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.

Transient reemergence of Bordetella parapertussis in France in 2022.

Between March and October 2022, a peak of detection of Bordetella parapertussis by qPCR, real-time PCR was observed in France.Hypothesis/Gap Statement. Whether this peak was due to resurgence from previous circulating lineages or reintroduction into the country was unknown.Objective. The objective of this study is to understand B. parapertussis-transient increase observed in France in 2022 whereas it had virtually stopped being reported since the start of the COVID-19 pandemic in 2020.Methods. We analysed real-time PCR (qPCR) data from the two largest French outpatient laboratories performing whooping cough diagnosis and characterized all B. parapertussis isolates collected in the 2016-2022 period by the French National Reference Centre for Whooping Cough.Results. Microbiological analyses reveal that 13 of 18 bacterial isolates collected in 2022 produce the vaccine antigen pertactin, whereas none of the 22 isolates collected in the 2016-2021 period did.Conclusion. We hypothesize a re-introduction of B. parapertussis from regions of the world where whole-cell vaccines are still in use.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

High purity and recovery of native filamentous hemagglutinin (FHA) from Bordetella pertussis using affinity chromatography.

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.

Comparative structural and immunological analysis of outer membrane proteins and dermonecrotic toxin in Bordetella bronchiseptica canine isolate.

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals' welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components.

fim3-24/ptxP-3 genotype is associated to whooping cough outbreak in Brazilian Midwest: The selection of Bordetella pertussis strains driven by vaccine immunization.

Whopping cough (or Pertussis) is an acute infectious respiratory disease caused by Bordetella pertussis bacteria. The disease is highly transmissible and can be fatal in children under two years old. Since the introduction of vaccine immunization in 1940, Pertussis incidence decreased worldwide. In Brazil, the immunization was introduced in 1977 using the whole cell (wP) vaccine. Despite the high vaccination coverage, an unexpected increase in the number of observed Pertussis cases was observed in 2012. In this year, 2257 cases were reported exceeding the average incidence rate of <1000 cases per year until 2010. This outbreak reached a peak level in 2014 and ended in 2018 according to the Brazilian National Surveillance System (SINAN). To understand the relationship between the outbreak and the vaccination, bacterial isolates (n = 136) from the Brazilian Midwest region obtained during the outbreak were submitted to genotyping of two vaccine loci: ptxP and fim3. Most of isolates (102) were obtained from nursing children (29 days to 2 years old). Genotyping of 94 isolates revealed that fim3-24/ptxP-3 was the most prevalent genotype (68%) associated with the outbreak peak. Two additional genotypes were also observed: fim3-1/ptxP-3 (15%) and fim3-3/ptxP-3 (17%). Conversely, the fim3-1/ptxP-2 genotype, which is harbored by the strain used in the wP vaccine (Bp137), was not observed. These results showed that B. pertussis circulating strains in the outbreak analyzed were different from the strain used for Pertussis immunization in Brazil. These observations provide insights that could be used to target vaccination programs to prevent future whooping cough outbreaks in Brazil.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

Bordetella pertussis isolates in Finland after acellular vaccination: serotype change and biofilm formation.

OBJECTIVES: In Finland, whole cell pertussis vaccine (wP) was introduced in 1952 and was replaced by acellular pertussis vaccine (aP) without fimbrial (FIM) antigen in 2005. We aimed to analyse the changes in serotypes of circulating Bordetella pertussis before and after acellular vaccination and to explore the relationship between biofilm formation and serotype diversity after the introduction of aP vaccine. METHODS: Serotyping of 1399 B. pertussis isolates collected at the Finnish National Reference Laboratory for Pertussis and Diphtheria in Turku, Finland, from 1974 to 2023 was performed by slide agglutination or indirect ELISA. Of 278 isolates collected after 2005, 53 were selected, genotyped for fim3 and fim2 alleles, and tested for biofilm formation. The selection criteria included maintaining a relatively equal distribution of isolates per time interval, ensuring approximately a 50:50 ratio of FIM2 (N = 26) and FIM3 (N = 27) serotypes. The reference strain Tohama I was used as a control. RESULTS: During the wP era, the majority of circulating B. pertussis exhibited the FIM2 serotype. However, FIM3 strains have appeared since 1999 and become prevalent. After the implementation of aP vaccines, the distribution of serotypes has exhibited substantial variability. FIM3 isolates displayed an enhanced biofilm formation compared to FIM2 isolates (Geometric mean value (95% CI): 0.90 (0.79-1.03) vs. 0.75 (0.65-0.85); p < 0.05). Of the 27 FIM3 isolates, 8 harboured fim3-1 and 19 fim3-2 alleles. FIM3 isolates with fim3-2 allele were significantly associated with increased biofilm formation when compared to those with fim3-1 (1.07 (0.96-1.19) vs. 0.61 (0.52-0.72); p < 0.0001). CONCLUSION: Following the implementation of aP vaccines, the distribution of serotypes in Finland has exhibited substantial variability. FIM3 isolates with the fim3-2 allele displayed an enhanced biofilm formation capability compared to FIM2 isolates.

RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

Novel mouse monoclonal antibodies against Bordetella pertussis pertactin antigen with versatile applications.

BACKGROUND: Pertussis, or whooping cough, is a highly contagious respiratory disease caused by Bordetella pertussis (BP). Pertactin (PRN) is one of the main immunogenic components of BP and is employed in many commercialized acellular pertussis vaccines (aPVs). Purification of this protein by conventional chromatography methods is challenging and commonly requires multiple laborious processes with low recovery. Using specific monoclonal antibodies (mAbs) for the purification of PRN antigen is expected to yield high purity and recovery of the target molecule. METHODS: Recombinant PRN antigen was used to produce mouse mAbs using hybridoma technology. Structural and functional characteristics of the mAbs were assessed by ELISA, immunoblotting, and flow cytometry. Selected mAbs were employed to purify PRN by affinity chromatography, and the purity and recovery of the purified protein were analyzed by ELISA, SDS-PAGE, and immunoblotting. Moreover, ELISA and flow cytometry techniques were designed using these mAbs to detect PRN in different strains of BP. RESULTS: Five mAbs were produced and selected based on their reactivity with native PRN. Our results demonstrate that purification of PRN by affinity chromatography resulted in a highly pure antigen with 75-85 percent recovery. In addition, ELISA and flow cytometry results indicated that these mAbs could recognize PRN in the bacterial cell walls of different BP strains. CONCLUSION: We successfully produced PRN-specific mAbs and designed an affinity chromatography method to purify PRN antigen with higher purity and recovery than conventional methods. These mAbs could be employed as valuable tools for the detection and purification of PRN for vaccine manufacturing.

Biological differences between FIM2 and FIM3 fimbriae of Bordetella pertussis: not just the serotype.

INTRODUCTION: Bordetella pertussis still circulates worldwide despite vaccination. Fimbriae are components of some acellular pertussis vaccines. Population fluctuations of B. pertussis fimbrial serotypes (FIM2 and FIM3) are observed, and fim3 alleles (fim3-1 [clade 1] and fim3-2 [clade 2]) mark a major phylogenetic subdivision of B. pertussis. OBJECTIVES: To compare microbiological characteristics and expressed protein profiles between fimbrial serotypes FIM2 and FIM3 and genomic clades. METHODS: A total of 19 isolates were selected. Absolute protein abundance of the main virulence factors, autoagglutination and biofilm formation, bacterial survival in whole blood, induced blood cell cytokine secretion, and global proteome profiles were assessed. RESULTS: Compared to FIM3, FIM2 isolates produced more fimbriae, less cellular pertussis toxin subunit 1 and more biofilm, but auto-agglutinated less. FIM2 isolates had a lower survival rate in cord blood, but induced higher levels of IL-4, IL-8 and IL-1ß secretion. Global proteome comparisons uncovered 15 differentially produced proteins between FIM2 and FIM3 isolates, involved in adhesion and metabolism of metals. FIM3 isolates of clade 2 produced more FIM3 and more biofilm compared to clade 1. CONCLUSION: FIM serotype and fim3 clades are associated with proteomic and other biological differences, which may have implications on pathogenesis and epidemiological emergence.

Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain.

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.

Uncovering the folding mechanism of pertactin: A comparative study of isolated and vectorial folding.

Autotransporters are a large family of virulence factors found in Gram-negative bacteria that play important roles in their pathogenesis. The passenger domain of autotransporters is almost always composed of a large ß-helix, with only a small portion of it being relevant to its virulence function. This has led to the hypothesis that the folding of the ß-helical structure aids the secretion of the passenger domain across the Gram-negative outer membrane. In this study, we used molecular dynamics simulations and enhanced sampling methods to investigate the stability and folding of the passenger domain of pertactin, an autotransporter from Bordetella pertussis. Specifically, we employed steered molecular dynamics to simulate the unfolding of the entire passenger domain as well as self-learning adaptive umbrella sampling to compare the energetics of folding rungs of the ß-helix independently ("isolated folding") versus folding rungs on top of a previously folded rung ("vectorial folding"). Our results showed that vectorial folding is highly favorable compared with isolated folding; moreover, our simulations showed that the C-terminal rung of the ß-helix is the most resistant to unfolding, in agreement with previous studies that found the C-terminal half of the passenger domain to be more stable than the N-terminal one. Overall, this study provides new insights into the folding process of an autotransporter passenger domain and its potential role in secretion across the outer membrane.

Pasteurian Contributions to the Study of Bordetella pertussis Toxins.

As a tribute to Louis Pasteur on the occasion of the 200th anniversary of his birth, this article summarizes the main contributions of scientists from Pasteur Institutes to the current knowledge of toxins produced by Bordetella pertussis. The article therefore focuses on publications authored by researchers from Pasteur Institutes and is not intended as a systematic review of B. pertussis toxins. Besides identifying B. pertussis as the causative agent of whooping cough, Pasteurians have made several major contributions with respect to the structure-function relationship of the Bordetella lipo-oligosaccharide, adenylyl cyclase toxin and pertussis toxin. In addition to contributing to the understanding of these toxins' mechanisms at the molecular and cellular levels and their role in pathogenesis, scientists at Pasteur Institutes have also exploited potential applications of the gathered knowledge of these toxins. These applications range from the development of novel tools to study protein-protein interactions over the design of novel antigen delivery tools, such as prophylactic or therapeutic vaccine candidates against cancer and viral infection, to the development of a live attenuated nasal pertussis vaccine. This scientific journey from basic science to applications in the field of human health matches perfectly with the overall scientific objectives outlined by Louis Pasteur himself.

Filamentous Hemagglutinin of Bordetella pertussis Does Not Interact with the ß2 Integrin CD11b/CD18.

The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the ß2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the ß2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.

Blockade of the Adenylate Cyclase Toxin Synergizes with Opsonizing Antibodies to Protect Mice against Bordetella pertussis.

Bordetella produces an array of virulence factors, including the adenylate cyclase toxin (ACT), which is essential, immunogenic in humans, and highly conserved. Despite mediating immune-evasive functions as a leukotoxin, ACT's potential role as a protective antigen is unclear. To better understand the contributions of humoral anti-ACT immunity, we evaluated protection against Bordetella pertussis by antibodies binding structurally defined ACT epitopes in a mouse pneumonia model. An ACT-neutralizing antibody, but not a nonneutralizing antibody or an isotype control, significantly increased mouse survival after lethal challenge with B. pertussis. When modified to impair Fc effector functions, the neutralizing antibody retained protective capabilities, indicating that protection was mediated by the blockade of the interactions of ACT with its αMß2 integrin receptor. After infection with a lower bacterial dose, ACT neutralization synergistically reduced lung bacterial colonization levels when combined with an opsonic antibody binding the surface antigen pertactin. Notably, protection was significantly enhanced when antibodies were administered intranasally as opposed to systemically, indicating that local immune responses are key to antibody-mediated protection against ACT and pertactin. These data reconcile previous conflicting reports to indicate that neutralizing anti-ACT antibodies support the phagocytosis of opsonized B. pertussis and thereby contribute to pertussis protection in vivo. IMPORTANCE Despite high vaccine coverage in developed countries, the incidence of pertussis has increased in recent decades, often leading to severe consequences for sensitive groups, including infants. For this reason, improving the efficacy of pertussis vaccines is critical, and the addition of new antigens is a leading strategy to achieve this goal. The Bordetella pertussis adenylate cyclase toxin (ACT) acts to disarm host immunity and is considered a promising vaccine candidate since it is found in all Bordetella species. In this work, we show that antibodies neutralizing ACT offer protection against pertussis. Using a murine infection model, we show that antibodies neutralizing ACT can contribute to protection against infection through synergistic interactions with antibodies recognizing current vaccine antigens. Our data can help guide the design of future vaccines, whereby the inclusion of ACT-based immunogens might increase protection against pertussis infection.

Pertactin-Deficient Bordetella pertussis with Unusual Mechanism of Pertactin Disruption, Spain, 1986-2018.

Bordetella pertussis not expressing pertactin has increased in countries using acellular pertussis vaccines (ACV). The deficiency is mostly caused by pertactin gene disruption by IS481. To assess the effect of the transition from whole-cell vaccine to ACV on the emergence of B. pertussis not expressing pertactin in Spain, we studied 342 isolates collected during 1986-2018. We identified 93 pertactin-deficient isolates. All were detected after introduction of ACV and represented 38% of isolates collected during the ACV period; 58.1% belonged to a genetic cluster of isolates carrying the unusual prn::del(-292, 1340) mutation. Pertactin inactivation by IS481 insertion was identified in 23.7% of pertactin-deficient isolates, arising independently multiple times and in different phylogenetic branches. Our findings support the emergence and dissemination of a cluster of B. pertussis with an infrequent mechanism of pertactin disruption in Spain, probably resulting from introduction of ACV.

The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model.

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 108 viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity.

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors.