Publications by authors named "Nicholas H Carbonetti"

33 Publications

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo.

Sci Rep 2021 Mar 8;11(1):5429. Epub 2021 Mar 8.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.
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http://dx.doi.org/10.1038/s41598-021-84817-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940712PMC
March 2021

Highlights of the 12th International Bordetella Symposium.

Clin Infect Dis 2020 12;71(9):2521-2526

Laboratory of Vaccinology and Mucosal Immunity, Université Libre de Bruxelles, Brussels, Belgium.

To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.
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http://dx.doi.org/10.1093/cid/ciaa651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713684PMC
December 2020

Age-Dependent Effects of Type I and Type III IFNs in the Pathogenesis of Infection and Disease.

J Immunol 2020 04 9;204(8):2192-2202. Epub 2020 Mar 9.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; and

Type I and III IFNs play diverse roles in bacterial infections, being protective for some but deleterious for others. Using RNA-sequencing transcriptomics we investigated lung gene expression responses to infection in adult mice, revealing that type I and III IFN pathways may play an important role in promoting inflammatory responses. In infected mice, lung type I/III IFN responses correlated with increased proinflammatory cytokine expression and with lung inflammatory pathology. In mutant mice with increased type I IFN receptor (IFNAR) signaling, infection exacerbated lung inflammatory pathology, whereas knockout mice with defects in type I IFN signaling had lower levels of lung inflammation than wild-type mice. Curiously, -infected IFNAR1 knockout mice had wild-type levels of lung inflammatory pathology. However, in response to infection these mice had increased levels of type III IFN expression, neutralization of which reduced lung inflammation. In support of this finding, -infected mice with a knockout mutation in the type III IFN receptor (IFNLR1) and double IFNAR1/IFNLR1 knockout mutant mice had reduced lung inflammatory pathology compared with that in wild-type mice, indicating that type III IFN exacerbates lung inflammation. In marked contrast, infant mice did not upregulate type I or III IFNs in response to infection and were protected from lethal infection by increased type I IFN signaling. These results indicate age-dependent effects of type I/III IFN signaling during infection and suggest that these pathways represent targets for therapeutic intervention in pertussis.
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http://dx.doi.org/10.4049/jimmunol.1900912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141952PMC
April 2020

Peptidoglycan Recognition Protein 4 Suppresses Early Inflammatory Responses to and Contributes to Sphingosine-1-Phosphate Receptor Agonist-Mediated Disease Attenuation.

Infect Immun 2019 02 24;87(2). Epub 2019 Jan 24.

Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore, Maryland, USA.

Incidence of whooping cough (pertussis), a bacterial infection of the respiratory tract caused by the bacterium , has reached levels not seen since the 1950s. Antibiotics fail to improve the course of disease unless administered early in infection. Therefore, there is an urgent need for the development of antipertussis therapeutics. Sphingosine-1-phosphate receptor (S1PR) agonists have been shown to reduce pulmonary inflammation during infection in mouse models. However, the mechanisms by which S1PR agonists attenuate pertussis disease are unknown. We report the results of a transcriptome sequencing study examining pulmonary transcriptional responses in -infected mice treated with S1PR agonist AAL-R or vehicle control. This study identified peptidoglycan recognition protein 4 (PGLYRP4) as one of the most highly upregulated genes in the lungs of infected mice following S1PR agonism. PGLYRP4, a secreted, innate mediator of host defenses, was found to limit early inflammatory pathology in knockout mouse studies. Further, S1PR agonist AAL-R failed to attenuate pertussis disease in PGLYRP4 knockout (KO) mice. virulence factor tracheal cytotoxin (TCT), a secreted peptidoglycan breakdown product, induces host tissue damage. TCT-oversecreting strains were found to drive an early inflammatory response similar to that observed in PGLYRP4 KO mice. Further, TCT-oversecreting strains induced significantly greater pathology in PGLYRP4-deficient animals than their wild-type counterparts. Together, these data indicate that S1PR agonist-mediated protection against pertussis disease is PGLYRP4 dependent. Our data suggest PGLYRP4 functions, in part, by preventing TCT-induced airway damage.
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http://dx.doi.org/10.1128/IAI.00601-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346131PMC
February 2019

Fatal Pertussis in the Neonatal Mouse Model Is Associated with Pertussis Toxin-Mediated Pathology beyond the Airways.

Infect Immun 2017 11 18;85(11). Epub 2017 Oct 18.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA

In infants, can cause severe disease, manifested as pronounced leukocytosis, pulmonary hypertension, and even death. The exact cause of death remains unknown, and no effective therapies for treating fulminant pertussis exist. In this study, a neonatal mouse model of critical pertussis is characterized, and a central role for pertussis toxin (PT) is described. PT promoted colonization, leukocytosis, T cell phenotypic changes, systemic pathology, and death in neonatal but not adult mice. Surprisingly, PT inhibited lung inflammatory pathology in neonates, a result which contrasts dramatically with observed PT-promoted pathology in adult mice. Infection with a PT-deficient strain induced severe pulmonary inflammation but not mortality in neonatal mice, suggesting that death in these mice was not associated with impaired lung function. Dissemination of infection beyond the lungs was also detected in neonatal mice, which may contribute to the observed systemic effects of PT. We propose that it is the systemic activity of pertussis toxin and not pulmonary pathology that promotes mortality in critical pertussis. In addition, we observed transmission of infection between neonatal mice, the first report of transmission in mice. This model will be a valuable tool to investigate causes of pertussis pathogenesis and identify potential therapies for critical pertussis.
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http://dx.doi.org/10.1128/IAI.00355-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649019PMC
November 2017

Reduction of Pertussis Inflammatory Pathology by Therapeutic Treatment With Sphingosine-1-Phosphate Receptor Ligands by a Pertussis Toxin-Insensitive Mechanism.

J Infect Dis 2017 01;215(2):278-286

Department of Microbiology and Immunology.

Recent data have demonstrated the potential of sphingosine 1-phosphate (S1P) receptor (S1PR) agonism in the treatment of infectious diseases. A previous study used a murine model of Bordetella pertussis infection to demonstrate that treatment with the S1PR agonist AAL-R reduces pulmonary inflammation during infection. In the current study, we showed that this effect is mediated via the S1PR1 on LysM+ (myeloid) cells. Signaling via this receptor results in reduced lung inflammation and cellular recruitment as well as reduced morbidity and mortality in a neonatal mouse model of disease. Despite the fact that S1PRs are pertussis toxin-sensitive G protein-coupled receptors, the effects of AAL-R were pertussis toxin insensitive in our model. Furthermore, our data demonstrate that S1PR agonist administration may be effective at therapeutic time points. These results indicate a role for S1P signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis.
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http://dx.doi.org/10.1093/infdis/jiw536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853922PMC
January 2017

Highlights of the 11th International Bordetella Symposium: from Basic Biology to Vaccine Development.

Clin Vaccine Immunol 2016 Nov 4;23(11):842-850. Epub 2016 Nov 4.

CINDEFI (CONICET-UNLP), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.

Pertussis is a severe respiratory disease caused by infection with the bacterial pathogen Bordetella pertussis The disease affects individuals of all ages but is particularly severe and sometimes fatal in unvaccinated young infants. Other Bordetella species cause diseases in humans, animals, and birds. Scientific, clinical, public health, vaccine company, and regulatory agency experts on these pathogens and diseases gathered in Buenos Aires, Argentina from 5 to 8 April 2016 for the 11th International Bordetella Symposium to discuss recent advances in our understanding of the biology of these organisms, the diseases they cause, and the development of new vaccines and other strategies to prevent these diseases. Highlights of the meeting included pertussis epidemiology in developing nations, genomic analysis of Bordetella biology and evolution, regulation of virulence factor expression, new model systems to study Bordetella biology and disease, effects of different vaccines on immune responses, maternal immunization as a strategy to prevent newborn disease, and novel vaccine development for pertussis. In addition, the group approved the formation of an International Bordetella Society to promote research and information exchange on bordetellae and to organize future meetings. A new Bordetella.org website will also be developed to facilitate these goals.
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http://dx.doi.org/10.1128/CVI.00388-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098017PMC
November 2016

Pertussis leukocytosis: mechanisms, clinical relevance and treatment.

Pathog Dis 2016 10 7;74(7). Epub 2016 Sep 7.

Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St., HSF-I 380, Baltimore, MD 21201, USA

The significant and sometimes dramatic rise in the number of circulating white blood cells (leukocytosis) in infants suffering from pertussis (whooping cough) has been recognized for over a century. Although pertussis is a disease that afflicts people of all ages, it can be particularly severe in young infants, and these are the individuals in whom leukocytosis is most pronounced. Very high levels of leukocytosis are associated with poor outcome in infants hospitalized with pertussis and modern treatments are often aimed at reducing the number of leukocytes. Pertussis leukocytosis is caused by pertussis toxin, a soluble protein toxin released by Bordetella pertussis during infection, but the exact mechanisms by which this occurs are still unclear. In this minireview, I discuss the history of clinical and experimental findings on pertussis leukocytosis, possible contributing mechanisms causing this condition and treatments aimed at reducing leukocytosis in hospitalized infants. Since recent studies have detailed significant associations between specific levels of pertussis leukocytosis and fatal outcome, this is a timely review that may stimulate new thinking on how to understand and combat this problem.
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http://dx.doi.org/10.1093/femspd/ftw087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761200PMC
October 2016

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

PLoS Pathog 2016 08 2;12(8):e1005803. Epub 2016 Aug 2.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.
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http://dx.doi.org/10.1371/journal.ppat.1005803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970705PMC
August 2016

Intracellular disassembly and activity of pertussis toxin require interaction with ATP.

Pathog Dis 2016 08 30;74(6). Epub 2016 Jun 30.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.
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http://dx.doi.org/10.1093/femspd/ftw065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761283PMC
August 2016

Bordetella pertussis: new concepts in pathogenesis and treatment.

Curr Opin Infect Dis 2016 06;29(3):287-94

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Purpose Of Review: The purpose of this review is to summarize and discuss recent findings and selected topics of interest in Bordetella pertussis virulence and pathogenesis and treatment of pertussis. It is not intended to cover issues on immune responses to B. pertussis infection or problems with currently used pertussis vaccines.

Recent Findings: Studies on the activities of various B. pertussis virulence factors include the immunomodulatory activities of filamentous hemagglutinin, fimbriae, and adenylate cyclase toxin. Recently emerging B. pertussis strains show evidence of genetic selection for vaccine escape mutants, with changes in vaccine antigen-expressing genes, some of which may have increased the virulence of this pathogen. Severe and fatal pertussis in young infants continues to be a problem, with several studies highlighting predictors of fatality, including the extreme leukocytosis associated with this infection. Treatments for pertussis are extremely limited, though early antibiotic intervention may be beneficial. Neutralizing pertussis toxin activity may be an effective strategy, as well as targeting two host proteins, pendrin and sphingosine-1-phosphate receptors, as novel potential therapeutic interventions.

Summary: Pertussis is reemerging as a major public health problem and continued basic research is revealing information on bacterial virulence and disease pathogenesis, as well as potential novel strategies for vaccination and targets for therapeutic intervention.
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http://dx.doi.org/10.1097/QCO.0000000000000264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846492PMC
June 2016

Novel therapies for the treatment of pertussis disease.

Pathog Dis 2015 Nov 21;73(8):ftv074. Epub 2015 Sep 21.

Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore, MD 21201, USA

Whooping cough, or pertussis, incidence has reached levels not seen since the 1950s. Previous studies have shown that antibiotics fail to improve the course of disease unless diagnosed early. Early diagnosis is complicated by the non-diagnostic presentation of disease early in infection. This review focuses on previous attempts at developing novel host-directed therapies for the treatment of pertussis. In addition, two novel approaches from our group are discussed. Manipulation of the signaling pathway of sphingosine-1-phosphate, a lipid involved in many immune processes, has shown great promise, but is in its infancy. Pendrin, a host epithelial anion exchanger upregulated in the airways with B. pertussis infection, appears to drive mucus production and dysregulation of airway surface liquid pH and salinity. In addition to detailing these potential new therapeutic targets, the need for greater focus on the neonatal model of disease is highlighted.
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http://dx.doi.org/10.1093/femspd/ftv074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626598PMC
November 2015

Contribution of pertussis toxin to the pathogenesis of pertussis disease.

Pathog Dis 2015 Nov 21;73(8):ftv073. Epub 2015 Sep 21.

Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore, MD 21201, USA

Pertussis toxin (PT) is a multisubunit protein toxin secreted by Bordetella pertussis, the bacterial agent of the disease pertussis or whooping cough. PT in detoxified form is a component of all licensed acellular pertussis vaccines, since it is considered to be an important virulence factor for this pathogen. PT inhibits G protein-coupled receptor signaling through Gi proteins in mammalian cells, an activity that has led to its widespread use as a cell biology tool. But how does this activity of PT contribute to pertussis, including the severe respiratory symptoms of this disease? In this minireview, the contribution of PT to the pathogenesis of pertussis disease will be considered based on evidence from both human infections and animal model studies. Although definitive proof of the role of PT in humans is lacking, substantial evidence supports the idea that PT is a major contributor to pertussis pathology, including the severe respiratory symptoms associated with this disease.
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http://dx.doi.org/10.1093/femspd/ftv073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626579PMC
November 2015

Sphingosine-1-phosphate Receptor Agonism Reduces Bordetella pertussis-mediated Lung Pathology.

J Infect Dis 2015 Jun 23;211(12):1883-6. Epub 2014 Dec 23.

Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore.

Recent pertussis resurgence represents a major public health concern. Currently, there are no effective treatments for critical pertussis in infants. Recent data have demonstrated the potential of sphingosine-1-phosphate receptor (S1PR) agonism in the treatment of infectious diseases. We used the murine Bordetella pertussis model to test the hypothesis that treatment with S1PR agonist AAL-R reduces pulmonary inflammation during infection. AAL-R treatment resulted in reduced expression of inflammatory cytokines and chemokines and attenuated lung pathology in infected mice. These results demonstrate a role for sphingosine-1-phosphate (S1P) signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis.
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http://dx.doi.org/10.1093/infdis/jiu823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836722PMC
June 2015

Epithelial anion transporter pendrin contributes to inflammatory lung pathology in mouse models of Bordetella pertussis infection.

Infect Immun 2014 Oct 28;82(10):4212-21. Epub 2014 Jul 28.

Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore, Maryland, USA

Pertussis disease, characterized by severe and prolonged coughing episodes, can progress to a critical stage with pulmonary inflammation and death in young infants. However, there are currently no effective treatments for pertussis. We previously studied the role of pertussis toxin (PT), an important Bordetella pertussis virulence factor, in lung transcriptional responses to B. pertussis infection in mouse models. One of the genes most highly upregulated in a PT-dependent manner encodes an epithelial transporter of bicarbonate, chloride, and thiocyanate, named pendrin, that contributes to asthma pathology. In this study, we found that pendrin expression is upregulated at both gene and protein levels in the lungs of B. pertussis-infected mice. Pendrin upregulation is associated with PT production by the bacteria and with interleukin-17A (IL-17A) production by the host. B. pertussis-infected pendrin knockout (KO) mice had higher lung bacterial loads than infected pendrin-expressing mice but had significantly reduced levels of lung inflammatory pathology. However, reduced pathology did not correlate with reduced inflammatory cytokine expression. Infected pendrin KO mice had higher levels of inflammatory cytokines and chemokines than infected pendrin-expressing mice, suggesting that these inflammatory mediators are less active in the airways in the absence of pendrin. In addition, treatment of B. pertussis-infected mice with the carbonic anhydrase inhibitor acetazolamide reduced lung inflammatory pathology without affecting pendrin synthesis or bacterial loads. Together these data suggest that PT contributes to pertussis pathology through the upregulation of pendrin, which promotes conditions favoring inflammatory pathology. Therefore, pendrin may represent a novel therapeutic target for treatment of pertussis disease.
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http://dx.doi.org/10.1128/IAI.02222-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187853PMC
October 2014

Pertussis toxin exacerbates and prolongs airway inflammatory responses during Bordetella pertussis infection.

Infect Immun 2012 Dec 1;80(12):4317-32. Epub 2012 Oct 1.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Throughout infection, pathogenic bacteria induce dramatic changes in host transcriptional repertoires. An understanding of how bacterial factors influence host reprogramming will provide insight into disease pathogenesis. In the human respiratory pathogen Bordetella pertussis, the causative agent of whooping cough, pertussis toxin (PT) is a key virulence factor that promotes colonization, suppresses innate immune responses during early infection, and causes systemic disease symptoms. To determine the full extent of PT-associated gene regulation in the airways through the peak of infection, we measured global transcriptional profiles in the lungs of BALB/c mice infected with wild-type (WT) or PT-deficient (ΔPT) B. pertussis. ΔPT bacteria were inoculated at a dose equivalent to the WT dose and at a high dose (ΔPT(high)) to distinguish effects caused by higher bacterial loads achieved in WT infection from effects associated with PT. The results demonstrated that PT was associated with a significant upregulation of immune and inflammatory response genes as well as several other genes implicated in airway pathology. In contrast to the early, transient responses observed for ΔPT(high) infection, WT infection induced a prolonged expression of inflammatory genes and increased the extent and duration of lung histopathology. In addition, the administration of purified PT to ΔPT(high)-infected mice 1 day after bacterial inoculation exacerbated and prolonged inflammatory responses and airway pathology. These data indicate that PT not only is associated with exacerbated host airway responses during peak B. pertussis infection but also may inhibit host mechanisms of attenuating and resolving inflammation in the airways, suggesting possible links between PT and pertussis disease symptoms.
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http://dx.doi.org/10.1128/IAI.00808-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497438PMC
December 2012

Enhancement of Bordetella parapertussis infection by Bordetella pertussis in mixed infection of the respiratory tract.

FEMS Immunol Med Microbiol 2011 Oct 29;63(1):119-28. Epub 2011 Jul 29.

Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, MD, USA.

The epidemiological and pathogenic relationship between Bordetella pertussis and Bordetella parapertussis, the two causes of whooping cough (pertussis), is unclear. We hypothesized that B. pertussis, due to its immunosuppressive activities, might enhance B. parapertussis infection when the two species were present in a coinfection of the respiratory tract. The dynamics of this relationship were examined using the mouse intranasal inoculation model. Infection of the mouse respiratory tract by B. parapertussis was not only enhanced by the presence of B. pertussis, but B. parapertussis significantly outcompeted B. pertussis in this model. Staggered inoculation of the two organisms revealed that the advantage for B. parapertussis is established at an early stage of infection. Coadministration of PT enhanced B. parapertussis single infection, but had no effect on mixed infections. Mixed infection with a PT-deficient B. pertussis strain did not enhance B. parapertussis infection. Interestingly, the depletion of airway macrophages reversed the competitive relationship between these two organisms, but the depletion of neutrophils had no effect on mixed infection or B. parapertussis infection. We conclude that B. pertussis, through the action of PT, can enhance a B. parapertussis infection, possibly by an inhibitory effect on innate immunity.
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http://dx.doi.org/10.1111/j.1574-695X.2011.00836.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170498PMC
October 2011

Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity.

PLoS One 2011 Apr 20;6(4):e19016. Epub 2011 Apr 20.

Department of Microbiology and Immunology, University of Maryland Medical School, Baltimore, Maryland, United States of America.

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019016PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080395PMC
April 2011

Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools.

Future Microbiol 2010 Mar;5(3):455-69

Department of Microbiology & Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Pertussis toxin and adenylate cyclase toxin are two important virulence factors of Bordetella pertussis, the bacterial cause of the respiratory disease pertussis or whooping cough. In addition to studies on the structure, function and role in pathogenesis of these two toxins, they are both used as cell biology tools for a variety of applications owing to their ability to enter mammalian cells, perform enzymatic activities and modify cell signaling events. In this article, recent data from the research literature that enhance our understanding of the nature of these two toxins, their role in the pathogenesis of B. pertussis infection and disease, particularly in modulating host immune responses, and their use as tools for other areas of research will be outlined.
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http://dx.doi.org/10.2217/fmb.09.133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851156PMC
March 2010

Pertussis toxin stimulates IL-17 production in response to Bordetella pertussis infection in mice.

PLoS One 2009 Sep 17;4(9):e7079. Epub 2009 Sep 17.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.

In a mouse model of respiratory tract infection by Bordetella pertussis, bacteria multiply in the airways over the first week and are then cleared over the next 3-4 weeks by the host immune response. Pertussis toxin (PT), a virulence factor secreted exclusively by B. pertussis, promotes bacterial growth in the airways by suppression and modulation of host immune responses. By comparison of wild type and PT-deficient strains, we examined the role of PT in modulating airway cytokine and chemokine responses affecting neutrophil recruitment during B. pertussis infection in mice. We found that, despite early inhibition of neutrophil recruitment by PT, high numbers of neutrophils were recruited to the airways by 4 days post-infection with the wild type strain, but not with the PT-deficient strain, and that this correlated with upregulation of neutrophil-attracting chemokine gene expression. In addition, there was similar upregulation of genes expressing the cytokines IL-17A (IL-17), TNF-alpha and IFN-gamma, indicating a mixed Th1/Th17 response. Expression of IL-6, a cytokine involved in Th17 induction, was upregulated earlier than the IL-17 response. We showed that PT, rather than bacterial numbers, was important for induction of these responses. Flow cytometric analysis revealed that the IL-17-producing cells were macrophages and neutrophils as well as T cells, and were present predominantly in the airways rather than the lung tissue. Antibody neutralization of IL-17 significantly reduced chemokine gene expression and neutrophil recruitment to the airways, but only modestly increased peak bacterial loads. These data indicate that PT stimulates inflammatory responses by induction of Th1- and Th17-associated cytokines, including IL-17, during B. pertussis infection in mice, but a role for IL-17 in protection against the infection remains to be established.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007079PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738961PMC
September 2009

Role of neutrophils in response to Bordetella pertussis infection in mice.

Infect Immun 2009 Mar 22;77(3):1182-8. Epub 2008 Dec 22.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

Pertussis is an acute respiratory disease caused by the bacterium Bordetella pertussis, for which humans are the only known reservoir. During infection, B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play roles in promoting bacterial growth during early infection in a mouse model. Furthermore, in vitro and in vivo studies suggest that PT and ACT affect neutrophil chemotaxis and/or function, thereby altering the innate immune response. In this study we depleted animals of neutrophils to investigate whether neutrophils play a protective role during B. pertussis infection in mice. In addition, by infection with toxin-deficient strains, we investigated whether neutrophils are the main targets for PT and/or ACT activity in promoting bacterial growth. Surprisingly, we found no role for neutrophils during B. pertussis infection in naïve mice. However, in previously infected (immune) mice or in mice receiving immune serum, we observed a significant role for neutrophils during infection. Furthermore, in this immune mouse model our evidence indicates that neutrophils appear to be the main target cells for ACT, but not for PT.
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http://dx.doi.org/10.1128/IAI.01150-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643619PMC
March 2009

Pertussis toxin inhibits early chemokine production to delay neutrophil recruitment in response to Bordetella pertussis respiratory tract infection in mice.

Infect Immun 2008 Nov 2;76(11):5139-48. Epub 2008 Sep 2.

Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 W. Redwood St., HH 324, Baltimore, MD 21201, USA.

Pertussis is an acute respiratory disease of humans caused by the bacterium Bordetella pertussis. Pertussis toxin (PT) plays a major role in the virulence of this pathogen, including important effects that it has soon after inoculation. Studies in our laboratory and other laboratories have indicated that PT inhibits early neutrophil influx to the lungs and airways in response to B. pertussis respiratory tract infection in mice. Previous in vitro and in vivo studies have shown that PT can affect neutrophils directly by ADP ribosylating G(i) proteins associated with surface chemokine receptors, thereby inhibiting neutrophil migration in response to chemokines. However, in this study, by comparing responses to wild-type (WT) and PT-deficient strains, we found that PT has an indirect inhibitory effect on neutrophil recruitment to the airways in response to infection. Analysis of lung chemokine expression indicated that PT suppresses early neutrophil recruitment by inhibiting chemokine upregulation in alveolar macrophages and other lung cells in response to B. pertussis infection. Enhancement of early neutrophil recruitment to the airways in response to WT infection by addition of exogenous keratinocyte-derived chemokine, one of the dominant neutrophil-attracting chemokines in mice, further revealed an indirect effect of PT on neutrophil chemotaxis. Additionally, we showed that intranasal administration of PT inhibits lipopolysaccharide-induced chemokine gene expression and neutrophil recruitment to the airways, presumably by modulation of signaling through Toll-like receptor 4. Collectively, these results demonstrate how PT inhibits early inflammatory responses in the respiratory tract, which reduces neutrophil influx in response to B. pertussis infection, potentially providing an advantage to the pathogen in this interaction.
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http://dx.doi.org/10.1128/IAI.00895-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2573337PMC
November 2008

Retrograde transport of pertussis toxin in the mammalian cell.

Cell Microbiol 2008 May 31;10(5):1130-9. Epub 2007 Dec 31.

Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 W. Redwood St., HH 324, Baltimore, MD 21201, USA.

Pertussis toxin (PT), an AB5 exotoxin and important virulence factor of Bordetella pertussis, is hypothesized to traffic along a retrograde transport pathway in mammalian cells. This pathway includes endosomal uptake, transport to the Golgi complex and endoplasmic reticulum (ER), dissociation of the holotoxin in the ER and translocation of the A subunit (S1) to the cytosol, where it ADP-ribosylates its G protein targets. In this study, PT was visualized in the Golgi complex by immunofluorescence microscopy, but transport beyond the Golgi could not be detected by this method. To gain evidence for the retrograde pathway, peptide tags with target sites for tyrosine sulfation (a trans-Golgi network-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained in vitro enzymatic and cellular activity as assessed by ADP-ribosylation assays. Peptide-tagged PT subunits were found to be modified by tyrosine sulfation, and, at later time points, by N-glycosylation. Appearance of sulfated PT subunits was inhibited by pretreatment of cells with brefeldin A. In some cell types, much of the S4 glycosylation, but not that of S1, was resistant to endoglycosidase H, suggesting that, subsequent to core N-glycosylation in the ER, S4 was transported anterograde to the Golgi, where further glycosylation occurred. When cells were pretreated with methyl-beta-cyclodextrin, sulfation of PT subunits and PT cytotoxicity were reduced, suggesting that PT transport is dependent on cellular cholesterol content. These data support a retrograde pathway for PT intracellular transport.
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http://dx.doi.org/10.1111/j.1462-5822.2007.01115.xDOI Listing
May 2008

Evading the proteasome: absence of lysine residues contributes to pertussis toxin activity by evasion of proteasome degradation.

Infect Immun 2007 Jun 9;75(6):2946-53. Epub 2007 Apr 9.

Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 W. Redwood Street, Baltimore, MD 21201, USA.

Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.
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http://dx.doi.org/10.1128/IAI.02011-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932868PMC
June 2007

Immunomodulation in the pathogenesis of Bordetella pertussis infection and disease.

Curr Opin Pharmacol 2007 Jun 5;7(3):272-8. Epub 2007 Apr 5.

Department of Microbiology & Immunology, University of Maryland School of Medicine, HH 324 Baltimore, MD 21201, USA.

Bordetella pertussis infection of the airways causes the disease pertussis (or whooping cough). The infection can be fatal in infants, but in older children, adolescents and adults usually results in a chronic cough of varying severity that persists long after clearance of the infection. The cause of the cough is unknown, but is presumably a result of the pathogenic effects of one or more of the various virulence factors produced by this bacterium. Accumulating recent evidence indicates that the majority of the virulence-associated effects of these factors is devoted to suppression and modulation of the host immune response, which can be skewed towards the recently described Th17 profile. Although the interplay between virulence factors and immune mechanisms might have evolved to benefit both partners in the host-pathogen interaction, it could also contribute to the severe disease pathology associated with this infection.
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http://dx.doi.org/10.1016/j.coph.2006.12.004DOI Listing
June 2007

Pertussis toxin targets airway macrophages to promote Bordetella pertussis infection of the respiratory tract.

Infect Immun 2007 Apr 22;75(4):1713-20. Epub 2007 Jan 22.

Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 W. Redwood St., HH 324, Baltimore, MD 21201, USA.

Pertussis toxin (PT), a secreted virulence factor of Bordetella pertussis, ADP ribosylates mammalian G(i) proteins and plays an important early role in respiratory tract infection by this pathogen in a mouse intranasal infection model. To test the hypothesis that PT targets resident airway macrophages (AM) to promote this infection, we depleted AM by intranasal administration of liposome-encapsulated clodronate prior to bacterial inoculation. This treatment enhanced respiratory tract infection by B. pertussis, even though it also induced a rapid influx of neutrophils to the airways. Strikingly, AM depletion also enhanced infection by mutant strains deficient in PT production or activity to the same level as the wild-type infection, indicating that AM may be the primary target cells for PT in promoting infection. The enhancing effect of clodronate-liposome treatment on infection (i) was shown to be due to macrophage depletion rather than neutrophil influx; (ii) was observed for both tracheal infection and lung infection; (iii) was observed during the early and peak phases of the infection but was lost by day 14 postinoculation, during clearance of the infection; (iv) persisted for at least 1 week (prior to bacterial inoculation); and (v) was equivalent in magnitude to the effect of PT pretreatment and the effects were not additive, consistent with the idea that PT targets AM. We found that PT efficiently ADP ribosylated AM G proteins both in vitro and after intranasal administration of PT in mice and that the duration of G protein modification in vivo was equivalent to the duration of the enhancing effect of PT treatment on the bacterial infection. Collectively, these observations indicate that PT targets AM to promote early infection of the respiratory tract by B. pertussis.
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http://dx.doi.org/10.1128/IAI.01578-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865687PMC
April 2007

Pertussis toxin as an adjuvant suppresses the number and function of CD4+CD25+ T regulatory cells.

Eur J Immunol 2006 Mar;36(3):671-80

Basic Research Program, SAIC-Frederick, Inc., Laboratory of Molecular Immunoregulation, National Cancer Institute-Frederick. Frederick, MD 21702-1201, USA.

We observed a remarkable reduction in the frequency and immunosuppressive activity of splenic CD4+CD25+ T cells in C57BL/6 mice with MOG33-55-induced experimental autoimmune encephalomyelitis (EAE). Our study revealed that pertussis toxin (PTx), one component of the immunogen used to induce murine EAE, was responsible for down-regulating splenic CD4+CD25+ cells. Treatment of normal BALB/c mice with PTx in vivo reduced the frequency, suppressive activity and FoxP3 expression by splenic CD4+CD25+ T cells. However, PTx treatment did not alter the expression of characteristic phenotypic markers (CD45RB, CD103, GITR and CTLA-4) and did not increase the expression of CD44 and CD69 by the residual splenic and lymph node CD4+CD25+ T cells. This property of PTx was attributable to its ADP-ribosyltransferase activity. PTx did not inhibit suppressive activity of purified CD4+CD25+ T regulatory (Treg) cells in vitro, but did so in vivo, presumably due to an indirect effect. Although the exact molecular target of PTx that reduces Treg activity remains to be defined, our data suggests that alteration of both distribution and function of splenic immunocytes should play a role. This study concludes that an underlying cause for the immunological adjuvanticity of PTx is down-regulation of Treg cell number and function.
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http://dx.doi.org/10.1002/eji.200535353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153960PMC
March 2006

Pertussis toxin and adenylate cyclase toxin provide a one-two punch for establishment of Bordetella pertussis infection of the respiratory tract.

Infect Immun 2005 May;73(5):2698-703

Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 W. Baltimore St., BRB13-009, Baltimore, MD 21201, USA.

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 (DeltaCYA) 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 log(10)). In mixed-infection experiments, the DeltaCYA strain was significantly outcompeted by the wild-type strain, and intranasal administration of purified ACT did not increase colonization by DeltaCYA. 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 DeltaCYA, 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.
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http://dx.doi.org/10.1128/IAI.73.5.2698-2703.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087369PMC
May 2005

Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells.

BMC Microbiol 2005 Feb 3;5. Epub 2005 Feb 3.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Background: Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2-S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.

Results: Protease inhibitor studies suggested that S1 processing may involve a metalloprotease, and processing does not involve furin, a mammalian cell protease that cleaves several other bacterial toxins. However, inhibitor studies showed a general lack of correlation of S1 processing with PT cellular activity. A combination of replacement, insertion and deletion mutations in the C-terminal region of S1, as well as mass spectrometry data, suggested that the cleavage site is located around residue 203-204, but that cleavage is not strongly sequence-dependent. Processing of S1 was abolished by each of 3 overlapping 8 residue deletions just downstream of the putative cleavage site, but not by smaller deletions in the same region. Processing of the various mutant forms of PT did not correlate with cellular activity of the toxin, nor with the ability of the bacteria producing them to infect the mouse respiratory tract. In addition, S1 processing was not detected in transfected cells expressing S1, even though S1 was fully active in these cells.

Conclusions: S1 processing is not essential for the cellular activity of PT. This distinguishes it from the processing of various other bacterial toxins, which has been shown to be important for their cytotoxicity. S1 processing may be mediated primarily by a metalloprotease, but the cleavage site on S1 is not sequence-dependent and processing appears to depend on the general topology of the protein in that region, indicating that multiple proteases may contribute to this cleavage.
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http://dx.doi.org/10.1186/1471-2180-5-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC550662PMC
February 2005