• Characterization of differential regulation and its role in the pathogenesis of Bordetella pertussis

      Kinnear, Susan Marie Jung; Carbonetti, Nicholas H. (2000)
      Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvgAS regulatory locus. It has been shown by transcription activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed within 10 minutes following an inducing signal, while ptx is not transcribed until 2-4 hours later. We present data indicating that, while fim2 is transcribed early similar to fha, prn is transcribed after 1 hour, an intermediate time. We have identified cis-acting sequences necessary for transcription of prn in B. pertussis using prn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of prn to activate transcription from the promoter. Our genetic data indicate that the region critical for prn activation extends upstream to -84. We used a number of methods including prn-lac fusion analysis, RT-PCR, and 5' RACE to identify the bvg-dependent 5' end of the prn transcript as the cytosine at -125 with respect to the previously published transcription start site. We have also provided evidence that there may be an inhibitory effect of the binding of high concentrations of BvgA at the prn promoter. Although there have been many insightful studies into the mechanisms of BvgAS-mediated regulation, the role that differential regulation of virulence genes plays in B. pertussis pathogenesis has not been characterized. We provide evidence that suggests that alterations to the promoter regions of bvg-activated genes can alter the kinetic pattern of expression of these genes without changing steady-state transcription levels. In addition, B. pertussis strains containing ptx promoter alterations that express ptx at an early time, demonstrate a significant reduction in the ability to colonize respiratory tracts in an intranasal mouse model of infection. The colonization defect can be mimicked by the maintenance of the wild type B. pertussis inoculum at 37°C prior to inoculation, thus suppressing modulation of the BvgAS system and down-regulation of ptx expression. This data suggests a role for differential regulation of bvg-activated genes, and subsequently for the BvgAS regulatory system, in the pathogenesis of B. pertussis.
    • The effect of pertussis toxin on the innate immune response to Bordetella pertussis infection in mice

      Andreasen, Charlotte; Carbonetti, Nicholas H. (2008)
      Bordetella pertussis is a small Gram-negative bacterium that infects the human respiratory tract and causes the disease pertussis, also known as whooping cough. B. pertussis binds to ciliated cells in the trachea and proliferates within the upper and lower respiratory tract, where several toxins are released. Pertussis toxin (PT), produced exclusively by B. pertussis, and adenylate cyclase toxin (ACT) are important virulence factors of B. pertussis; however, their complete role during infection is not yet clear. Our studies showed that PT inhibits early neutrophil recruitment to the lung tissue (d1) and airways (d1-2) in response to infection with 5x105 CFU of B. pertussis by inhibiting the early production of neutrophil-attracting chemokines (6 hrs). However, at the peak of infection, a strong inflammatory Th1/Th17 response is produced, in part by PT and ACT activity. The production of IL-17 results in the induction of chemokine production as well as a neutrophil recruitment to the airways. Neutrophil depletion experiments suggested that neutrophils are not important in response to B. pertussis in naive mice. However, in an immune mouse model, neutrophils played a role in controlling the infection. Furthermore, in contrast to findings in naive mice, neutrophils appear to be the main target for ACT activity in immune mice, suggesting that B. pertussis may have evolved to evade immune responses in previously infected (or vaccinated) individuals.
    • Genetic analysis of BvgA activaton of virulence gene promoters in Bordetella pertussis

      Marques, Ryan Robert; Carbonetti, Nicholas H. (1998)
      Bordetella pertussis, the causative agent of whooping cough, regulates its virulence factors coordinately in response to external stimuli. The locus responsible for this regulation is the bvg locus which encodes a two component signal transduction system made up of a sensor protein, BvgS and a transcriptional activator protein, BvgA. The bvg locus regulates the expression of several virulence factors such as filamentous hemagglutinin (Fha), pertussis toxin (Ptx) and adenylate cyclase/hemolysin toxin (Cya). Previously it was hypothesized that BvgA directly activated the fha and bvg promoters and that a secondary activator was responsible for activation of the ptx promoter. This was based on the observations that bvg and fha promoter-lac fusions on the E. coli chromosome were activated in the presence of the bvg locus whereas a ptx promoter-lac fusion was inactive. Furthermore, BvgA was shown to bind directly upstream of the promoters of fha and bvg at heptanucleotide repeat sequences, but no such binding was observed upstream of the ptx promoter. However, attempts to identify a secondary activator for ptx were unsuccessful and mutants that had a Fha+Ptx- phenotype were found to possess mutations in either the rpoA gene, that encodes the alpha subunit of RNA polymerase (RNAP) or the bvgA gene itself In this study we determined that Bvg alone was responsible for the transcriptional activation of the ptx promoter and that compared to the fha promoter, a higher concentration of BvgA was necessary for ptx promoter activation. We also genetically analyzed the mechanism of ptx promoter activation and showed that a heptanucleotide inverted repeat sequence upstream of the ptx promoter that shared identity with the inverted repeat present upstream of the fha promoter, was important for promoter activation. This inverted repeat sequence has subsequently been shown to be within the primary binding site for BvgA at the ptx promoter in in vitro footprinting studies. The intervening sequence between the repeat region and the ptx promoter was analyzed. We determined in this study that this intervening region played an important role in ptx promoter activation. Replacement of this region as well as deletions of 10, 42 and 63 bp within this region resulted in the inactivation of the ptx promoter. Nevertheless, any of three adjacent 21 bp deletions from this region did not significantly reduce ptx promoter activity. These data support the cooperative-binding model of ptx promoter activation, in which this region provides weaker binding sites for cooperative binding of BvgA, probably as dimers, which places it in a location where it can assist in the binding of RNAP at the ptx promoter. The above result, as well as the fact that mutations with a Fha +Ptx- phenotype mapped to the alpha subunit of RNAP, suggests that there is some interaction between RNAP and BvgA. We tested this hypothesis by attempting to isolate positive control (pc) mutants of BvgA that were defective in their ability to activate the ptx and fha promoters but still retained wild type binding to DNA. One of the mutants obtained demonstrated pc mutant characteristics. This mutant will have to be characterized further but it strengthens the hypothesis that there is interaction between the transcriptional activator, BvgA and RNAP.
    • Retrograde transport of pertussis toxin in the mammalian cell

      Plaut, Roger; Carbonetti, Nicholas H. (2007)
      Pertussis toxin (PT), an AB5 exotoxin and an important virulence factor of Bordetella pertussis, has been hypothesized to traffic along a retrograde transport pathway in mammalian cells. This model includes endosomal uptake, transport to the Golgi apparatus and endoplasmic reticulum (ER), ATP-stimulated dissociation of the holotoxin in the ER, and translocation of the A subunit (S1) to the cytosol. PT has been detected in the Golgi apparatus by immunofluorescence microscopy and cell fractionation, but transport beyond the Golgi has not been detected by these methods. To gain biochemical evidence for the retrograde pathway, target sites for tyrosine sulfation (a trans-Golgi network (TGN)-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained enzymatic activity as assessed by in vitro and cellular 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 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. To investigate the importance of the interaction of PT with ATP, a form of PT was engineered with changes to two amino acid residues in S5 thought to be involved in the interaction. This form of PT was reduced in its ability to be dissociated by interaction with ATP in vitro; it was also substantially reduced in activity in an in vitro enzymatic assay and in a cellular ADP-ribosylation assay. When cells were treated with PT possessing the ATP-binding mutations and the tyrosine sulfation and N-glycosylation target sequences, the N-glycosylation patterns of the S1 and S4 subunits were altered, consistent with an inability of the holotoxin to dissociate in the ER. This form of PT also colocalized substantially with ER markers when observed under indirect immunofluorescence microscopy. Overall, these results comprise evidence in support of the hypothesized retrograde pathway of PT intracellular transport.
    • The Role of Type I and III Interferons in the Pathogenesis of Bordetella pertussis Infection and Disease.

      Ardanuy, Jeremy; Carbonetti, Nicholas H. (2020)
      Bordetella pertussis is a Gram-negative bacterial pathogen that infects human respiratory tracts and is the causative agent for the disease pertussis, otherwise known as whooping cough. In 2012, there were 48,277 reported cases in the United States, the most since 1955. Symptoms build up to severe paroxysmal coughing, often for 10+ weeks after onset. For infants, pertussis can be fatal due to complications including pulmonary hypertension, pneumothorax, high-level circulating lymphocytosis, and pneumonia. In adults, the disease is severe too, with long-lasting cough, lung damage, and serious symptoms resulting in hospitalization. A difficulty in treatment/prevention of pertussis is a suboptimal vaccine that confers waning immunity, and a lack of effective treatments available. Antibiotics are administered to patients to prevent transmission, but usually don’t change the clinical course of disease. Host directed therapeutics treating pertussis could benefit individuals with severe cough and save the lives of infected infants. Using RNA-sequencing transcriptomics we investigated lung gene expression responses to Bordetella pertussis infection in adult mice, revealing that type I and III interferon (IFN) responses and signaling pathways may play an important role in promoting inflammatory responses. In B. pertussis infected mice, lung type I/III IFN responses correlated with increased proinflammatory cytokine expression and lung inflammatory pathology. In mutant mouse models with increased type I IFN signaling, B. pertussis exacerbated lung inflammatory pathology, whereas knockout mice with deficiencies in type I/III IFN signaling had reduced lung inflammation compared to wild-type mice. In direct contrast, infant mice didn’t upregulate type I/III IFNs in response to B. pertussis infection and were protected from lethal infection by increased type I IFN signaling, indicating age dependent effects of type I/III IFN signaling during B. pertussis infection. The induction of type I/III IFNs was found to be MyD88 dependent, and TLR9 and STING were identified as DNA sensing pattern recognition receptors required for type I/III IFN responses, as well as for typical levels of lung inflammatory pathology. This observation, coupled with results showing DNase treatment of B. pertussis-infected mice causing reduced lung pathology, indicated a DNA dependent induction of type I/III IFNs, making these targets for therapeutic intervention.