scholarly journals Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Shihono Teruya ◽  
Yukihiro Hiramatsu ◽  
Keiji Nakamura ◽  
Aya Fukui-Miyazaki ◽  
Kentaro Tsukamoto ◽  
...  
Keyword(s):  
Nervous System ◽  
Adenylate Cyclase ◽  
Virulence Factor ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Neural Cells ◽  
Intracerebral Injection ◽  
Content Type ◽  
Voltage Gated ◽  
Dermonecrotic Toxin

ABSTRACT Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis. The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed. IMPORTANCE Bordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

scholarly journals Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Mariette Barbier ◽  
Dylan T. Boehm ◽  
Emel Sen-Kilic ◽  
Claire Bonnin ◽  
Theo Pinheiro ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Mutant Strain ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Sigma Factor ◽  
Negative Regulator ◽  
Host Cells ◽  
Sequencing Analysis ◽  
Western Blots ◽  
Content Type

ABSTRACT Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

scholarly journals The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins ofBordetella bronchisepticaandB. parapertussis, but NotB. pertussis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Aya Fukui-Miyazaki ◽  
Hirono Toshima ◽  
Yukihiro Hiramatsu ◽  
Keisuke Okada ◽  
Keiji Nakamura ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Bordetella Pertussis ◽  
Host Factor ◽  
Target Cells ◽  
Content Type ◽  
Functional Differences ◽  
Adenylate Cyclase Toxin

ABSTRACTBordetella pertussis,Bordetella bronchiseptica, andBordetella parapertussisshare highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles inBordetellainfections. We herein demonstrate that CyaAs function as virulence factors differently betweenB. bronchiseptica/B. parapertussisandB. pertussis.B.bronchisepticaCyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly toB.pertussisCyaA. The hemolytic activity ofB.bronchisepticaCyaA on sheep erythrocytes was also preserved. However, in nucleated target cells,B.bronchisepticaCyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects ofB.bronchisepticaCyaA based on its enzyme activity were markedly attenuated.B.parapertussisCyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly toB.bronchisepticaCyaA; however,B.pertussisCyaA has Phe375instead of Ser, and thus, was not affected by 14-3-3. In addition,B.pertussisCyaA impaired the barrier function of epithelial cells, whereasB.bronchisepticaCyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity betweenB. bronchiseptica/B.parapertussisandB. pertussis.IMPORTANCEBordetella pertussis,B. bronchiseptica, andB. parapertussisare bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by eachBordetellaspecies. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor ofBordetella. The toxins ofB. bronchisepticaand presumablyB. parapertussiswere inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas theB. pertussistoxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences inBordetellaare attributed to the different activities of adenylate cyclase toxins.

scholarly journals Bordetella pertussis Virulence Factors Affect Phagocytosis by Human Neutrophils

2000 ◽  
Vol 68 (3) ◽  
pp. 1735-1739 ◽  
Author(s):  
Christine L. Weingart ◽  
Alison A. Weiss
Keyword(s):  
Adenylate Cyclase ◽  
Virulence Factors ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Human Neutrophils ◽  
Wild Type ◽  
Dermonecrotic Toxin ◽  
Adenylate Cyclase Toxin

ABSTRACT The interaction between human neutrophils and wild-typeBordetella pertussis or mutants expressing altered lipopolysaccharide or lacking virulence factors—pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, filamentous hemagglutinin (FHA), pertactin, or BrkA—was examined. In the absence of antibodies, the wild-type strain and the mutants, with the exception of mutants lacking FHA, attached efficiently to neutrophils. The addition of opsonizing antibodies caused a significant reduction (approximately 50%) in attachment of the wild-type strain and most of the mutants expressing FHA, suggesting that bacterium-mediated attachment is more efficient than Fc-mediated attachment. Phagocytosis was also examined. In the absence of antibodies, about 12% of the wild-type bacteria were phagocytosed. Opsonization caused a statistically significant reduction in phagocytosis (to 3%), possibly a consequence of reduced attachment. Phagocytosis of most of the mutants was similar to that of the wild type, with the exception of the mutants lacking adenylate cyclase toxin. About 70% of the adenylate cyclase toxin mutants were phagocytosed, but only in the presence of opsonizing antibody, suggesting that Fc receptor-mediated signaling may be needed for phagocytosis. These studies indicate that FHA mediates attachment ofB. pertussis to neutrophils, but adenylate cyclase toxin blocks phagocytosis.

scholarly journals Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Jason M. Warfel ◽  
Tod J. Merkel ◽  
Erik L. Hewlett
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunologic Response ◽  
Serum Samples ◽  
Content Type ◽  
Adenylate Cyclase Toxin ◽  
Strong Candidate ◽  
Candidate Antigen

ABSTRACT Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

scholarly journals BordetellaAdenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Jawid Nazir Ahmad ◽  
Jana Holubova ◽  
Oldrich Benada ◽  
Olga Kofronova ◽  
Ludek Stehlik ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Immune Evasion ◽  
Bordetella Pertussis ◽  
Alveolar Macrophages ◽  
Human Monocyte ◽  
Content Type ◽  
Macrophage Cells ◽  
Human Alveolar Macrophages ◽  
Adenylate Cyclase Toxin

ABSTRACTMonocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells.Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producingB. pertussisbacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression onin vitrodifferentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiatedin vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCEMacrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agentBordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion ofB. pertussisby suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

scholarly journals Pertussis Toxin and Adenylate Cyclase Toxin Provide a One-Two Punch for Establishment of Bordetella pertussis Infection of the Respiratory Tract

2005 ◽  
Vol 73 (5) ◽  
pp. 2698-2703 ◽  
Author(s):  
Nicholas H. Carbonetti ◽  
Galina V. Artamonova ◽  
Charlotte Andreasen ◽  
Nicholas Bushar
Keyword(s):  
Adenylate Cyclase ◽  
Respiratory Tract ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Infection Model ◽  
Wild Type ◽  
Adenylate Cyclase Toxin ◽  
Tract Infections

ABSTRACT Previously we found that pertussis toxin (PT), an exotoxin virulence factor produced by Bordetella pertussis, plays an important early role in colonization of the respiratory tract by this pathogen, using a mouse intranasal infection model. In this study, we examined the early role played by another exotoxin produced by this pathogen, adenylate cyclase toxin (ACT). By comparing a wild-type strain to a mutant strain (ΔCYA) with an in-frame deletion of the cyaA gene encoding ACT, we found that the lack of ACT confers a significant peak (day 7) colonization defect (1 to 2 log10). In mixed-infection experiments, the ΔCYA strain was significantly outcompeted by the wild-type strain, and intranasal administration of purified ACT did not increase colonization by ΔCYA. These data suggest that ACT benefits the bacterial cells that produce it and, unlike PT, does not act as a soluble factor benefiting the entire infecting bacterial population. Comparison of lower respiratory tract infections over the first 4 days after inoculation revealed that the colonization defect of the PT deletion strain was apparent earlier than that of ΔCYA, suggesting that PT plays an earlier role than ACT in the establishment of B. pertussis infection. Examination of cells in the bronchoalveolar lavage fluid of infected mice revealed that, unlike PT, ACT does not appear to inhibit neutrophil influx to the respiratory tract early after infection but may combat neutrophil activity once influx has occurred.

scholarly journals Overexpression of the RNA Polymerase Alpha Subunit Reduces Transcription of Bvg-Activated Virulence Genes inBordetella pertussis

2000 ◽  
Vol 182 (2) ◽  
pp. 529-531 ◽  
Author(s):  
Nicholas H. Carbonetti ◽  
Alla Romashko ◽  
Teresa J. Irish
Keyword(s):  
Rna Polymerase ◽  
Virulence Factor ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Virulence Genes ◽  
Transcriptional Activator ◽  
Alpha Subunit ◽  
Terminal Domain

ABSTRACT Overexpression of the RNA polymerase alpha subunit inBordetella pertussis reduces expression of the virulence factor pertussis toxin. Here we show that this reduction is at the level of transcription, is reversed by overexpression of the transcriptional activator BvgA, and is dependent on the C-terminal domain of alpha.

scholarly journals Cyclic AMP-Mediated Suppression of Neutrophil Extracellular Trap Formation and Apoptosis by the Bordetella pertussis Adenylate Cyclase Toxin

2014 ◽  
Vol 82 (12) ◽  
pp. 5256-5269 ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Erik L. Hewlett
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Neutrophil Function ◽  
Neutrophil Extracellular Trap ◽  
Target Cells ◽  
Content Type ◽  
Extracellular Traps ◽  
Adenylate Cyclase Toxin ◽  
Insight Into

ABSTRACTThe adenylate cyclase toxin (ACT) ofBordetella pertussisintoxicates target cells by generating supraphysiologic levels of intracellular cyclic AMP (cAMP). Since ACT kills macrophages rapidly and potently, we asked whether ACT would also kill neutrophils. In fact, ACT prolongs the neutrophil life span by inhibiting constitutive apoptosis and preventing apoptosis induced by exposure to liveB. pertussis. Imaging ofB. pertussis-exposed neutrophils revealed thatB. pertussislacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-typeB. pertussisdoes not, suggesting that ACT suppresses NET formation. Indeed, ACT inhibits formation of NETs by generating cAMP and consequently inhibiting the oxidative burst. Convalescent-phase serum from humans following clinical pertussis blocks the ACT-mediated suppression of NET formation. These studies provide novel insight into the phagocyte impotence caused by ACT, which not only impairs neutrophil function but also inhibits death of neutrophils by apoptosis and NETosis.

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