of the M2 anti-inflammatory macrophage phenotype in growth of human gliomas. J Pathol 216:15–24. doi:10.1002/path.2370 PubMed DOI

Unlu G, Prizer B, Erdal R, Yeh HW, Bayraktar EC, Birsoy K. 2022. Metabolic-scale gene activation screens identify SLCO2B1 as a heme transporter that enhances cellular iron availability. Molecular Cell 82:2832–2843. doi:10.1016/j.molcel.2022.05.024 PubMed DOI PMC

Ma Y, Abbate V, Hider RC. 2015. Iron-sensitive fluorescent probes: monitoring intracellular iron pools. Metallomics 7:212–222. doi:10.1039/c4mt00214h PubMed DOI

Walkinshaw DR, Weist R, Xiao L, Yan K, Kim GW, Yang XJ. 2013. Dephosphorylation at a conserved SP motif governs cAMP sensitivity and nuclear localization of class IIa histone deacetylases. J Biol Chem 288:5591–5605. doi:10.1074/jbc.M112.445668 PubMed DOI PMC

Helmstadter KG, Ljubojevic-Holzer S, Wood BM, Taheri KD, Sedej S, Erickson JR, Bossuyt J, Bers DM. 2021. CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes. Basic Res Cardiol 116:11. doi:10.1007/s00395-021-00850-2 PubMed DOI PMC

Ha CH, Kim JY, Zhao J, Wang W, Jhun BS, Wong C, Jin ZG. 2010. PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A 107:15467–15472. doi:10.1073/pnas.1000462107 PubMed DOI PMC

Bridges KR, Cudkowicz A. 1984. Effect of iron chelators on the transferrin receptor in K562 cells. J Biol Chem 259:12970–12977. doi:10.1016/S0021-9258(18)90642-3 PubMed DOI

Bomford A, Isaac J, Roberts S, Edwards A, Young S, Williams R. 1986. The effect of desferrioxamine on transferrin receptors, the cell cycle and growth rates of human leukaemic cells. Biochem J 236:243–249. doi:10.1042/bj2360243 PubMed DOI PMC

Xing M, Post S, Ostrom RS, Samardzija M, Insel PA. 1999. Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells. J Biol Chem 274:10035–10038. doi:10.1074/jbc.274.15.10035 PubMed DOI

de Figueiredo P, Doody A, Polizotto RS, Drecktrah D, Wood S, Banta M, Strang MS, Brown WJ. 2001. Inhibition of transferrin recycling and endosome tubulation by phospholipase A2 antagonists. J Biol Chem 276:47361–47370. doi:10.1074/jbc.M108508200 PubMed DOI

Bate C, Williams A. 2015. cAMP-inhibits cytoplasmic phospholipase A2 and protects neurons against amyloid-β-induced synapse damage. Biology 4:591–606. doi:10.3390/biology4030591 PubMed DOI PMC

Gautam N, Hedqvist P, Lindbom L. 1998. Kinetics of leukocyte-induced changes in endothelial barrier function. Br J Pharmacol 125:1109–1114. doi:10.1038/sj.bjp.0702186 PubMed DOI PMC

Gautam N, Herwald H, Hedqvist P, Lindbom L. 2000. Signaling via β2 integrins triggers neutrophil-dependent alteration in endothelial barrier function. J Exp Med 191:1829–1839. doi:10.1084/jem.191.11.1829 PubMed DOI PMC

Zimmerman LI, Papin JF, Warfel J, Wolf RF, Kosanke SD, Merkel TJ. 2018. Histopathology of Bordetella pertussis in the baboon model. Infect Immun 86:e00511-18. doi:10.1128/IAI.00511-18 PubMed DOI PMC

Ahmad JN, Cerny O, Linhartova I, Masin J, Osicka R, Sebo P. 2016. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes. Cell Microbiol 18:384–398. doi:10.1111/cmi.12519 PubMed DOI

Adkins I, Kamanova J, Kocourkova A, Svedova M, Tomala J, Janova H, Masin J, Chladkova B, Bumba L, Kovar M, Ross PJ, Tuckova L, Spisek R, Mills KHG, Sebo P. 2014. Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells. PLoS One 9:e104064. doi:10.1371/journal.pone.0104064 PubMed DOI PMC

Leppla SH. 1984. Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells. Adv Cyclic Nucleotide Protein Phosphorylation Res 17:189–198. PubMed

Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW. 1998. ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc Natl Acad Sci U S A 95:13899–13904. doi:10.1073/pnas.95.23.13899 PubMed DOI PMC

Byrne JD, Betancourt T, Brannon-Peppas L. 2008. Active targeting schemes for nanoparticle systems in cancer therapeutics. Adv Drug Deliv Rev 60:1615–1626. doi:10.1016/j.addr.2008.08.005 PubMed DOI

Li JL, Wang L, Liu XY, Zhang ZP, Guo HC, Liu WM, Tang SH. 2009. In vitro cancer cell imaging and therapy using transferrin-conjugated gold nanoparticles. Cancer Lett 274:319–326. doi:10.1016/j.canlet.2008.09.024 PubMed DOI

White S, Taetle R, Seligman PA, Rutherford M, Trowbridge IS. 1990. Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in vitro and in vivo: evidence for synergistic antiproliferative effects. Cancer Res 50:6295–6301. PubMed

Lesley JF, Schulte RJ. 1985. Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies. Mol Cell Biol 5:1814–1821. doi:10.1128/mcb.5.8.1814-1821.1985 PubMed DOI PMC