Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

Osička, Radim; Osičková, Adriana; Hasan, Shakir; Bumba, Ladislav; Černý, Jiří; Šebo, Peter

doi:10.7554/elife.10766

Cited by 69 publications

(129 citation statements)

References 84 publications

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“…The ACT species selectivity and truncation data suggested a role for RTX blocks II–III, while earlier work suggested that the receptor-binding region lies between RTX residues 1166–1287 that span blocks II–III. 15, 30 We amplified codons 751–1358 (about two-thirds of RTX 751 ) under error-prone conditions. A library with 2.1 × 10 7 clones was constructed using homologous recombination of Nhe I/ Sph I linearized vector backbone and error prone PCR product with overhangs on both ends.…”

Section: Resultsmentioning

confidence: 99%

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Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin

et al. 2017

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Adenylate cyclase toxin (ACT) is an important B. pertussis virulence factor that is not included in current acellular pertussis vaccines. We previously demonstrated that immunization with the repeat-in-toxin (RTX) domain of ACT elicits neutralizing antibodies in mice and discovered the first two antibodies to neutralize ACT activities by occluding the receptor-binding site. Here, we fully characterized these antibodies and their epitopes. Both antibodies bind ACT with low nanomolar affinity and cross-react with ACT homologs produced by B. parapertussis and B. bronchiseptica. Antibody M1H5 binds B. pertussis RTX751 ~100-fold tighter than RTX751 from the other two species, while antibody M2B10 has similar affinity for all three variants. To initially map the antibody epitopes, we generated a series of ACT chimeras and truncation variants, which implicated the repeat blocks II–III. To identify individual epitope residues, we displayed randomly mutated RTX751 libraries on yeast and isolated clones with decreased antibody binding by flow cytometry. Next-generation sequencing identified candidate epitope residues based on enrichment of clones with mutations at specific positions. These epitopes form two adjacent surface patches on a model of the RTX751 domain, one for each antibody. Notably, the cellular receptor also binds within blocks II–III and shares at least one residue with the M1H5 epitope. A predicted structural model of RTX751 supports the notion that the antibody and receptor epitopes overlap. These data provide insight into mechanisms of ACT neutralization and guidance for engineering more stable RTX variants that may be more appropriate vaccine antigens.

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Section: Resultsmentioning

confidence: 99%

“…The sole shared residue is indicated by bold and purple. Bold, underlined and italized residues indicate those implicated in receptor binding 30 . The boxes indicate the first three Gly-Asp rich repeat blocks.…”

Section: Figurementioning

confidence: 99%

Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin

et al. 2017

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“…It is worth noting that, besides interfering with the bottleneck step at the level of PLC activation, the CyaA-generated cAMP signaling can provoke inhibition of the ROS-inducing pathways also upstream of PLC, at the level of signaling of opsonin receptors. Indeed, we have recently found that CyaA-generated cAMP signaling provokes inactivation of Syk in human monocytes, thereby interfering with the iC3b-activated prophagocytic and ROS-inducing signaling of CR3 through Syk (10).…”

Section: Discussionmentioning

confidence: 99%

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

Černý

Anderson

Stephens

et al. 2017

The Journal of Immunology

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The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in immune evasion and virulence of the whooping cough agent Bordetella pertussis. CyaA penetrates the complement receptor 3–expressing phagocytes and ablates their bactericidal capacities by catalyzing unregulated conversion of cytosolic ATP to the key second messenger molecule cAMP. We show that signaling of CyaA-generated cAMP blocks the oxidative burst capacity of neutrophils by two converging mechanisms. One involves cAMP/protein kinase A–mediated activation of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and limits the activation of MAPK ERK and p38 that are required for assembly of the NADPH oxidase complex. In parallel, activation of the exchange protein directly activated by cAMP (Epac) provokes inhibition of the phospholipase C by an as yet unknown mechanism. Indeed, selective activation of Epac by the cell-permeable analog 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate counteracted the direct activation of phospholipase C by 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide. Hence, by inhibiting production of the protein kinase C–activating lipid, diacylglycerol, cAMP/Epac signaling blocks the bottleneck step of the converging pathways of oxidative burst triggering. Manipulation of neutrophil membrane composition by CyaA-produced signaling of cAMP thus enables B. pertussis to evade the key innate host defense mechanism of reactive oxygen species–mediated killing of bacteria by neutrophils.

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“…Upon initial interaction with N-linked oligosaccharides (31,32), CyaA specifically binds a loop outside the I domain of the CD11b subunit of the ␣ M ␤ 2 integrin (CR3) (5), and the toxin delivers the AC enzyme into phagocyte cytosol in two steps (33). Compelling indirect evidence suggests that the AC-translocating and pore-forming activities of CyaA are mutually independent and are accomplished by two distinct subpopulations of CyaA conformers that employ the same transmembrane CyaA segments in an alternative manner (34).…”

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Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

et al. 2017

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The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, ␣ M ␤ 2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b ϩ ) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b Ϫ cells. The nonhemolytic AC ϩ Hly Ϫ bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC ϩ Hly Ϫ mutant also infected mouse lungs as efficiently as the parental AC ϩ Hly ϩ strain. Hence, elevation of cAMP in CD11b Ϫ cells and/or the poreforming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (Ͼ10 7 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.KEYWORDS Bordetella pertussis, adenylate cyclase toxin-hemolysin, cAMP intoxication, lung colonization, pore-forming activity, virulence T he adenylate cyclase toxin-hemolysin is produced by all three Bordetella species pathogenic to mammals, and it plays a prominent role in the early phases of respiratory tract infection by the whooping cough agent, Bordetella pertussis (1-3). The toxin specifically binds the CD11b subunit of the complement receptor 3 (CR3) (4, 5) and exerts an array of immunosubversive and cytotoxic activities on myeloid phagocytes. CyaA delivers a cell-invasive adenylyl cyclase (AC) enzyme domain into cytosol of CD11b ϩ cells, where the AC is activated by calmodulin and converts cytosolic ATP to the signaling molecule cyclic AMP (cAMP). The generated supraphysiological levels of cAMP then nearly instantly ablate the bactericidal oxidative burst and opsonophago-

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Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

Cited by 69 publications

References 84 publications

Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin

Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

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