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.

• Klíčová slova
• Bordetella pertussis, adenylate cyclase toxin, cyclic AMP, differentiation, iron acquisition, macrophages, monocytes,
• MeSH
• adenylátcyklasový toxin * metabolismus   genetika   MeSH
• AMP cyklický * metabolismus   MeSH
• antigeny CD14 * metabolismus   MeSH
• antigeny diferenciační myelomonocytární MeSH
• Bordetella pertussis * MeSH
• buněčná diferenciace * MeSH
• CD antigeny metabolismus   genetika   MeSH
• lidé MeSH
• monocyty * metabolismus   imunologie   mikrobiologie   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• receptory buněčného povrchu metabolismus   genetika   MeSH
• signální transdukce * MeSH
• upregulace MeSH
• železo metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• AMP cyklický * MeSH
• antigeny CD14 * MeSH
• antigeny diferenciační myelomonocytární MeSH
• CD antigeny MeSH
• CD14 protein, human MeSH Prohlížeč
• CD163 Antigen MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• receptory buněčného povrchu MeSH
• železo MeSH

INTRODUCTION: Tuberculosis (TB) remains the first cause of death from infection caused by a bacterial pathogen. Chemotherapy does not eradicate Mycobacterium tuberculosis (Mtb) from human lungs, and the pathogen causes a latent tuberculosis infection that cannot be prevented by the currently available Bacille Calmette Guerin (BCG) vaccine, which is ineffective in the prevention of pulmonary TB in adults. HLA-E-restricted CD8+ T lymphocytes are essential players in protective immune responses against Mtb. Hence, expanding this population in vivo or ex vivo may be crucial for vaccination or immunotherapy against TB. METHODS: The enzymatically inactive Bordetella pertussis adenylate cyclase (CyaA) toxoid is an effective tool for delivering peptide epitopes into the cytosol of antigen-presenting cells (APC) for presentation and stimulation of specific CD8+ T-cell responses. In this study, we have investigated the capacity of the CyaA toxoid to deliver Mtb epitopes known to bind HLA-E for the expansion of human CD8+ T cells in vitro. RESULTS: Our results show that the CyaA-toxoid containing five HLA-E-restricted Mtb epitopes causes significant expansion of HLA-E-restricted antigen-specific CD8+ T cells, which produce IFN-γ and exert significant cytotoxic activity towards peptide-pulsed macrophages. DISCUSSION: HLA-E represents a promising platform for the development of new vaccines; our study indicates that the CyaA construct represents a suitable delivery system of the HLA-E-binding Mtb epitopes for ex vivo and in vitro expansion of HLA-E-restricted CD8+ T cells inducing a predominant Tc1 cytokine profile with a significant increase of IFN-γ production, for prophylactic and immunotherapeutic applications against Mtb.

• Klíčová slova
• Bordetella pertussis adenylate cyclase, HLA-E, Mycobacterium tuberculosis, cytotoxic t lymphocytes, immunotherapy, peptides, vaccine,
• MeSH
• adenylátcyklasy MeSH
• antigeny HLA-E MeSH
• Bordetella pertussis MeSH
• CD8-pozitivní T-lymfocyty MeSH
• epitopy MeSH
• histokompatibilita - antigeny třídy I MeSH
• lidé MeSH
• Mycobacterium tuberculosis * MeSH
• peptidy MeSH
• toxoidy MeSH
• tuberkulóza * prevence a kontrola   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• antigeny HLA-E MeSH
• epitopy MeSH
• histokompatibilita - antigeny třídy I MeSH
• peptidy MeSH
• toxoidy 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
• dendritické buňky patologie   MeSH
• lymfatické uzliny patologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• pertuse * MeSH
• pertusový toxin MeSH
• plíce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pertusový 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
• adenylátcyklasový toxin MeSH
• bakteriální adheziny * metabolismus   MeSH
• Bordetella pertussis * genetika   MeSH
• faktory virulence rodu Bordetella genetika   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nosní dutina mikrobiologie   MeSH
• pertuse * přenos   MeSH
• pertusová vakcína MeSH
• protein MyD88 MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• bakteriální adheziny * MeSH
• faktory virulence rodu Bordetella MeSH
• pertusová vakcína MeSH
• protein MyD88 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.

• Klíčová slova
• Bordetella pertussis, adenylate cyclase toxin, dedifferentiation, macrophages, monocytes,
• MeSH
• adenylátcyklasový toxin farmakologie   fyziologie   MeSH
• alveolární makrofágy cytologie   účinky léků   MeSH
• AMP cyklický fyziologie   MeSH
• biologické modely MeSH
• Bordetella pertussis fyziologie   MeSH
• buněčná diferenciace MeSH
• dediferenciace buněk účinky léků   MeSH
• dýchací soustava účinky léků   imunologie   mikrobiologie   MeSH
• fagocytóza MeSH
• interakce hostitele a patogenu imunologie   MeSH
• lidé MeSH
• monocyty cytologie   účinky léků   MeSH
• myši MeSH
• prezentace antigenu účinky léků   MeSH
• přirozená imunita účinky léků   MeSH
• slizniční imunita účinky léků   MeSH
• systémy druhého messengeru účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• AMP cyklický MeSH

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

• MeSH
• AMP cyklický metabolismus   MeSH
• Bordetella pertussis metabolismus   MeSH
• cytoskeletální proteiny metabolismus   MeSH
• dendritické buňky metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• histondeacetylasy metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• molekuly buněčné adheze metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pertuse mikrobiologie   MeSH
• signální transdukce fyziologie   MeSH
• talin metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AMP cyklický MeSH
• CRTC3 protein, mouse MeSH Prohlížeč
• cytoskeletální proteiny MeSH
• Enah protein, mouse MeSH Prohlížeč
• fosfoproteiny MeSH
• Hdac5 protein, mouse MeSH Prohlížeč
• histondeacetylasy MeSH
• mikrofilamentové proteiny MeSH
• molekuly buněčné adheze MeSH
• talin MeSH
• Tln1 protein, mouse MeSH Prohlížeč
• transkripční faktory MeSH
• Vasodilator-Stimulated Phosphoprotein MeSH

Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

• Klíčová slova
• T-helper cells, immune response, intracellular pathways, phagocytosis,
• MeSH
• adenylátcyklasový toxin imunologie   MeSH
• AMP cyklický chemie   MeSH
• Bordetella pertussis MeSH
• buněčná imunita MeSH
• dendritické buňky imunologie   MeSH
• dýchací soustava imunologie   mikrobiologie   MeSH
• fagocytóza MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• neutrofily imunologie   MeSH
• pertuse imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• signální transdukce MeSH
• slizniční imunita MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• AMP cyklický MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 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+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b- cells. The nonhemolytic AC+ Hly- 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+ Hly- mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b- cells and/or the pore-forming 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 (>107 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.

• Klíčová slova
• Bordetella pertussis, adenylate cyclase toxin-hemolysin, cAMP intoxication, lung colonization, pore-forming activity, virulence,
• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• Bordetella pertussis patogenita   MeSH
• buněčná membrána metabolismus   MeSH
• dendritické buňky imunologie   MeSH
• fagocyty imunologie   MeSH
• hemolyziny metabolismus   MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pertuse mikrobiologie   MeSH
• plíce mikrobiologie   patologie   MeSH
• T-lymfocyty imunologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• AMP cyklický MeSH
• antigeny CD11b MeSH
• hemolyziny MeSH
• makrofágový antigen 1 MeSH

The mechanisms of immunogenicity underlying mild heat-shock (mHS) treatment < 42°C of tumor cells are largely attributed to the action of heat-shock proteins; however, little is known about the immunogenicity of tumor cells undergoing severe cytotoxic heat-shock treatment (sHS > 43°C). Here, we found that sHS, but not mHS (42°C), induces immunogenic cell death in human cancer cell lines as defined by the induction of ER stress response and ROS generation, cell surface exposure of calreticulin, HSP70 and HSP90, decrease of cell surface CD47, release of ATP and HMGB1. Only sHS-treated tumor cells were efficiently killed and phagocytosed by dendritic cells (DCs), which was partially dependent on cell surface calreticulin. DCs loaded with mHS or sHS-treated tumor cells displayed similar level of maturation and stimulated IFNγ-producing CD8+ T cells without any additional adjuvants in vitro. However, only DCs loaded with sHS-treated tumor cells stimulated antigen-specific CD4+ T cells and induced higher CD8+ T-cell activation and proliferation. sHS-treated murine cells also exposed calreticulin, HSP70 and HSP90 and activated higher DC maturation than mHS treated cells. Vaccination with sHS-treated tumor cells elicited protective immunity in mice. In this study, we defined specific conditions for the sHS treatment of human lung and ovarian tumor cells to arrive at optimal ratio between effective cell death, immunogenicity and content of tumor antigens for immunotherapeutic vaccine generation.

• Klíčová slova
• Antitumor immunity, calreticulin, cancer immunotherapy, dendritic cells, heat-shock treatment, hyperthermia, immunogenic cell death,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH