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.

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.

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.

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.

Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture.

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

Evolution of Bordetella pertussis over a 23-year period in France, 1996 to 2018.

BackgroundBordetella pertussis is the main agent of whooping cough. Vaccination with acellular pertussis vaccines has been largely implemented in high-income countries. These vaccines contain 1 to 5 antigens: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and/or fimbrial proteins (FIM2 and FIM3). Monitoring the emergence of B. pertussis isolates that might partially escape vaccine-induced immunity is an essential component of public health strategies to control whooping cough.AimWe aimed to investigate temporal trends of fimbriae serotypes and vaccine antigen-expression in B. pertussis over a 23-year period in France (1996-2018).MethodsIsolates (n = 2,280) were collected through hospital surveillance, capturing one third of hospitalised paediatric pertussis cases. We assayed PT, FHA and PRN production by Western blot (n = 1,428) and fimbriae production by serotyping (n = 1,058). Molecular events underlying antigen deficiency were investigated by genomic sequencing.ResultsThe proportion of PRN-deficient B. pertussis isolates has increased steadily from 0% (0/38) in 2003 to 48.4% (31/64) in 2018 (chi-squared test for trend, p < 0.0001), whereas only 5 PT-, 5 FHA- and 9 FIM-deficient isolates were found. Impairment of PRN production was predominantly due to IS481 insertion within the prn gene or a 22 kb genomic inversion involving the prn promoter sequence, indicative of convergent evolution. FIM2-expressing isolates have emerged since 2011 at the expense of FIM3.ConclusionsB. pertussis is evolving through the rapid increase of PRN-deficient isolates and a recent shift from FIM3 to FIM2 expression. Excluding PRN, the loss of vaccine antigen expression by circulating B. pertussis isolates is epidemiologically insignificant.

Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic.

Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg- mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg- mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

Pertactin contributes to shedding and transmission of Bordetella bronchiseptica.

Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis.

Naturally circulating pertactin-deficient Bordetella pertussis strains induce distinct gene expression and inflammatory signatures in human dendritic cells.

Respiratory infections caused by Bordetella pertussis are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating B. pertussis strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating B. pertussis strains are deficient in Prn. To better understand the resurgence of pertussis, we investigated the response of human monocyte-derived dendritic cells (moDCs) to naturally circulating Prn-expressing (Prn-Pos) and Prn-deficient (Prn-Neg) B. pertussis strains from 2016 in the Netherlands. Transcriptome analysis of moDC showed enriched IFNα response-associated gene expression after exposure to Prn-Pos B. pertussis strains, whereas the Prn-Neg strains induced enriched expression of interleukin- and TNF-signaling genes, as well as other genes involved in immune activation. Multiplex immune assays confirmed enhanced proinflammatory cytokine secretion by Prn-Neg stimulated moDC. Comparison of the proteomes from the Prn-Pos and Prn-Neg strains revealed, next to the difference in Prn, differential expression of a number of other proteins including several proteins involved in metabolic processes. Our findings indicate that Prn-deficient B. pertussis strains induce a distinct and stronger immune activation of moDCs than the Prn-Pos strains. These findings highlight the role of pathogen adaptation in the resurgence of pertussis as well as the effects that vaccine pressure can have on a bacterial population.

DegP Initiates Regulated Processing of Filamentous Hemagglutinin in Bordetella bronchiseptica.

Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis, the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica. FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA' (the main product of FhaB processing) and an accumulation of full-length FhaB in whole-cell lysates. Also, FHA' was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica. Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria. IMPORTANCE Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.

Construction and evaluation of a pertactin-deficient live attenuated pertussis vaccine candidate BPZE1 derivative.

Pertussis, mainly caused by Bordetella pertussis, is a severe respiratory disease that can be fatal, especially in young infants. Vaccines, massively implemented since the middle of the last century, have substantially reduced the pertussis incidence, but have not been able to fully control the disease. One of the shortcomings of current pertussis vaccines is their inability to prevent infection by and transmission of B. pertussis, in contrast to immunity following natural infection. We have developed the live attenuated nasal vaccine BPZE1 and have shown that it prevents both disease and B. pertussis infection in preclinical models. This vaccine is now in clinical development. However, the initial clinical studies have suggested that vaccine take is hampered by pre-existing antibodies to pertactin. Here, we have constructed a pertactin-deficient BPZE1 derivative called BPZE1P in order to overcome this limitation. BPZE1P colonized the murine respiratory tract as efficiently as BPZE1 and induced antibodies at levels similar to those elicited by BPZE1. In the presence of pre-existing antibodies induced by acellular pertussis vaccination, BPZE1P colonized the mouse respiratory tract more efficiently than BPZE1. Both vaccines protected equally well the murine lungs and noses from challenge with laboratory and clinical strains of B. pertussis, including pertactin-deficient strains, against which current acellular pertussis vaccines are less efficient. BPZE1P may thus be an interesting alternative to BPZE1 to overcome vaccine take limitations due to pre-existing antibodies to pertactin.

Production of Highly Active Recombinant Dermonecrotic Toxin of Bordetella Pertussis.

Pathogenic Bordetella bacteria release a neurotropic dermonecrotic toxin (DNT) that is endocytosed into animal cells and permanently activates the Rho family GTPases by polyamination or deamidation of the glutamine residues in their switch II regions (e.g., Gln63 of RhoA). DNT was found to enable high level colonization of the nasal cavity of pigs by B. bronchiseptica and the capacity of DNT to inhibit differentiation of nasal turbinate bone osteoblasts causes atrophic rhinitis in infected pigs. However, it remains unknown whether DNT plays any role also in virulence of the human pathogen B. pertussis and in pathogenesis of the whooping cough disease. We report a procedure for purification of large amounts of LPS-free recombinant DNT that exhibits a high biological activity on cells expressing the DNT receptors Cav3.1 and Cav3.2. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the DNT molecule adopts a V-shaped structure with well-resolved protein domains. These results open the way to structure-function studies on DNT and its interactions with airway epithelial layers.

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles.

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

Multiple weak interactions between BvgA~P and ptx promoter DNA strongly activate transcription of pertussis toxin genes in Bordetella pertussis.

Pertussis toxin is the preeminent virulence factor and major protective antigen produced by Bordetella pertussis, the human respiratory pathogen and etiologic agent of whooping cough. Genes for its synthesis and export are encoded by the 12 kb ptx-ptl operon, which is under the control of the pertussis promoter, Pptx. Expression of this operon, like that of all other known protein virulence factors, is regulated by the BvgAS two-component global regulatory system. Although Pptx has been studied for years, characterization of its promoter architecture vis-à-vis BvgA-binding has lagged behind that of other promoters, mainly due to its lower affinity for BvgA~P. Here we take advantage of a mutant BvgA protein (Δ127-129), which enhances ptx transcription in B. pertussis and also demonstrates enhanced binding affinity to Pptx. By using this mutant protein labeled with FeBABE, binding of six head-to-head dimers of BvgA~P was observed, with a spacing of 22 bp, revealing a binding geometry similar to that of other BvgA-activated promoters carrying at least one strong binding site. All of these six BvgA-binding sites lack sequence features associated with strong binding. A genetic analysis indicated the degree to which each contributes to Pptx activity. Thus the weak/medium binding affinity of Pptx revealed in this study explains its lower responsiveness to phosphorylated BvgA, relative to other promoters containing a high affinity binding site, such as that of the fha operon.

Allele frequencies of Bordetella pertussis virulence-associated genes identified from pediatric patients with severe respiratory infections.

Bordetella pertussis (B. pertussis) infection occasionally causes severe respiratory infections in children. Potential association between virulence-associated gene alleles and severe clinical outcomes has been suggested; however, frequencies of these alleles in pediatric patients with severe pertussis have not been clarified. We retrospectively tested stored respiratory samples collected from B. pertussis-positive patients by polymerase chain reaction targeting for major virulence-associated genes; fimbrae (fim) serotype 3 (fim3), pertactin (prn), pertussis toxin A (ptxA), and pertussis toxin promotor (ptxP). Based on the identified sequences, multilocus sequence typing (MLST) was conducted. Association of gene allele frequency and clinical outcomes such as management in pediatric intensive care unit, intubation, and mortality was analyzed. Out of 25 patients with available samples, the most prevalent allele for each virulence-associated gene was fim3A (17/21, 83%), ptxA1 (20/23, 87%), prn2 (13/16, 81%), and ptxP3 (14/17, 82%). In the study, total of 3 MLST types were identified; MLST-1 from 3 patients (19%), MLST-2 from 10 patients (63%), and MLST-4 from 3 patients (19%). Detection rate of the major MLST type; MLST-2 was significantly higher for patients who required intubation compared to those who did not (6/6, 100% vs 4/10, 40%; p = 0.034). In conclusion, MLST-2 was the most common MLST type in B. pertussis isolated from Japanese children with severe pertussis. Further studies investigating its causal association with disease severity is warranted.

Expression of small RNAs of Bordetella pertussis colonizing murine tracheas.

We performed RNA sequencing on Bordetella pertussis, the causative agent of whooping cough, and identified nine novel small RNAs (sRNAs) that were transcribed during the bacterial colonization of murine tracheas. Among them, four sRNAs were more strongly expressed in vivo than in vitro. Moreover, the expression of eight sRNAs was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. The present results suggest a BvgAS-independent gene regulatory system involving the sRNAs that is active during B. pertussis infection.

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

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

Population dynamics and antigenic drift of Bordetella pertussis following whole cell vaccine replacement, Barcelona, Spain, 1986-2015.

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.