Bordetella pertussis, which is the causative agent of whooping cough, has re-emerged as a public health threat despite broad vaccine coverage.
Re-emergence of this disease correlates with a transition from the use of whole-cell pertussis vaccines to acellular component vaccines, and the epidemiology of pertussis has shifted while the overall number of pertussis cases has increased.
Bordetella spp. are paradigms for studying global virulence-control systems, bacterial toxins, type V secretion proteins and the evolution of pathogens.
Bordetella spp. produce many virulence factors that contribute to pathogenesis, including toxins, adhesion factors, iron-acquisition systems and surface structures.
Considering the fact that Bordetella pertussis is a strict human pathogen, the development of appropriate animal models has been challenging. However, a novel baboon infection and transmission model provides a promising new research avenue.
Future goals include improving the efficacy of vaccines, protecting unvaccinated infants from infection and developing better treatment strategies for infants who become infected with B. pertussis. Achieving these goals requires a more thorough understanding of the mechanisms that are used by B. pertussis to establish infection and cause disease.
Pertussis, also known as whooping cough, has recently re-emerged as a major public health threat despite high levels of vaccination against the aetiological agent Bordetella pertussis. In this Review, we describe the pathogenesis of this disease, with a focus on recent mechanistic insights into B. pertussis virulence-factor function. We also discuss the changing epidemiology of pertussis and the challenges facing vaccine development. Despite decades of research, many aspects of B. pertussis physiology and pathogenesis remain poorly understood. We highlight knowledge gaps that must be addressed to develop improved vaccines and therapeutic strategies.
Pertussis is a highly contagious respiratory disease that is transmitted directly from human to human1, probably via aerosolized respiratory droplets. The primary causative agent, Bordetella pertussis, is a Gram-negative bacterium that was first described by Bordet and Gengou in 1906 (Ref. 2). The closely related bacterium Bordetella parapertussis Hu is responsible for a minority of cases (approximately 14%) and is less capable of causing severe disease3. Both B. pertussis and B. parapertussisHu are human-specific, and phylogenetic analyses indicate that they evolved from Bordetella bronchiseptica or a B. bronchiseptica-like ancestor4,5 (Box 1). B. bronchiseptica infects a broad range of mammals, including humans, and although it can cause overt disease such as kennel cough in dogs and atrophic rhinitis in pigs, it typically colonizes its hosts chronically and asymptomatically6. Despite differences in host range and disease-causing propensity, B. pertussis, B. parapertussisHu and B. bronchiseptica are so closely related that they are now considered to be subspecies. Together, these organisms provide a paradigm for understanding bacterial adaptation to humans and the dichotomy between acute disease and chronic asymptomatic infection4,5. Although other Bordetella species have been isolated from humans, they seem to be primarily opportunistic human pathogens.
In the pre-vaccine era, pertussis was widespread and mainly affected young children (1–9 years old)7. The classical manifestation of the disease in this age group, is characterized by three phases: the catarrhal phase, the paroxysmal phase and the convalescent phase8. Clinical observations, combined with results from studies using animal models (Box 2), suggest that classic pertussis is initiated by the adherence of bacteria to the ciliated respiratory epithelium in the nasopharynx and trachea9,10. Adherent bacteria survive innate host defences, such as mucociliary clearance and the action of antimicrobial peptides, multiply locally and resist elimination by inflammatory cells. Symptoms during this catarrhal phase are similar to those of many upper respiratory infections, such as the common cold. After 1–2 weeks, the disease progresses to the paroxysmal phase, which can persist for 1–10 weeks and is characterized by periods of normal airway function that are interspersed with multiple severe spasmodic coughing fits, followed by characteristic inspiratory whoops, and often, emesis. The onset of adaptive immunity coincides with bacterial clearance but not with the cessation of symptoms, which typically decline gradually over another month but can persist for much longer (this is known as the convalescent phase)8. In infants (<1 year old), pertussis can take a more serious course, in which bacteria disseminate into the lungs and cause necrotizing bronchiolitis, intra-alveolar haemorrhage and fibrinous oedema10. In severe cases, extreme lymphocytosis occurs, which positively correlates with intractable pulmonary hypertension, respiratory failure and death10.
The introduction of whole-cell pertussis (wP) vaccines in the late 1940s resulted in a rapid reduction in both the incidence of pertussis and the number of deaths caused by the infection. However, the success of these vaccines was undermined by concerns about their safety (Box 3); thus, they were replaced with acellular pertussis (aP) vaccines in the late 1990s in many developed countries11. Since then, the number of pertussis cases has increased and dramatic epidemic cycles have returned. In 2012, 48,277 cases of pertussis and 18 deaths associated with the infection were reported to the US Centers for Disease Control and Prevention (CDC); this represents the greatest burden of pertussis in the United States in 60 years, and similar outbreaks are occurring in other countries12,13,14. However, the epidemiology of contemporary pertussis does not replicate that of the pre-vaccine era. Disease is now more common in infants and older children (aged 9–19) and, strikingly, older children who develop pertussis are often fully vaccinated according to current recommendations15,16. Ominously, studies that have analysed the incidence of pertussis among children who were born and vaccinated during the transition to aP vaccines have found that the rate of infection is significantly higher among children who have been vaccinated with only aP vaccines compared with those who have been vaccinated with even a single dose of the wP vaccine17. To combat the rise of infections in this group, regulatory agencies have called for boosters to be administered earlier18. However, the benefit of boosting with aP vaccines is unclear as it is unknown whether the re-emergence of pertussis is due simply to waning immunity or whether it is due to fundamental differences in the nature of the immune response that is induced by aP vaccines compared with wP vaccines or with natural infection.
The increased incidence of disease among older children and adults is especially worrying because of the corresponding risk of transmission to incompletely or non-immunized infants1. Compounding the problem, antibiotic treatment has minimal efficacy by the time most diagnoses are made, and severe cases can be unresponsive to standard therapies for respiratory distress10. Therefore, the re-emergence of pertussis as a global public health problem presents two challenges: first, the development of vaccines that have an acceptable safety profile, provide long-lasting immunity, reduce the burden of infection and prevent transmission; and second, the development of therapeutic agents and treatment strategies that reduce morbidity and mortality in vulnerable populations. Both goals require a better understanding of the aetiological agents of pertussis and the mechanisms by which they cause disease.
In this Review, we discuss our current understanding of the mechanisms that are used by Bordetella spp. to cause respiratory disease, focusing on the roles and functions of virulence factors in pathogenesis. For the interested reader, more specialized recent reviews on pertussis toxin biology19,20, virulence gene regulation21, immunity22,23 and vaccines24, as well as an earlier comprehensive review on Bordetella spp. pathogenesis25 are available.
Bordetella virulence regulation
Several Bordetella virulence factors were identified and biochemically characterized before genetic tools became available, including pertussis toxin (PT), adenylate cyclase toxin (ACT), dermonecrotic toxin (DNT), filamentous haemagglutinin (FHA) and fimbriae (Fim). The first transposon mutagenesis screen of B. pertussis identified the genes that encode these factors, as well as a locus — now known as bvgAS — which encodes a two-component regulatory system that is required for their expression26. Reasoning that BvgAS also activates the expression of genes that encode additional unknown Bordetella virulence factors, mutagenesis screens using Tn5lac and Tn5phoA were conducted27,28. These, together with subsequent genome-wide analyses, showed that BvgAS controls hundreds of genes in response to changing environmental conditions, including genes that encode surface structures and secreted proteins involved in pathogenesis, factors required for survival outside the mammalian host, enzymes involved in cellular metabolism and physiology and additional regulatory systems29,30.
The BvgAS phosphorelay. BvgA is a typical response-regulator protein with a receiver domain at its amino terminus and a DNA-binding helix–turn–helix domain at its carboxyl terminus21 (Fig. 1a). BvgS is a polydomain sensor kinase that contains two N-terminal venus flytrap (VFT) domains, which are located in the periplasm31. C-terminal to the VFT domains is a membrane-spanning region, followed by a cytoplasmically located PAS domain, a histidine kinase (HK) domain, a receiver domain and a histidine phosphotransferase (Hpt) domain. During growth in standard medium at 37 °C, BvgAS is active and uses ATP to phosphorylate a conserved histidine in the HK domain32. The phosphoryl group is subsequently relayed to an aspartate in the receiver domain, then to a histidine in the Hpt domain and, finally, to an aspartate in the receiver domain of the response regulator BvgA32. Phosphorylated BvgA is competent for dimerization and binds to specific DNA sequences to either activate or repress transcription33,34. Although the signal (or signals) to which BvgS responds in nature is unknown, growth at a low temperature (∼25 °C) or in the presence of magnesium sulphate (MgSO4) or nicotinic acid (which are 'chemical modulators' of BvgS) inactivates BvgS; thus, BvgA remains unphosphorylated and is unable to regulate transcription.
BvgAS controls multiple phenotypic phases. The genes that are regulated by the BvgAS phosphorelay belong to four classes and their differential regulation results in at least three distinct phenotypic phases (Fig. 1b). Class 1 genes include the ptx–ptl operon (which encodes PT and its transport system), cyaA–E (which encodes ACT) and the bsc operon (which encodes a type III Secretion System (T3SS)). These genes are maximally expressed when BvgAS is fully active (known as the Bvg+ phase). Class 2 genes are maximally expressed in both the Bvg-intermediate (Bvgi) and Bvg+ phases. The Bvgi phase occurs when bacteria are grown in the presence of low concentrations of chemical modulators or within the first few hours following a switch from Bvg− phase conditions to Bvg+ phase conditions. Class 2 genes include fhaB (which encodes filamentous haemagglutinin (FHA)), fim genes (which encode fimbriae) and bvgAS itself; thus, bvgAS is positively autoregulated. Class 3 genes, of which only one (bipA, which encodes an outer membrane protein of unknown function) has been characterized so far35,36, are maximally expressed in the Bvgi phase. Class 4 genes, which are also known as vrg (virulence-repressed genes) loci, are maximally expressed in the Bvg− phase and include genes that are required for flagella synthesis and motility in B. bronchiseptica.
Role of BvgAS-mediated gene regulation. The conservation of BvgAS among Bordetella spp. and its ability to control multiple phenotypic phases in response to environmental cues suggests that it has an important and conserved role in the infectious cycle. As B. pertussis and B. parapertussisHu strains are unable to survive for extended periods of time outside the human host (unpublished observations from various research groups), it was hypothesized that BvgAS-mediated gene regulation must occur in the mammalian respiratory tract. Experiments with mutants that were locked in either the Bvg+ phase or the Bvg− phase, or that ectopically expressed Bvg− phase factors in the Bvg+ phase, showed that the Bvg+ phase is necessary and sufficient for respiratory infection, that cells in the Bvg− phase are unable to survive in vivo and that failure to repress Bvg− phase factors (such as flagella) is detrimental to the development of infection37,38,39,40. Moreover, recent studies using sensitive reporter systems have provided strong evidence that switching to the Bvg− phase does not occur in vivo41,42. In B. bronchiseptica, the Bvg− phase is required for survival under nutrient-limiting conditions, such as those that might be encountered in an external environment43. It has been hypothesized that the Bvgi phase is important for transmission, and with the development of the baboon model (Box 2), this hypothesis is now testable. Although additional regulatory systems are undoubtedly important during the infectious cycle of Bordetella spp., their precise roles have not yet been determined.
Pertussis toxin. PT, which is sometimes referred to as lymphocytosis-promoting factor owing to its ability to induce lymphocytosis in mammals, was one of the first identified, and is one of the most extensively characterized, B. pertussis virulence factors44. The presumed requirement of PT for the development of infection and the observed positive correlation between PT-specific immunity and bacterial clearance led to the hypothesis that pertussis, like cholera and diphtheria, is a toxin-mediated disease45. However, although PT is important for pathogenesis, it is now clear that pertussis results from the coordinated function of many different virulence factors46.
PT is an ADP-ribosylating AB5-type toxin47 (Fig. 2a). The holotoxin is composed of one catalytic subunit (the A subunit) and five membrane-binding or transport subunits (B subunits; which form the B pentamer), which are assembled in the periplasm and then exported by the type IV secretion system that is encoded by the ptl locus48. PT holotoxin can bind to almost any sialic acid-containing glycoprotein49, and thus, multiple receptors have been identified and characterized in a broad range of cell types in vitro20; however, the specific cell types that are targeted by PT in vivo are unknown. After binding, PT enters the host cell by receptor-mediated endocytosis and follows a retrograde transport pathway to the Golgi apparatus and then the endoplasmic reticulum (ER)50 (Fig. 2b). The A subunit exits the ER, possibly by 'hijacking' the ER-associated degradation pathway that normally expels misfolded proteins51. In the cytoplasm, the A subunit catalyses the transfer of ADP–ribose from NAD+ to a cysteine residue near the C terminus of the α-subunit of heterotrimeric G proteins, some of which are inhibitory G proteins. Among other downstream effects, this modification eliminates the ability of these inhibitory G proteins to inhibit adenylate cyclase activity (resulting in increased cyclic AMP (cAMP) levels in the cell) and blocks other G protein-regulated enzymes and pathways20,52, leading to dysregulation of the immune response.
PT has an extraordinarily broad range of pharmacological effects in cell culture and animal models, which has confounded efforts aimed at identifying its precise role (or roles) during human infection. PT inhibits the migration of cells that express G protein-coupled chemokine receptors in vitro, such as neutrophils, monocytes and lymphocytes53. In mouse models, the production of PT by B. pertussis correlates with decreased pro-inflammatory chemokine and cytokine production, decreased recruitment of neutrophils to the lungs and increased bacterial burdens early in infection54,55. Experiments in mice, in which alveolar macrophages are depleted using clodronate, suggest that PT initially targets these cells56. PT production at the peak of infection correlates with exacerbated inflammation and pathology in the airways57. Although these and other observations in animal models suggest that PT contributes to the establishment of infection by suppressing early inflammation and inhibiting the microbicidal action of inflammatory cells, in addition to contributing to inflammatory pathology at the peak of infection, it is not known whether PT induces these effects during human infection. However, it has been shown that the production of PT positively correlates with the extreme lymphocytosis that occurs in primary human pertussis cases58, and antibodies against PT protect against severe disease59.
Adenylate cyclase toxin. ACT (Fig. 2c, d), which is a member of the RTX (repeats in toxin) toxin family, is encoded by cyaA and is produced by all Bordetella subspecies that infect mammals19. ACT is secreted by the cyaBDE-encoded type I secretion system and is palmitoylated by the product of cyaC (Refs 60, 61). The toxin contains two distinct functional modules: the C-terminal domain, which contains the RTX repeats, mediates binding to target cells and forms cation-selective pores in plasma membranes62,63; and the N-terminal domain, which is a calmodulin-dependent adenylate cyclase that converts ATP to cyclic AMP (cAMP)64,65. Recent studies indicate that ACT can adopt multiple conformations and that these forms are distinct in their ability to effect pore formation or the translocation of adenylate cyclase into the host cell66. Thus, the observed effects of ACT on different cell types are the result of a combination of ion permeability, increased levels of cAMP (which leads to the perturbation of downstream signalling events) and possibly, the depletion of intracellular ATP.
Although ACT can intoxicate many cell types, it binds with high affinity to complement receptor 3 (CR3; also known as αMβ2 integrin or CD11b/CD18), which is present on neutrophils, macrophages and dendritic cells67, and early work correlated ACT-dependent cAMP production in human neutrophils with the inhibition of phagocytosis and oxidative burst68. More recent studies have shown that ACT blocks complement-dependent phagocytosis by macrophages69. In addition, this toxin also suppresses the activation and chemotaxis of T cells70. The importance of these in vitro observations is unclear; however, a recent study using the baboon model and clinical samples from humans showed that the concentrations of ACT in B. pertussis-infected respiratory tissues are considerably lower than the concentrations of purified protein that are used in most in vitro studies71. In mouse models, ACT-deficient bacteria are cleared faster than wild-type bacteria, and studies using immunodeficient and neutropenic mice suggest that ACT has a crucial role in enabling bacteria to resist neutrophil-mediated clearance72,73. These data, in addition to the fact that ACT is one of the few virulence factors that is conserved and produced by all pathogenic Bordetella species5, suggest that ACT has the potential to be an effective antigen in future vaccine formulations74.
Type III secretion. For reasons of experimental tractability, the Bordetella Bsc type III secretion system (T3SS) is most extensively studied in B. bronchiseptica and induces caspase-independent necrotic death in a diverse range of cell types in vitro75. Mutations that eliminate T3SS activity decrease bacterial persistence in the lower respiratory tracts of rats and mice, following intranasal inoculation76,77. Infection of mice with T3SS-defective B. bronchiseptica mutants also results in a more robust antibody response, and re-stimulated splenocytes from animals that have been infected with these mutants show increased production of pro-inflammatory interferon-γ (IFNγ) and decreased production of anti-inflammatory interleukin-10 (IL-10)78. Consistent with these data, IFNγ has been shown to facilitate clearance of B. bronchiseptica from the lower respiratory tract, whereas IL-10 delays clearance78. Together, these observations suggest that the Bsc T3SS has an immunomodulatory role that promotes persistence in the lower respiratory tract; however, the mechanistic basis for this phenomenon remains to be determined.
Remarkably, and despite concerted efforts by several research teams, only a single effector protein, BteA, has been definitively identified as a translocated substrate of the Bsc T3SS79,80. BopN — a homologue of outer membrane protein YopN (which regulates type III secretion in pathogenic Yersinia spp.) — has been proposed to be a second effector81, but thus far, evidence that BopN is translocated by the Bsc system is lacking. BteA is both necessary and sufficient for cytotoxicity in vitro, and mutations in bteA recapitulate the phenotypes that are associated with eliminating T3SS activity in vitro and in vivo77,79. Following translocation into host cells, the N-terminal targeting domain results in BteA localization to the ezrin-rich lipid rafts that underlie sites of bacterial attachment82. However, the mechanisms responsible for the potent cytotoxicity of BteA remain unclear.
Type III secretion is tightly regulated in Bordetella spp. The bteA and bsc genes are transcriptionally activated by the alternative sigma factor BtrS, which is activated by BvgAS83. Expression of the bcs genes is also upregulated by iron starvation84. In addition to these regulatory mechanisms, the partner-switching proteins BtrU, BtrV and BtrW mediate a cycle of serine phosphorylation and dephosphorylation events, which regulate secretion activity83,85.
Perhaps the most pressing question regarding the Bsc T3SS relates to its potential role during human infection. A requirement for T3SS activity in B. pertussis cytotoxicity has not been documented, despite the fact that T3SS genes are intact and are highly conserved, transcribed and regulated, in addition to the observation that bteA alleles are functionally interchangeable between subspecies82,83. Fortunately, recent studies are beginning to shed light on this paradox. Although Bsc activity is not generally observed in laboratory-adapted B. pertussis strains, the tip complex of the T3SS, Bsp22, is secreted by clinical isolates in vitro, and mutations in the ATPase gene, bscN, result in elevated production of pro-inflammatory cytokines and accelerated clearance of B. pertussis from the lungs of aerosol-infected mice86. Furthermore, T3SS activity seems to be lost following laboratory passage of B. pertussis and regained after passage in mice86,87.
Tracheal cytotoxin. Tracheal cytotoxin (TCT) is a disaccharide–tetrapeptide monomer of peptidoglycan that is produced during cell wall remodelling88. Although most Gram-negative bacteria recycle this molecule89,90, B. pertussis does so inefficiently and releases a large amount of TCT into the extracellular environment. TCT is the only known B. pertussis virulence factor that is not regulated by BvgAS. In hamster tracheal rings, TCT functions synergistically with lipo-oligosaccharide to stimulate the production of pro-inflammatory cytokines (such as tumour necrosis factor α (TNFα), IL-1α, IL-1β and IL-6) and inducible nitric oxide synthase (iNOS), resulting in the destruction and extrusion of ciliated cells from the epithelial surface91,92. The biological activity of TCT depends on NOD1 (nucleotide-binding oligomerization domain-containing protein 1), which is a cytosolic pattern recognition receptor that senses bacterial peptidoglycan and induces the production of pro-inflammatory mediators93. NOD1-dependent detection of TCT seems to be host specific, as human NOD1 poorly detects TCT, whereas mouse NOD1 detects TCT efficiently93. Although it has been postulated that TCT-mediated cytopathology contributes to the characteristic cough in pertussis, the lack of appropriate animal models has prevented this hypothesis from being tested. Thus, the contribution of TCT to pertussis pathogenesis in humans remains unclear.
Dermonecrotic toxin. Subcutaneous injection of B. pertussis or B. bronchiseptica cells into mice results in the formation of necrotic lesions, owing to the activity of dermonecrotic toxin (DNT)94. Consistent with a role in infection, the production of DNT is positively regulated by BvgAS26,29 and there is evidence that DNT contributes to the ability of B. bronchiseptica to induce turbinate atrophy and lung pathology in swine95. DNT has transglutaminase activity, can activate Rho GTPases96,97 and inhibits osteogenic cell differentiation in vitro, which suggests that the toxin acts directly on host cells98,99. However, as DNT lacks a signal sequence for export and is not secreted from bacterial cells grown in culture94,100, it may actually function in the bacterial cytoplasm during infection — possibly by facilitating bacterial survival within a specific host niche and hence indirectly functioning in pathogenesis.
Filamentous haemagglutinin. Filamentous haemagglutinin (FHA) (Fig. 3a) is a large rod-shaped protein and, together with FhaC (filamentous haemagglutinin transporter protein), it functions as a prototypical member of the two-partner secretion pathway (TPS pathway)101. It is initially synthesized as a ∼370 kDa pre-pro-protein (FhaB) that undergoes processing to produce the mature ∼250 kDa FHA as it is translocated across the cytoplasmic membrane by the Sec translocation system and across the outer membrane by FhaC102. The N-terminal signal peptide is probably removed by leader peptidase and the C-terminal prodomain is processed by SphB1 (autotransporter subtilisin-like protease) and other, as yet unidentified, factors103,104. Mature FHA is oriented with its mature C terminus (the MCD) distal to the bacterial surface, and a substantial amount of FHA is also released into culture supernatants when the bacteria are grown in vitro104.
FHA is both necessary and sufficient to mediate bacterial adherence to several eukaryotic cell types in vitro105,106. However, FHA is only one of several factors that contribute to the adherence of bacteria to tracheal explants9,107, which suggests that additional adhesins are important for adherence in vivo. Studies using cultured, non-ciliated cells have reported that FHA binds to CR3, very late antigen V (VLA-5) and leukocyte response integrin–integrin-associated protein (LRI–IAP) complexes, and an RGD (Arg-Gly-Asp) motif that is located in the centre of the FHA molecule is implicated in this process108,109,110. More recent studies that have examined B. bronchiseptica infection using animal and cell culture models have shown that the FHA molecules that are produced by B. pertussis and B. bronchiseptica are functionally interchangeable. These studies have also shown that the production of an FHA protein that contains an RAE (Arg-Ala-Asp) motif instead of an RGD motif results in no observable differences and that the MCD is required for function111. Whether FHA interacts with CR3, VLA-5, LRI–IAP or other mammalian receptors during infection has yet to be determined.
Experiments in which B. bronchiseptica is delivered in a small volume to the nasal cavities of rats and pigs have shown that FHA is essential for progression of the infection from the upper to the lower respiratory tract111,112. In mouse models, in which large numbers of bacteria are directly delivered into the lungs, FHA-deficient B. bronchiseptica strains induce a more robust inflammatory response than wild-type bacteria73,111. This response is characterized by increased production of pro-inflammatory cytokines and chemokines (such as TNFα, keratinocyte-derived chemokine (KC), CC-motif ligand 2 (CCL2; also known as MCP-1) and IL-17) in lung tissue and increased recruitment of neutrophils to the lungs during the first 4 days post-inoculation73. Animals that do not succumb to inflammation-mediated pulmonary damage clear the FHA-deficient bacteria from their lungs much faster than animals that are inoculated with wild-type bacteria73,111. These data suggest that FHA enables B. bronchiseptica to modulate inflammation during the establishment of infection, thereby facilitating bacterial persistence. It is currently not known whether FHA exerts these effects by binding directly to host receptors while it is attached to the bacterial cell surface or whether these effects occur after FHA release from the bacterial cell. Furthermore, it has been suggested that FHA functions as a scaffold to direct the delivery of other virulence factors (such as ACT113); however, the in vivo relevance of this activity has not been determined.
Fimbriae. Bordetella spp. produce type I pili, which are also known as fimbriae (Fig. 3b). The putative chaperone (FimB), usher (FimC) and tip adhesin (FimD) are encoded by the fimBCD operon, which is located between the fhaB and fhaC genes114. The fim2 and fim3 genes, which encode the two major fimbrial subunits, are located elsewhere on the chromosome and can undergo phase variation115. Alternative major fimbrial subunit genes (fimA, fimN and fimX) have also been identified116,117,118. Although in vitro adherence assays using cultured cells have yielded variable results119,120, studies with tracheal explants indicate a role for fimbriae in mediating adherence to ciliated respiratory epithelium9,107. Studies with both B. pertussis and B. bronchiseptica have shown a requirement for fimbriae during colonization of the lower respiratory tract in rodents120,121, and mice that have been inoculated with Fim-deficient B. pertussis show a more robust inflammatory response than mice that have been inoculated with wild-type bacteria122. Similarly to FHA, fimbriae seem to be involved in adherence and/or suppression of the initial inflammatory response to infection, potentially contributing to persistence.
Pertactin. Pertactin (PRN) is a member of the classical autotransporter family of outer membrane proteins123 (Fig. 3c). The surface-localized 'passenger' domain forms a β-helix in which β-strands are connected by short turns or, in a few cases, large extrahelical loops124. Similarly to fimbriae, studies using non-ciliated mammalian cells to investigate a role for PRN in adherence or invasion have yielded equivocal results112,125. Studies using ciliated rabbit tracheal explant cultures suggest that PRN contributes to the adherence of B. pertussis to ciliated respiratory epithelium9, although experiments in mice failed to identify a role for PRN in vivo126. However, in the case of B. bronchiseptica, studies indicate that PRN is involved in resistance to neutrophil-mediated clearance and promoting persistence in the lower respiratory tract112,125. In recent years, B. pertussis strains that do not produce PRN have been isolated from patients127, raising the concern that such strains have been selected owing to the presence of PRN-specific antibodies, which are generated in response to immunization with PRN-containing aP vaccines. Whether vaccine-driven evolution of B. pertussis strains is actually occurring is currently under investigation, as it has decisive implications for the development of new and improved vaccines.
Lipopolysaccharide. B. pertussis, B. parapertussisHu and B. bronchiseptica produce different forms of lipopolysaccharide (LPS). B. pertussis produces a penta-acylated lipid A that is linked to a complex core trisaccharide, B. bronchiseptica produces a hexa-acylated lipid A that is linked to a similar, if not identical, complex core trisaccharide and O-antigen repeats, and B. parapertussisHu produces a hexa-acylated lipid A that is linked to an altered core structure and O-antigen repeats128,129,130. Because it lacks O-antigen, B. pertussis LPS is often referred to as lipooligosaccharide (LOS)131. The genes that are required for the synthesis of O-antigen in B. bronchiseptica and B. parapertussisHu are repressed by BvgAS132; however, some O-antigen is produced under Bvg+ phase conditions, and mutants that are unable to produce O-antigen show defective virulence in mouse models132,133.
In mice, B. bronchiseptica LPS is sensed by Toll-like receptor 4 (TLR4), resulting in an early TNFα response and recruitment of neutrophils to the lungs134,135. Although B. parapertussisHu LPS and B. pertussis LOS can stimulate mouse TLR4, they do so less efficiently, and Tlr4−/− mice are only modestly impaired in their ability to control infection by these organisms136,137,138. In addition, it has been reported that B. pertussis LOS-mediated stimulation of mouse dendritic cells results in the development of anti-inflammatory regulatory T cells136. On the basis of these observations, it has been suggested that B. pertussis and B. parapertussisHu have evolved to be less inflammatory than B. bronchiseptica and that diminished inflammation might facilitate persistence during human infection136,137. However, subsequent studies have shown that human and mouse TLR4–MD-2–CD14 complexes differ in their ability to recognize different forms of lipid A. Although mouse TLR4–MD-2–CD14 responds similarly to both penta- and hexa-acylated lipid A, human TLR4–MD-2–CD14 responds robustly to hexa-acylated lipid A but only weakly to penta-acylated lipid A139. Furthermore, in contrast to mouse TLR4–MD-2–CD14, which responds to B. pertussis lipid A regardless of whether the phosphate groups are modified or not, human TLR4–MD-2–CD14 responds more robustly to lipid A that contains glucosamine (GlcN)-modified phosphate groups than to lipid A that contains unmodified phosphate groups140. Although it seems that most B. pertussis LOS contains GlcN-modified phosphate groups141, the fact that it is penta-acylated suggests that its ability to stimulate TLR4 in humans is even weaker than its ability to stimulate TLR4 in mice. These data provide additional support for the hypothesis that B. pertussis and B. parapertussisHu strains have evolved to be relatively non-inflammatory in humans. However, these data also raise concerns about extrapolating conclusions that have been drawn from mouse studies to humans, as the TLR4–MD-2–CD14-dependent immune responses clearly differ in these hosts.
Additional surface proteins. Many additional BvgAS-activated genes encode known or predicted surface-localized or secreted proteins, which are suspected to have roles in pathogenesis29,142. BrkA, TcfA, BapC, BatB, Vag8, SphB1 and Phg are BvgAS-activated classical autotransporter proteins, and their putative roles in pathogenesis include mediating adherence, serum resistance, the evasion of antibody-mediated clearance and the proteolytic processing of other surface proteins103,143,144,145,146,147,148. BipA and BcfA are BvgAS-regulated members of the intimin–invasin family and, although their roles in pathogenesis are unknown35,149, immunization of mice with BcfA can accelerate clearance of B. bronchiseptica following intranasal challenge150. These data suggest that these poorly characterized surface molecules should be considered for the development of new vaccines containing different or alternative antigens.
Many BvgAS-regulated genes encode proteins that are probably involved in metabolism, respiration and other physiological processes29,142, which presumably reflects the diversity of environmental conditions encountered by Bordetella spp. as they travel within and outside the mammalian respiratory tract. Among these factors, those that are involved in the acquisition and use of iron have been the focus of most studies. In addition to producing and using the siderophore alcaligin151, B. pertussis and B. bronchiseptica can use various xenosiderophores (including enterobactin152) and haem–iron sources, such as haemoglobin153. Most, if not all, of these iron-acquisition mechanisms are required during respiratory infection in mice154,155, which demonstrates the necessity of iron for bacterial survival, the variety of mechanisms that are used by the host to sequester iron and the reciprocal array of mechanisms that are used by the bacterium to acquire this essential element.
In addition, accumulating evidence suggests that biofilm production by pathogenic Bordetella spp. in vitro and during infection may contribute to colonization of the respiratory tract. Biofilm formation is regulated by a complex programme of both Bvg-dependent and Bvg-independent gene expression156,157,158,159; genes that promote this process are maximally expressed in the Bvgi phase156. Bvg-independent production of an exopolysaccharide via expression of the bps locus and the presence of extracellular DNA are also required for biofilm formation157,160,161. Recent evidence suggests that the second messenger cyclic-di-GMP is also crucial for the regulation of biofilm formation162.
Current and future challenges
Despite high rates of immunization with aP vaccines, epidemics of pertussis have recently occurred in the United States, Europe, Australia and Japan (US Centers for Disease Control and Prevention (CDC), Australian Government Department of Health and Ageing and Japanese National Institute of Infectious Diseases; see further information)163,164 and similar outbreaks seem to be imminent in developed countries throughout the world. Moreover, irrespective of socioeconomic status, the highest rates of mortality are in infants, who are also the most difficult population to treat and protect. In considering these challenges and looking ahead, we suggest three priorities for future studies.
The first priority is to improve the robustness and duration of protection that is conferred by vaccination, which will require further study of the immunological responses to infection and vaccination (Box 4). The deficiencies of current aP vaccines are well documented, including the striking observation that aP vaccination of baboons protects against disease symptoms but not against colonization or transmission165. Many efforts are in progress to overcome these deficiencies24, such as the inclusion of additional antigens in aP vaccines, reformulation with adjuvants that favour T helper 1 (TH1) cell and TH17 cell responses, as opposed to the TH2 cell-type immunity that is generated by alum-adjuvanted vaccines, as well as the development of live attenuated B. pertussis vaccines166. The development of live attenuated vaccines has considerable advantages, including the ability to generate mucosal immunity, but the issue of public acceptance looms large. Similarly, it is interesting to note that outside North America, Europe and parts of Asia, wP vaccines remain in widespread use, and approaches to decrease their reactogenicity while retaining their immunogenicity should be considered167. The known efficacy of wP vaccines, combined with the cost-effectiveness of this approach, might be of more benefit to people than the development of improved but more costly vaccines that are composed of purified proteins. It is important to remember that the development and approval of novel vaccines will be a prolonged process. In light of recent findings concerning the lack of protection against colonization or transmission by aP vaccination165, maximizing the efficacy of current vaccines by prenatal vaccination, additional boosting and alternative strategies is imperative.
A second priority is to mitigate infant mortality. Nearly 90% of all deaths from pertussis occur in infants who are less than 4 months old168, and the most frequent cause is intractable pulmonary hypertension associated with marked lymphocytosis and bronchopneumonia. Currently, the only efficacious therapy for severe cases is rapid leukodepletion, which is only available at advanced critical care centres169,170. Respiratory samples that have been obtained during autopsies show luminal aggregates of leukocytes occluding small pulmonary arteries, along with an abundance of B. pertussis10,171. The pathology of fatal pertussis pneumonia seems to be mostly caused by PT. Thus, in addition to protecting susceptible infants by maternal vaccination or by vaccination at birth, it is also imperative to pursue approaches for limiting PT activity during infection. Potential therapeutic modalities include the development of humanized monoclonal antibodies and small molecules that inhibit PT interactions with host cell receptors or the enzymatic activity of PT, as well as therapeutic compounds designed to target regulatory factors such as the BvgAS system.
Finally, although animal models have proven to be useful, we need to improve our understanding of human disease. Decades of research on B. pertussis virulence determinants have primarily been based on tissue culture models and mouse infections. These studies have shown what adhesins, toxins and other virulence factors can do under laboratory conditions, but very little, if anything, is known about what they really do during human disease. Specificity is the rule for human-adapted pathogens and it can manifest at several levels, including gene expression, virulence-factor delivery, binding-specificity and activity. Perhaps the most vivid illustration of our lack of understanding of B. pertussis is that we still don't know why infection makes people cough!
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The authors thank N. Carbonetti, P. Sebo and R. Fernandez for their comments on the manuscript and their help preparing figures. The authors apologize to colleagues whose work could not be cited owing to space limitations. This work was supported by the US National Institute of Allergy and Infectious Disease (NIAID) of the US National Institutes of Health (NIH) grant numbers T32 AI007151 (J.A.M.) and R01 AI094991 (P.A.C.).
The authors declare no competing financial interests.
- Bordetella parapertussis Hu
Two clades of bacteria have been classified as B. parapertussis. Those isolated from humans are designated B. parapertussisHu and those isolated from sheep are designated B. parapertussisOv.
An increase in the number of lymphocytes (that is, T cells, B cells and NK cells) in the bloodstream.
- Insertion sequence elements
Short, transposable DNA sequences.
- Multilocus sequence typing
A molecular method that is used to characterize microorganisms by comparing the sequences of housekeeping genes.
- Paroxysmal coughing
Multiple, rapid coughing attacks that are characteristic of pertussis.
- Type IV secretion system
A protein complex that is homologous to the conjugative machinery that spans the cell envelope. Type IV secretion systems (T4SSs) are capable of both secretion and uptake of DNA and proteins.
The bisphosphonate drug clodronate is a chemical that, when delivered to macrophages via phagocytosis of clodronate-containing liposomes, causes apoptotic cell death.
- Type I secretion system
A protein complex that spans the inner and outer membranes of Gram-negative bacteria and that consists of an ATP-binding cassette (ABC)transporter protein, a membrane fusion protein and an outer membrane protein; it is responsible for the secretion of various molecules.
- Oxidative burst
(Also known as respiratory burst). The rapid production of reactive oxygen species, such as hydrogen peroxide and superoxide radicals (typically by neutrophils and monocytes), which assist in combating bacterial and fungal infections.
- Alternative sigma factor
A protein factor that associates with RNA polymerase and is important for promoter recognition and the initiation of transcription. They are typically induced under different environmental conditions. Alternative sigma factors differ from primary sigma factors, which are required for the transcription of essential genes.
- Pattern recognition receptor
A protein of the innate immune system that recognizes pathogen-associated molecular patterns (PAMPs); for example, the Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide.
- Rho GTPases
A family of small signalling GTPases within the Ras superfamily. Rho GTPases are found in eukaryotic cells and are involved in a broad range of cellular activities.
- Two-partner secretion pathway
(TPS pathway). A secretion pathway that consists of two proteins: a large exoprotein (TspA) and an outer membrane pore-forming β-barrel protein (TspB).
- Sec translocation system
A protein complex that transports molecules across the cytoplasmic membrane.
- Phase variation
A mechanism by which bacteria vary the expression of genes that encode extracellular structures, such as flagella and fimbriae, typically as a means to avoid host immunity. This is frequently accomplished by DNA rearrangement or slip-strand mispairing.
- Autotransporter family
A family of proteins that are found in Gram-negative bacteria and that consist of an amino-terminal passenger domain and a carboxy-terminal β-barrel. The β-barrel is responsible for transporting the passenger domain into the extracellular space and anchoring the protein to the outer membrane.
- Intimin–invasin family
A family of virulence factors that mediate adherence and invasion.
An iron-chelating compound that is secreted by microorganisms to scavenge iron for cellular metabolism. Xenosiderophores are siderophores that can be utilized across different species.
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Melvin, J., Scheller, E., Miller, J. et al. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol 12, 274–288 (2014). https://doi.org/10.1038/nrmicro3235
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