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 is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

• Keywords
• Bordetella, calcium homeostasis, effector protein BteA, host cell death mechanism, type III secretion system (T3SS),
• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Bordetella bronchiseptica genetics   metabolism   drug effects   MeSH
• Bordetella pertussis genetics   pathogenicity   metabolism   drug effects   MeSH
• Cell Death * drug effects   MeSH
• Endoplasmic Reticulum metabolism   drug effects   MeSH
• Homeostasis * MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Mitochondria metabolism   drug effects   MeSH
• Type III Secretion Systems metabolism   genetics   MeSH
• Calcium * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Type III Secretion Systems MeSH
• Calcium * 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 Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient 'contact weapons' that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections.

• Keywords
• Kingella kingae, RTX toxin, RtxA, membrane, pore-forming, β2 integrins,
• Publication type
• Journal Article MeSH
• Review MeSH

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMβ2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.

• Keywords
• Bordetella pertussis, RTX toxin, adenylate cyclase toxin, cAMP intoxication, lung colonization, lung inflammation, pore-forming activity, virulence,
• MeSH
• Adenylate Cyclase Toxin metabolism   MeSH
• Cyclic AMP metabolism   MeSH
• Bordetella pertussis pathogenicity   physiology   MeSH
• Phagocytes metabolism   microbiology   MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Sheep MeSH
• Cell Membrane Permeability MeSH
• Whooping Cough metabolism   microbiology   pathology   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Cyclic AMP 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

The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5'-untranslated region (5'-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways.IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.

• Keywords
• Bordetella pertussis, host-pathogen interactions, intracellular bacteria, macrophages, two-component regulatory systems, virulence regulation,
• Publication type
• Journal Article MeSH

Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis adenylate cyclase toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

• Keywords
• Bordetella pertussis, adenylate cyclase toxin, dedifferentiation, macrophages, monocytes,
• MeSH
• Adenylate Cyclase Toxin pharmacology   physiology   MeSH
• Macrophages, Alveolar cytology   drug effects   MeSH
• Cyclic AMP physiology   MeSH
• Models, Biological MeSH
• Bordetella pertussis physiology   MeSH
• Cell Differentiation MeSH
• Cell Dedifferentiation drug effects   MeSH
• Respiratory System drug effects   immunology   microbiology   MeSH
• Phagocytosis MeSH
• Host-Pathogen Interactions immunology   MeSH
• Humans MeSH
• Monocytes cytology   drug effects   MeSH
• Mice MeSH
• Antigen Presentation drug effects   MeSH
• Immunity, Innate drug effects   MeSH
• Immunity, Mucosal drug effects   MeSH
• Second Messenger Systems drug effects   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Cyclic AMP MeSH