Architecture and matrix assembly determinants of Bordetella pertussis biofilms on primary human airway epithelium.

Traditionally, whooping cough or pertussis caused by the obligate human pathogen Bordetella pertussis (Bp) is described as an acute disease with severe symptoms. However, many individuals who contract pertussis are either asymptomatic or show very mild symptoms and yet can serve as carriers and sources of bacterial transmission. Biofilms are an important survival mechanism for bacteria in human infections and disease. However, bacterial determinants that drive biofilm formation in humans are ill-defined. In the current study, we show that Bp infection of well-differentiated primary human bronchial epithelial cells leads to formation of bacterial aggregates, clusters, and highly structured biofilms which are colocalized with cilia. These findings mimic observations from pathological analyses of tissues from pertussis patients. Distinct arrangements (mono-, bi-, and tri-partite) of the polysaccharide Bps, extracellular DNA, and bacterial cells were visualized, suggesting complex heterogeneity in bacteria-matrix interactions. Analyses of mutant biofilms revealed positive roles in matrix production, cell cluster formation, and biofilm maturity for three critical Bp virulence factors: Bps, filamentous hemagglutinin, and adenylate cyclase toxin. Adherence assays identified Bps as a new Bp adhesin for primary human airway cells. Taken together, our results demonstrate the multi-factorial nature of the biofilm extracellular matrix and biofilm development process under conditions mimicking the human respiratory tract and highlight the importance of model systems resembling the natural host environment to investigate pathogenesis and potential therapeutic strategies.

Reemergence of Bordetella parapertussis, United States, 2019-2023.

To determine changes in Bordetella pertussis and B. parapertussis detection rates, we analyzed 1.43 million respiratory multiplex PCR test results from US facilities from 2019 through mid-2023. From mid-2022 through mid-2023, Bordetella spp. detection increased 8.5-fold; 95% of detections were B. parapertussis. While B. parapertussis rates increased, B. pertussis rates decreased.

BPP0974 is a Bordetella parapertussis adhesin expressed in the avirulent phase, implicated in biofilm formation and intracellular survival.

B. parapertussis is a bacterium that causes whooping cough, a severe respiratory infection disease, that has shown an increased incidence in the population. Upon transmission through aerosol droplets, the initial steps of host colonization critically depend on the bacterial adhesins. We here described BPP0974, a B. parapertussis protein that exhibits the typical domain architecture of the large repetitive RTX adhesin family. BPP0974 was found to be retained in the bacterial membrane and secreted into the culture medium. This protein was found overexpressed in the avirulent phase of B. parapertussis, the phenotype proposed for initial host colonization. Interestingly, BPP0974 was found relevant for the biofilm formation as well as involved in the bacterial attachment to and survival within the respiratory epithelial cells. Taken together, our results suggest a role for BPP0974 in the early host colonization and pathogenesis of B. parapertussis.

Current understanding of Bordetella-induced cough.

Typical pathogenic bacteria of the genus Bordetella cause respiratory diseases, many of which are characterized by severe coughing in host animals. In human infections with these bacteria, such as whooping cough, coughing imposes a heavy burden on patients. The pathophysiology of this severe coughing had long been uncharacterized because convenient animal models that reproduce Bordetella-induced cough have not been available. However, rat and mouse models were recently shown as useful for understanding, at least partially, the causative factors and the mechanism of Bordetella-induced cough. Many types of coughs are induced under various physiological conditions, and the neurophysiological pathways of coughing are considered to vary among animal species, including humans. However, the neurophysiological mechanisms of the coughs in different animal species have not been entirely understood, and, accordingly, the current understanding of Bordetella-induced cough is still incomplete. Nevertheless, recent research findings may open the way for the development of prophylaxis and therapeutic measures against Bordetella-induced cough.

Resurgence of Bordetella pertussis, including one macrolide-resistant isolate, France, 2024.

As other European countries, France is experiencing a resurgence of pertussis in 2024. Between 1 January and 31 May 2024, 5,616 (24.9%) positive Bordetella pertussis qPCR tests were identified, following a 3-year period of almost null incidence. Of 67 cultured and whole genome sequenced B. pertussis isolates, 66 produced pertactin and 56 produced FIM2, in contrast to pre-COVID-19 years. One isolate of genotype Bp-AgST4 was resistant to macrolides. Pertussis resurgence may favour isolates that produce FIM2 and pertactin.

Protective Activity and Safety of Experimental Acellular Pertussis Vaccines Based on Antigenic Complexes Isolated from Biofilm and Planktonic Cultures of Bordetella pertussis.

Continued circulation of the whooping cough pathogen, even in countries with high vaccine coverage, can be related to persistence of Bordetella pertussis biofilms in the respiratory tract. The films differ from planktonic cells by increased resistance to the host immune system and antibacterial drugs. The available acellular pertussis vaccines (aPV) containing antigens isolated from planktonic cultures of B. pertussis protect from severe forms of whooping cough, but do not effectively influence circulation of virulent strains in the subclinical forms of the disease and asymptomatic carriage. It is promising to create new generation aPV based on antigens isolated from biofilm cultures of B. pertussis capable of more effectively controlling the entire infectious cycle of whooping cough, including colonization, persistence, and transmission of the pathogen. From antigenic complexes isolated from the culture medium of biofilm and planktonic cultures of the strain B. pertussis No. 317 (serotype 1.2.3), experimental aPV were made: aPV-B and aPV-P, respectively. In intracerebral infection of mice with a virulent strain of B. pertussis, aPV-B demonstrated 2.5-fold higher protective activity than aPV-P and also more effectively reduced colonization of the lungs by B. pertussis cells in mice after intranasal infection with a virulent strain. Both vaccine preparations were safe and did not cause death in mice after administration of histamine.

Inhibition of Pertussis Toxin by Human α-Defensins-1 and -5: Differential Mechanisms of Action.

Whooping cough is a severe childhood disease, caused by the bacterium Bordetella pertussis, which releases pertussis toxin (PT) as a major virulence factor. Previously, we identified the human antimicrobial peptides α-defensin-1 and -5 as inhibitors of PT and demonstrated their capacity to inhibit the activity of the PT enzyme subunit PTS1. Here, the underlying mechanism of toxin inhibition was investigated in more detail, which is essential for developing the therapeutic potential of these peptides. Flow cytometry and immunocytochemistry revealed that α-defensin-5 strongly reduced PT binding to, and uptake into cells, whereas α-defensin-1 caused only a mild reduction. Conversely, α-defensin-1, but not α-defensin-5 was taken up into different cell lines and interacted with PTS1 inside cells, based on proximity ligation assay. In-silico modeling revealed specific interaction interfaces for α-defensin-1 with PTS1 and vice versa, unlike α-defensin-5. Dot blot experiments showed that α-defensin-1 binds to PTS1 and even stronger to its substrate protein Gαi in vitro. NADase activity of PTS1 in vitro was not inhibited by α-defensin-1 in the absence of Gαi. Taken together, these results suggest that α-defensin-1 inhibits PT mainly by inhibiting enzyme activity of PTS1, whereas α-defensin-5 mainly inhibits cellular uptake of PT. These findings will pave the way for optimization of α-defensins as novel therapeutics against whooping cough.

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.

Missed pertussis diagnosis during co-infection with Bordetella holmesii.

The purpose of this study is to identify predictive factors associated with missed diagnosis of B. pertussis-B. holmesii co-infection by assessing the analytical performance of a commercially available multiplexed PCR assay and by building a prediction model based on clinical signs and symptoms for detecting co-infections. This is a retrospective study on the electronic health records of all clinical samples that tested positive to either B. pertussis or B. holmesii from January 2015 to January 2018 at Geneva University Hospitals. Multivariate logistic regression was used to build a model for co-infection prediction based on the electronic health record chart review. Limit of detection was determined for all targets of the commercial multiplexed PCR assay used on respiratory samples. A regression model, developed from clinical symptoms and signs, predicted B. pertussis and B. holmesii co-infection with an accuracy of 82.9% (95% CI 67.9-92.8%, p value = .012), for respiratory samples positive with any of the two tested Bordetella species. We found that the LOD of the PCR reaction targeting ptxS1 is higher than that reported by the manufacturer by a factor 10. The current testing strategy misses B. pertussis and B. holmesii co-infections by reporting only B. holmesii infections. Thus, we advocate to perform serological testing for detecting a response against pertussis toxin whenever a sample is found positive for B. holmesii. These findings are important, both from a clinical and epidemiological point of view, as the former impacts the choice of antimicrobial drugs and the latter biases surveillance data, by underestimating B. pertussis infections during co-infections.

Clinical characteristics of 967 children with pertussis: a single-center analysis over an 8-year period in Beijing, China.

The purpose of this study is to understand children's clinical characteristics with pertussis and analyze risk factors on critical pertussis patients. Demographic data from patients with pertussis at Children's Hospital affiliated to the Capital Institute of Pediatrics between March 2011 and December 2018 were collected. We retrospectively gathered more information with the positive exposure, vaccination, antibiotic usage before diagnosis, clinical manifestation, laboratory tests, therapy, and complications for hospitalized children. We divided the patients into severe and non-severe groups, comparing related factors and clinical characteristics among each group. In particular, we summarize the clinical features of the severe patients before aggravation. A total of 967 pertussis cases were diagnosed, of which 227 were hospitalized. The onset age younger than 3 months old accounted for the highest proportion, and 126 patients received hospitalization. For those patients, the incidence of post-tussive vomiting, paroxysmal cyanosis, post-tussive heart rate decrease, hypoxemia, severe pneumonia, and mechanical ventilation was significantly higher than that in the ≥ 3-month-old group (p < 0.05). Among 227 hospitalized patients, 54 suffered from severe pertussis. Risk factors for severe patients included early age of onset, pathogen exposure, and unvaccinated status. Cough paroxysms, post-tussive vomiting, paroxysmal cyanosis, facial flushing/cyanosis/fever during cough, increased WBC, and chest X-ray revealing pneumonia/consolidation/atelectasis were important indications of severe pertussis. Unvaccinated status was an independent risk factor for severe pertussis. The most vulnerable population was infants < 3 months old to pertussis, and may be on the severe end of the disease. Pediatricians must detect and treat severe cases promptly and recommend timely vaccination for all eligible children.

Evolution of Bordetella pertussis in the acellular vaccine era in Norway, 1996 to 2019.

We described the population structure of Bordetella pertussis (B. pertussis) in Norway from 1996 to 2019 and determined if there were evolutionary shifts and whether these correlated with changes in the childhood immunization program. We selected 180 B. pertussis isolates, 22 from the whole cell vaccine (WCV) era (1996-1997) and 158 from the acellular vaccine (ACV) era (1998-2019). We conducted whole genome sequencing and determined the distribution and frequency of allelic variants and temporal changes of ACV genes. Norwegian B. pertussis isolates were evenly distributed across a phylogenetic tree that included global strains. We identified seven different allelic profiles of ACV genes (A-F), in which profiles A1, A2, and B dominated (89%), all having pertussis toxin (ptxA) allele 1, pertussis toxin promoter (ptxP) allele 3, and pertactin (prn) allele 2 present. Isolates with ptxP1 and prn1 were not detected after 2007, whereas the prn2 allele likely emerged prior to 1972, and ptxP3 before the early 1980s. Allele conversions of ACV genes all occurred prior to the introduction of ACV. Sixteen percent of our isolates showed mutations within the prn gene. ACV and its booster doses (implemented for children in 2007 and adolescents in 2013) might have contributed to evolvement of a more uniform B. pertussis population, with recent circulating strains having ptxA1, ptxP3, and prn2 present, and an increasing number of prn mutations. These strains clearly deviate from ACV strains (ptxA1, ptxP1, prn1), and this could have implications for vaccine efficiency and, therefore, prevention and control of pertussis.

The role of Bordetella pertussis in the development of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is one of the most common neurological disorders which main cause is not identified yet. Some studies mentioned the possible role of infectious agents such as chlamydia pneumonia, mycoplasma and also, B. pertussis via asymptomatic nasopharyngeal colonization. The current study aimed to investigate and compared the serum level of B. pertussis antibody and the rate of nasopharyngeal colonization by this pathogen in subjects with and without MS. METHODS: In this case-control study, 109 patients with MS and 114 subjects without MS referred to Sina Hospital in Hamadan in 2019 are studied and compared in terms of serum titer of B. pertussis antibody and nasopharyngeal colonization by this bacterium. Colonization was evaluated using culture and real-time PCR techniques. Data were analyzed using SPSS version 16 with a 95% confidence interval. RESULTS: The serum titer of B. pertussis antibody in case and control groups was 37.8 and 35.1%, respectively (P = 0.74). Culture and real-time PCR techniques revealed no case of nasopharyngeal colonization by B. pertussis. CONCLUSION: There was no difference between B. pertussis antibody titer and the rate of nasopharyngeal colonization between both MS patients and the healthy control group. Therefore, it seems that probably B. pertussis has not a role in MS development.

Genome Characteristic of Bordetella parapertussis Isolated from Iran.

Pertussis also known as whooping cough is a respiratory infection in humans particularly with severe symptoms in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome. The expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms and 13 short insertions and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.

Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) Contributes to Bordetella pertussis Inflammatory Pathology.

Whooping cough (pertussis) is a severe pulmonary infectious disease caused by the bacteria Bordetella pertussis. Pertussis infects an estimated 24 million people annually, resulting in >150,000 deaths. The NIH placed pertussis on the list of emerging pathogens in 2015. Antibiotics are ineffective unless administered before the onset of the disease characteristic cough. Therefore, there is an urgent need for novel pertussis therapeutics. We have shown that sphingosine-1-phosphate receptor (S1PR) agonists reduce pertussis inflammation without increasing bacterial burden. Transcriptomic studies were performed to identify this mechanism and allow for the development of pertussis therapeutics that specifically target problematic inflammation without sacrificing bacterial control. These data suggested a role for triggering receptor expressed on myeloid cells-1 (TREM-1). TREM-1 cell surface receptor functions as an amplifier of inflammatory responses. Expression of TREM-1 is increased in response to bacterial infection of mucosal surfaces. In mice, B. pertussis infection results in Toll-like receptor 9 (TLR9)-dependent increased expression of TREM-1 and its associated cytokines. Interestingly, S1PR agonists dampen pulmonary inflammation and TREM-1 expression. Mice challenged intranasally with B. pertussis and treated with ligand-dependent (LP17) and ligand-independent (GF9) TREM-1 inhibitors showed no differences in bacterial burden and significantly reduced tumor necrosis factor-α (TNF-α) and C-C motif chemokine ligand 2 (CCL-2) expression compared to controls. Mice receiving TREM-1 inhibitors showed reduced pulmonary inflammation compared to controls, indicating that TREM-1 promotes inflammatory pathology, but not bacterial control, during pertussis infection. This implicates TREM-1 as a potential therapeutic target for the treatment of pertussis.

Lack of evidence supporting a role of IFN-ß and TGF-ß in differential polarization of Bordetella pertussis specific-T cell responses.

Bordetella Pertussis (BP) vaccine-induced immunity is waning worldwide despite excellent vaccine coverage. Replacement of the whole-cell inactivated vaccine (wP) by an acellular subunit vaccine (aP) is thought to play a major role and to be associated with the recurrence of whooping cough. Previously, we detected that the polarization towards a Th2 and Th1/Th17 response in aP and wP vaccinees, respectively, persists upon aP boosting in adolescents and adults. Additionally, IL-9 and TGF-ß were found to be up-regulated in aP-primed donors and network analysis further identified IFN-ß as a potential upstream regulator of IL-17 and IL-9. Based on these findings, we hypothesized that IFN-ß produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production. Also, due to the well characterized role of TGF-ß in both Th17 and Th9 differentiation, we put forth that TGF-ß addition to BP-stimulated CD4 + T cells might modulate IL-17 and IL-9 production. To test this hypothesis, we stimulated in vitro cultures of PBMC or isolated naive CD4 + T cells from aP vs wP donors with a pool of BP epitopes and assessed the effect of IFN-ß or TGF-ß in proliferative responses as well as in the cytokine secretion of IL-4, IL-9, IL-17, and IFN-γ. IFN-ß reduced BP-specific proliferation in PBMC as well as cytokine production but increased IL-9, IL-4, and IFN-γ cytokines in naïve CD4 + T cells. These effects were independent of the childhood vaccination received by the donors. Similarly, TGF-ß reduced BP-specific proliferation in PBMC but induced proliferation in naïve CD4 + T cells. However, stimulation was associated with a generalized inhibition of cytokine production regardless of the original aP or wP vaccination received by the donors. Our study suggests that key T cell functions such as cytokine secretion are under the control of antigen stimulation and environmental cues but molecular pathways different than the ones investigated here might underlie the long-lasting differential cytokine production associated with aP- vs wP-priming in childhood vaccination.

Effectiveness of experimental and commercial pertussis vaccines in the elimination of Bordetella pertussis isolates with different genetic profiles in murine model.

The aim of this study was to compare the elimination of Bordetella pertussis clinical isolates, representing different genotypes in relation to alleles encoding virulence factors (MLST-multi-locus antigen sequence typing), MLVA type (multi-locus variable-number tandem repeat analysis) and PFGE group (pulsed-field gel electrophoresis) from the lungs of naive mice or mice were immunised with the commercial whole-cell pertussis vaccine, the acellular pertussis vaccine and the experimental whole-cell pertussis vaccine. Molecular data indicate that the resurgence of pertussis in populations with high vaccine coverage is associated with genomic adaptation of B. pertussis, to vaccine selection pressure. Pertactin-negative B. pertussis isolates were suspected to contribute to the reduced vaccine effectiveness. It was shown that one of the isolates used is PRN deficient. The mice were intranasally challenged with bacterial suspension containing approximately 5 × 10 7 CFU/ml B. pertussis. The immunogenicity of the tested vaccines against PT (pertussis toxin), PRN (pertactin), FHA (filamentous haemagglutinin) and FIM (fimbriae types 2 and 3) was examined. The commercial whole-cell and acellular pertussis vaccines induced an immunity effective at eliminating the genetically different B. pertussis isolates from the lungs. However, the elimination of the PRN-deficient isolate from the lungs of mice vaccinated with commercial vaccines was delayed as compared to the PRN ( +) isolate, suggesting phenotypic differences with the circulating isolates and vaccine strains. The most effective vaccine was the experimental vaccine with the composition identical to that of the strains used for infection.

Clinical impact of respiratory syncytial virus infection on children hospitalized for pertussis.

BACKGROUND: Although Respiratory syncytial virus (RSV) is one of the common pathogens in children with pertussis and viral coinfection, the clinical impact of RSV infection on pertussis remains unclear. We compared clinical characteristics and sought differences between infants with single Bordetella pertussis (B. pertussis) infection and those with RSV coinfection. METHODS: We enrolled 80 patients with pertussis who were hospitalized in Shenzhen Children's Hospital from January 2017 to December 2019. Respiratory tract samples were tested for B. pertussis with real-time polymerase chain reaction and respiratory viruses with immunofluorescence assay. Clinical data were obtained from hospital records and collected using a structured questionnaire. RESULTS: Thirty-seven of 80 patients had B. pertussis infection alone (pertussis group) and 43 had RSV-pertussis coinfection (coinfection group). No significant differences were found with regard to sex, body weight, preterm birth history, pertussis vaccination, symptoms, presence of pneumonia, or lymphocyte count between the 2 groups. Univariate analysis showed patients with RSV coinfection were older (median, 4.57 months vs 4.03 months, p = 0.048); more commonly treated with ß-lactam antibiotics (21% vs 5%, p = 0.044); had higher rates of wheezes (40% vs 14%, p = 0.009) and rales (35% vs 14%, p = 0.028) on chest auscultation, a higher rate of readmission (40% vs 11%, p = 0.004), and a longer hospital stay (median, 10 days vs 7 days, p = 0.002). In the further binary logistic regression analysis, patients with RSV coinfection had higher rates of wheezes (OR = 3.802; 95% CI: 1.106 to 13.072; p = 0.034) and readmission (OR = 5.835; 95% CI: 1.280 to 26.610; p = 0.023). CONCLUSIONS: RSV coinfection increases readmission rate in children hospitalized for pertussis. RSV infection should be suspected when wheezes are present on auscultation of the chest in these patients. Early detection of RSV may avoid unnecessary antibiotic use.

Conserved Structural Features of Core Oligosaccharides among the Lipopolysaccharides of Respiratory Pathogens from the Genus Bordetella Analyzed Exclusively by NMR Spectroscopy.

Bacterial pathogens expose on the cell surface a variety of complex carbohydrate molecules. Gram-negative bacteria produce lipopolysaccharides, which are the main components of the outer membrane of bacterial envelopes and play a major role in host-pathogen interactions. B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii, are mammalian respiratory pathogens, having substantial economic impact on human health and agriculture. B. pertussis is responsible for whooping cough (pertussis) and B. holmesii is the second pertussis etiological factor, but the current anti-pertussis vaccines do not provide cross-protection. The structural data on any given hypothetical carbohydrate antigen is a prerequisite for further analysis of structure-related activities and their interaction with hosts. 1H NMR spectra constitute fingerprints of the analyzed glycans and provide unique identity information. The concept of structure-reporter groups has now been augmented by 1H,13C-correlation spectra of the Bordetella oligosaccharides. The comparative analysis of Bordetellae oligosaccharides (OS) revealed that the hexasaccharide, comprising the α-GlcpN, α-GlcpA, 4,6-disubstituted-ß-Glcp, 2,7-disubstituted-l-α-d-Hepp, 3,4-disubstituted-l-α-d-Hepp, and Kdo, constitute the least variable OS segment. This minimal common element in the structure of lipopolysaccharides of Bordetellae could be used to devise a universal cross-protective vaccine component against infections with various bacteria from the genus Bordetella.

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of Bordetella pertussis.

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMß2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein.

The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air-liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC- toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT- toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.