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1.
PLoS Pathog ; 18(6): e1010577, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35666769

RESUMO

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.


Assuntos
Bordetella pertussis , Coqueluche , Animais , Células Dendríticas/patologia , Pulmão , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Toxina Pertussis
2.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445770

RESUMO

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.


Assuntos
Toxina Adenilato Ciclase/toxicidade , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema Respiratório/metabolismo , Sistema Respiratório/microbiologia , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mucina-5AC/metabolismo , Coqueluche/metabolismo , Coqueluche/microbiologia
3.
Int J Mol Sci ; 21(23)2020 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-33260488

RESUMO

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the ß2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the ß2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted ß2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.


Assuntos
Toxinas Bacterianas/metabolismo , Antígenos CD18/metabolismo , Membrana Celular/metabolismo , Kingella kingae/metabolismo , Oligossacarídeos/metabolismo , Animais , Morte Celular , Linhagem Celular , Feminino , Glicosídeo Hidrolases/metabolismo , Glicosilação , Humanos , Macrófagos/metabolismo , Camundongos , Oligossacarídeos/química , Ligação Proteica
4.
J Mol Biol ; 432(20): 5696-5710, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32860773

RESUMO

Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight ß strands into blocks that fold into calcium-binding parallel ß-roll structures. The ß-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five ß-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV-V of CyaA (CyaA1372-1681) that consists of a contiguous assembly of two ß-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the "push-ratchet" mechanism of the T1SS-mediated secretion of very large RTX proteins.


Assuntos
Toxina Adenilato Ciclase/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Bordetella pertussis/metabolismo , Toxina Adenilato Ciclase/genética , Toxina Adenilato Ciclase/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Citosol/metabolismo , Bactérias Gram-Negativas/metabolismo , Conformação Proteica , Dobramento de Proteína , Sistemas de Secreção Tipo I
5.
J Biol Chem ; 295(28): 9349-9365, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32393579

RESUMO

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLß2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMß2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400-710 of CyaA as an "AC translocon" sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.


Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella , Citosol/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Animais , Células CHO , Cricetulus , Feminino , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Transporte Proteico , Células THP-1
6.
Toxins (Basel) ; 9(10)2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934122

RESUMO

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.


Assuntos
Toxina Adenilato Ciclase/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Coqueluche/imunologia , Animais , Bordetella pertussis , AMP Cíclico/química , Interações Hospedeiro-Patógeno , Humanos , Imunidade Celular , Imunidade nas Mucosas , Fagocitose , Sistema Respiratório/imunologia , Sistema Respiratório/microbiologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia
7.
Mol Cell ; 62(1): 47-62, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058787

RESUMO

Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into ß-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Cálcio/metabolismo , Bactérias Gram-Negativas/metabolismo , Sistemas de Secreção Tipo I/metabolismo , Toxina Adenilato Ciclase/química , Toxina Adenilato Ciclase/metabolismo , Animais , Bordetella pertussis/química , Bordetella pertussis/enzimologia , Linhagem Celular , Bactérias Gram-Negativas/química , Camundongos , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína , Transporte Proteico
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