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1.
Sci Rep ; 11(1): 19814, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615931

RESUMO

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines  by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.


Assuntos
Toxina Adenilato Ciclase/genética , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica , Bordetella pertussis , Animais , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/metabolismo , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Membrana Celular/metabolismo , Feminino , Hemólise , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células THP-1
2.
Infect Immun ; 85(6)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28396322

RESUMO

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMß2 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.


Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/patogenicidade , AMP Cíclico/metabolismo , Proteínas Hemolisinas/metabolismo , Antígeno de Macrófago 1/metabolismo , Coqueluche/microbiologia , Animais , Antígeno CD11b/metabolismo , Membrana Celular/metabolismo , Células Dendríticas/imunologia , Feminino , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fagócitos/imunologia , Linfócitos T/imunologia , Virulência
3.
J Immunol ; 194(10): 4901-13, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25876760

RESUMO

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates complement receptor 3-expressing phagocytes and catalyzes uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by complement-dependent oxidative burst and opsonophagocytic mechanisms. We show that cAMP signaling through the protein kinase A (PKA) pathway activates Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 and suppresses production of bactericidal NO in macrophage cells. Selective activation of PKA by the cell-permeable analog N(6)-benzoyladenosine-3',5'-cyclic monophosphate interfered with LPS-induced inducible NO synthase (iNOS) expression in RAW264.7 macrophages, whereas inhibition of PKA by H-89 largely restored the production of iNOS in CyaA-treated murine macrophages. CyaA/cAMP signaling induced SHP phosphatase-dependent dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and thereby inhibited TLR4-triggered induction of iNOS gene expression. Selective small interfering RNA knockdown of SHP-1, but not of the SHP-2 phosphatase, rescued production of TLR-inducible NO in toxin-treated cells. Finally, inhibition of SHP phosphatase activity by NSC87877 abrogated B. pertussis survival inside murine macrophages. These results reveal that an as yet unknown cAMP-activated signaling pathway controls SHP-1 phosphatase activity and may regulate numerous receptor signaling pathways in leukocytes. Hijacking of SHP-1 by CyaA action then enables B. pertussis to evade NO-mediated killing in sentinel cells of innate immunity.


Assuntos
Toxina Adenilato Ciclase/imunologia , Infecções por Bordetella/imunologia , Macrófagos/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Animais , Infecções por Bordetella/enzimologia , Bordetella pertussis/imunologia , Linhagem Celular , AMP Cíclico , Ativação Enzimática/imunologia , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia
4.
Infect Immun ; 81(11): 4081-90, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23980112

RESUMO

Bordetella pertussis is a Gram-negative pathogen causing the human respiratory disease called pertussis or whooping cough. Here we examined the role of the RNA chaperone Hfq in B. pertussis virulence. Hfq mediates interactions between small regulatory RNAs and their mRNA targets and thus plays an important role in posttranscriptional regulation of many cellular processes in bacteria, including production of virulence factors. We characterized an hfq deletion mutant (Δhfq) of B. pertussis 18323 and show that the Δhfq strain produces decreased amounts of the adenylate cyclase toxin that plays a central role in B. pertussis virulence. Production of pertussis toxin and filamentous hemagglutinin was affected to a lesser extent. In vitro, the ability of the Δhfq strain to survive within macrophages was significantly reduced compared to that of the wild-type (wt) strain. The virulence of the Δhfq strain in the mouse respiratory model of infection was attenuated, with its capacity to colonize mouse lungs being strongly reduced and its 50% lethal dose value being increased by one order of magnitude over that of the wt strain. In mixed-infection experiments, the Δhfq strain was then clearly outcompeted by the wt strain. This requirement for Hfq suggests involvement of small noncoding RNA regulation in B. pertussis virulence.


Assuntos
Bordetella pertussis/patogenicidade , Fator Proteico 1 do Hospedeiro/metabolismo , Fatores de Virulência/metabolismo , Animais , Carga Bacteriana , Bordetella pertussis/genética , Modelos Animais de Doenças , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Dose Letal Mediana , Pulmão/microbiologia , Camundongos , Toxina Pertussis/metabolismo , Análise de Sobrevida , Virulência , Fatores de Virulência/genética , Coqueluche/microbiologia , Coqueluche/patologia
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