Acylated domains (ADs), like that of the Bordetella pertussis adenylate cyclase toxin (CyaA), are structures found in all pore-forming toxins from the family of Repeat-in-ToXin (RTX) proteins. These AD segments are fatty-acylated on ε-amino groups of conserved lysine residues, such as the K860 and K983 residues of CyaA. The ε-amide-linked acyl chains are essential for toxin activity and promote irreversible membrane insertion of the CyaA molecule, thus enabling the toxin to translocate its N-terminal adenyl cyclase enzyme domain into the host cell cytoplasm. In parallel, the membrane-inserted CyaA molecules can oligomerize into cation-selective pores in the plasma membrane. Here, we show that the attached acyl chains are not only crucial for membrane insertion of the toxin but also play an important role in CyaA folding. We demonstrate that assembly of the noncanonical β-roll structure in the C-terminal segment of the AD of CyaA is cooperatively directed by the Ca2+-driven folding of the adjacent RTX domain. In contrast, the N-terminal AD segment consists of an α-helical structure that folds independently of Ca2+ ion binding and may form one or two acyl binding site(s) accommodating the acyl chains protruding from the C-terminal AD segment. This acyl-mediated interaction between the N- and C-terminal segments promotes local structural rearrangements within the AD that significantly enhances the stability of the toxin molecule. These findings highlight the critical role of the acyl modification in membrane interaction capacity and structural stability of the CyaA toxin.

• Keywords
• Bordetella pertussis, RTX toxin, acylation, adenylate cyclase toxin, protein folding,
• MeSH
• Acylation MeSH
• Adenylate Cyclase Toxin * metabolism   chemistry   genetics   MeSH
• Bordetella pertussis * metabolism   enzymology   genetics   MeSH
• Cell Membrane * metabolism   MeSH
• Humans MeSH
• Protein Domains MeSH
• Protein Folding MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• Calcium MeSH

Bordetella pertussis infects human upper airways and deploys an array of immunosuppressive virulence factors, among which the adenylate cyclase toxin (CyaA) plays a prominent role in disarming host phagocytes. CyaA binds the complement receptor-3 (CR3 aka αMβ2 integrin CD11b/CD18 or Mac-1) of myeloid cells and delivers into their cytosol an adenylyl cyclase enzyme that hijacks cellular signaling through unregulated conversion of cytosolic ATP to cAMP. We found that the action of as little CyaA as 22 pM (4 ng/mL) blocks macrophage colony-stimulating factor (M-CSF)-driven transition of migratory human CD14+ monocytes into macrophages. Global transcriptional profiling (RNAseq) revealed that exposure of monocytes to 22 pM CyaA for 40 hours in culture with 20 ng/mL of M-CSF led to upregulation of genes that exert negative control of monocyte to macrophage differentiation (e.g., SERPINB2, DLL1, and CSNK1E). The sustained CyaA action yielded downregulation of numerous genes involved in processes crucial for host defense, such as myeloid cell differentiation, chemotaxis of inflammatory cells, antigen presentation, phagocytosis, and bactericidal activities. CyaA-elicited signaling also promoted deacetylation and trimethylation of lysines 9 and 27 of histone 3 (H3K9me3 and H3K27me3) and triggered the formation of transcriptionally repressive heterochromatin patches in the nuclei of CyaA-exposed monocytes. These effects were partly reversed by the G9a methyltransferase inhibitor UNC 0631 and by the pleiotropic HDAC inhibitor Trichostatin-A, revealing that CyaA-elicited epigenetic alterations mediate transcriptional reprogramming of monocytes and play a role in CyaA-triggered block of monocyte differentiation into bactericidal macrophage cells.IMPORTANCETo proliferate on host airway mucosa and evade elimination by patrolling sentinel cells, the whooping cough agent Bordetella pertussis produces a potently immunosubversive adenylate cyclase toxin (CyaA) that blocks opsonophagocytic killing of bacteria by phagocytes like neutrophils and macrophages. Indeed, chemotactic migration of CD14+ monocytes to the infection site and their transition into bactericidal macrophages, thus replenishing the exhausted mucosa-patrolling macrophages, represents one of the key mechanisms of innate immune defense to infection. We show that the cAMP signaling action of CyaA already at a very low toxin concentration triggers massive transcriptional reprogramming of monocytes that is accompanied by chromatin remodeling and epigenetic histone modifications, which block the transition of migratory monocytes into bactericidal macrophage cells. This reveals a novel layer of toxin action-mediated hijacking of functional differentiation of innate immune cells for the sake of mucosal pathogen proliferation and transmission to new hosts.

• Keywords
• Bordetella pertussis, RTX toxins, cyclic AMP, differentiation, epigenetics, macrophages, monocytes,
• MeSH
• Adenylate Cyclase Toxin * metabolism   MeSH
• Bordetella pertussis * pathogenicity   enzymology   MeSH
• Cell Differentiation * drug effects   MeSH
• Macrophage Colony-Stimulating Factor MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Macrophages * drug effects   cytology   MeSH
• Monocytes * drug effects   cytology   physiology   MeSH
• Cellular Reprogramming * MeSH
• Chromatin Assembly and Disassembly * drug effects   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• Macrophage Colony-Stimulating Factor MeSH

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.

• Keywords
• Bordetella pertussis, adenylate cyclase toxin, cyclic AMP, differentiation, iron acquisition, macrophages, monocytes,
• MeSH
• Adenylate Cyclase Toxin * metabolism   genetics   MeSH
• Cyclic AMP * metabolism   MeSH
• CD163 Antigen MeSH
• Antigens, Differentiation, Myelomonocytic MeSH
• Bordetella pertussis * MeSH
• Cell Differentiation * MeSH
• Antigens, CD metabolism   genetics   MeSH
• Humans MeSH
• Lipopolysaccharide Receptors * metabolism   MeSH
• Monocytes * metabolism   immunology   microbiology   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• Receptors, Cell Surface metabolism   genetics   MeSH
• Signal Transduction * MeSH
• Up-Regulation MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• Cyclic AMP * MeSH
• CD163 Antigen MeSH
• Antigens, Differentiation, Myelomonocytic MeSH
• Antigens, CD MeSH
• CD14 protein, human MeSH Browser
• Lipopolysaccharide Receptors * MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• Receptors, Cell Surface MeSH
• Iron MeSH

The adenylate cyclase (ACT) and the pertussis (PT) toxins of Bordetella pertussis exert potent immunomodulatory activities that synergize to suppress host defense in the course of whooping cough pathogenesis. We compared the mouse lung infection capacities of B. pertussis (Bp) mutants (Bp AC- or Bp PT-) producing enzymatically inactive toxoids and confirm that ACT action is required for maximal bacterial proliferation in the first days of infection, whereas PT action is crucial for persistence of B. pertussis in mouse lungs. Despite accelerated and near complete clearance from the lungs by day 14 of infection, the PT- bacteria accumulated within the lymphoid tissue of lung-draining mediastinal lymph nodes (mLNs). In contrast, the wild type or AC- bacteria colonized the lungs but did not enter into mLNs. Lung infection by the PT- mutant triggered an early arrival of migratory conventional dendritic cells with associated bacteria into mLNs, where the PT- bacteria entered the T cell-rich paracortex of mLNs by day 5 and proliferated in clusters within the B-cell zone (cortex) of mLNs by day 14, being eventually phagocytosed by infiltrating neutrophils. Finally, only infection by the PT- bacteria triggered an early production of anti-B. pertussis serum IgG antibodies already within 14 days of infection. These results reveal that action of the pertussis toxin blocks DC-mediated delivery of B. pertussis bacteria into mLNs and prevents bacterial colonization of mLNs, thus hampering early adaptive immune response to B. pertussis infection.

• MeSH
• Bordetella pertussis * MeSH
• Dendritic Cells pathology   MeSH
• Lymph Nodes pathology   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Whooping Cough * MeSH
• Pertussis Toxin MeSH
• Lung MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Pertussis Toxin MeSH

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 108 viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.

• MeSH
• Adenylate Cyclase Toxin MeSH
• Adhesins, Bacterial * metabolism   MeSH
• Bordetella pertussis * genetics   MeSH
• Virulence Factors, Bordetella genetics   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Myeloid Differentiation Factor 88 MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Nasal Cavity microbiology   MeSH
• Whooping Cough * transmission   MeSH
• Pertussis Vaccine MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Adhesins, Bacterial * MeSH
• Virulence Factors, Bordetella MeSH
• Myeloid Differentiation Factor 88 MeSH
• Pertussis Vaccine MeSH

The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air-liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC- toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT- toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.

• Keywords
• Bordetella, CREB, adenylate cyclase toxin, cAMP, epithelium, mucin, pertussis toxin,
• MeSH
• Adenylate Cyclase Toxin toxicity   MeSH
• Bordetella pertussis metabolism   pathogenicity   MeSH
• Cell Line MeSH
• Respiratory System metabolism   microbiology   MeSH
• Epithelial Cells metabolism   microbiology   MeSH
• Humans MeSH
• Mucin 5AC metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Whooping Cough metabolism   microbiology   MeSH
• Cyclic AMP Response Element-Binding Protein metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Mucin 5AC MeSH
• Cyclic AMP Response Element-Binding Protein MeSH