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Department of Microbiology, Immunology, and Molecular Genetics,1 Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, California2
SUMMARY INTRODUCTION HISTORY PHYLOGENETIC RELATIONSHIPS BETWEEN BORDETELLA SUBSPECIES VIRULENCE DETERMINANTS AND MOLECULAR PATHOGENESIS Animal Models Bordetella Virulence Regulon Commonly Expressed Loci Differentially Expressed and Differentially Regulated Loci Virulence Determinants Filamentous hemagglutinin. Agglutinogens. Fimbriae. Pertactin and other autotransporters. Adenylate cyclase. Dermonecrotic toxin. Lipopolysaccharides. Type III secretion system. Tracheal cytotoxin. Pertussis toxin. PATHOLOGY B. pertussis Infection B. bronchiseptica Infection Dogs. Swine. Laboratory animals. (i) Guinea pigs. (ii) Rabbits. PATHOGENESIS AND IMMUNITY CLINICAL MANIFESTATIONS B. pertussis Classic illness. Mild illness and asymptomatic infection. Infants. Adults. B. parapertussishu B. bronchiseptica Swine. Dogs. Laboratory animals. Humans. B. holmesii DIAGNOSIS Differential Diagnosis of Bordetella Infections Infections in humans. Infection in animals. Specific Diagnosis of B. pertussis Infections Culture of B. pertussis. (i) Specimen collection. (ii) Specimen transport. (iii) Culture. (iv) DFA testing of nasopharyngeal secretions. (v) Detection of B. pertussis by PCR. (vi) Serologic diagnosis of B. pertussis infection. Specific Diagnosis of Other Bordetella Infections TREATMENT VACCINATION AND PREVENTION B. pertussis Vaccines Whole-cell DTP vaccines. (i) Reactogenicity. (ii) Vaccine efficacy. Acellular pertussis component DTP vaccines. (i) Reactogenicity. (ii) Vaccine efficacy. B. bronchiseptica Vaccines Dogs (kennel cough). Swine (atrophic rhinitis). EPIDEMIOLOGY: IMPLICATIONS FOR THE CONTROL OF HUMAN INFECTIONS B. pertussis Epidemiology Observed (reported pertussis). (i) Incidence. (ii) Mortality. (iii) Sex and race. (iv) Season and geographic areas. B. pertussis infection. (i) Percentage of cough illnesses due to B. pertussis in adolescents and adults. (ii) Rate of B. pertussis infections. (iii) Rate of cough illnesses due to B. pertussis infections. B. parapertussishu Epidemiology Incidence and mortality. Sex, race, season, and geographic areas. Interrelationship between B. parapertussishu and B. pertussis infections. B. bronchiseptica Epidemiology B. holmesii Epidemiology LONG-TERM GOALS OF PERTUSSIS PREVENTION Eradication of B. pertussis Circulation Lesser Goals Infant and Childhood DTaP Immunization Schedules FUTURE RESEARCH Pathogenesis of Disease Better Vaccines Role of B. holmesii in Human Disease More Complete Epidemiologic Study ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Although pertussis is relatively well controlled at present by extensive vaccination programs, it is evident that the circulation of B. pertussis throughout the world continues largely unabated (149). Whooping cough is still common in areas of the world where vaccine use is low. Recent studies suggest that there are presently 
48.5 million yearly cases of pertussis worldwide, with as many as 295,000 deaths (187). One effect of vaccination has been a shift in the incidence of reported pertussis from children aged 1-9 years in unvaccinated populations to infants, adolescents, and adults in vaccinated populations (149). Reasons for this shift include incomplete immunity in infants who have received fewer than three doses of vaccine, the relatively short-lived immunity that results from vaccination, and the recent greater awareness of pertussis in adolescents and adults. Although adolescent and adult pertussis is significant in terms of medical costs and lost work, the most worrisome consequence is epidemiological (149, 645). Numerous studies have shown that adults and adolescents provide a reservoir of B. pertussis and are the major source of transmission to partially immunized infants and children (35, 56, 135, 149, 186, 583).
During the last 20 years, many good reviews have been written relating to the microbiology of Bordetella species and the clinical and epidemiologic aspects of pertussis (136, 147, 149, 174, 418, 504, 802). The purpose of the present review is to consolidate data from the previous literature and new information, as well as to correlate clinical events with the latest molecular evidence.
| HISTORY |
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Bordet and Gengou reported the isolation of B. pertussis in 1906, although they had observed the organism microscopically in the sputum of a patient with pertussis in 1900 (63, 356). Since pertussis was such a severe disease in infants, vaccine development began soon after the growth of the organism in the laboratory (136, 141, 147). Initially, experimental vaccines were used to treat and prevent pertussis. Epidemic pertussis was brought under control in the United States with the widespread use of whole-cell pertussis vaccines in the 1940s and 1950s. Control of the disease has continued in the United States over the last decade with the use of acellular pertussis component DTP vaccines (diphtheria-tetanus toxoids, acellular pertussis vaccine, adsorbed vaccines) (referred to as DTaP vaccines) (149).
B. bronchiseptica was first isolated during the first decade of the 20th century by Ferry, McGowan and perhaps others in studies of dogs suffering from distemper (240, 242, 243, 283, 511, 658, 761, 831). Further studies in the early 20th century demonstrated B. bronchiseptica infections in many animals and also humans (79, 241, 283, 511, 658, 689, 761, 831).
B. parapertussis was first isolated from children with pertussis in the 1930s by Eldering and Kendrick (220, 221) and Bradford and Slavin (73). Pertussis associated with B. parapertussis infections was in general somewhat less severe than that due to B. pertussis and was not associated with lymphocytosis, a hallmark of B. pertussis infection in children.
B. holmesii was presented as a new gram-negative species associated with septicemia in 1995 (814). This organism was first isolated in 1983 but was not associated with respiratory illness until 1998. During the period from 1995 through 1998, B. holmesii was recovered from nasopharyngeal specimens of 33 patients in Massachusetts with pertussis-like symptoms (508, 839).
| PHYLOGENETIC RELATIONSHIPS BETWEEN BORDETELLA SUBSPECIES |
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B. parapertussishu strains are particularly interesting. They comprise a single electrophoretic type and, based on PCR-based RAPD fingerprinting and IS element analyses, are nearly identical regardless of their geographic origin or year of isolation (783, 842). A plausible hypothesis is that B. parapertussishu evolved relatively recently from a closely related B. bronchiseptica strain (Fig. 1). Given its long-standing position as a host-restricted human pathogen, the isolation of strains identified as B. parapertussis from asymptomatic and pneumonic sheep came as a considerable surprise and prompted speculation that cross-species transmission may occur. Subsequent studies, however, clearly demonstrated that human and ovine strains of B. parapertussis represent distinct clonal lineages that diverged independently from B. bronchiseptica (782). B. parapertussisov isolates are genetically diverse, and there appears to be little or no transmission between the sheep and human reservoirs.
Investigators at the Sanger Center recently sequenced the genomes of three Bordetella subspecies (B. pertussis strain Tohama 1, B. parapertussishu strain 12822, and B. bronchiseptica strain RB50) (608). The genome of RB50 is 5.34 Mb, while those of Tohama 1 and 12822 are 4.09 and 4.77 Mb, respectively. The differences in genome sizes and sequence comparison of the three genomes support the hypothesis that B. pertussis and B. parapertussis recently and independently evolved from B. bronchiseptica-like ancestors. Interestingly, this restriction to the human host included significant loss of DNA, perhaps corresponding to a more "streamlined" genome. In comparison with Tohama 1 and 12822, a large portion of the extra DNA in RB50 is attributed to prophage and prophage remnants (608). Other genes lost by B. pertussis and B. parapertussis include loci involved in small-molecule metabolism, membrane transport, and biosynthesis of surface structures. In addition to this substantial gene deletion, B. pertussis and B. parapertussis contain 358 and 200 pseudogenes, respectively, many of which have been inactivated by insertion of IS elements, in-frame stop codons, or frameshift mutations. Interestingly, very few genes known or suspected to be involved in pathogenicity are missing in the genomes of human-adapted bordetellae. It is interesting that while Bordetella subspecies have been studied extensively for years, full functional data are available for only a small portion of the Bordetella genomes. For instance, genome sequence analysis predicts that at least 30 genes are involved in biosynthesis of lipopolysaccharides (LPS) for B. bronchiseptica, but functional data are available for only 13 of these genes. A detailed list of the functional annotation for predicted proteins from the sequenced Bordetella strains is presented in Table 1.
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| VIRULENCE DETERMINANTS AND MOLECULAR PATHOGENESIS |
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10 mM nicotinic acid or 40 mM MgSO4 (527). Under these Bvg– phase conditions, BvgAS is unable to activate the transcription of vag genes and repression of vrg genes. The Bvg– phase corresponds to Lacey's C mode (Fig. 2A). The BvgS receiver is a pivotal component of the phosphorelay, acting as a biochemical checkpoint by mediating phosphorylation and dephosphorylation of the HPD and BvgA, as well as dephosphorylation of the transmitter. Mutational analyses of bvgAS have provided a number of tools for deciphering the structure of the virulence regulon and for investigating the role of Bvg-mediated signal transduction in vivo. Mutations that alter as well as those that completely abrogate signal transduction have been identified. The bvgS-C3 allele locks BvgS into an active form, rendering it insensitive to modulating signal (175, 544). Strains containing this mutation constitutively express all known Bvg-activated virulence factors. A deletion in bvgS, on the other hand, locks the bacteria in the Bvg– phase and renders them avirulent (544). The Bvg– phase of B. bronchiseptica is characterized by expression of motility and several metabolic processes involved in redox reactions and amino acid transport (8, 230, 268, 517). In contrast, B. pertussis and B. parapertussishu are nonmotile due to multiple frameshifted and transposon-disrupted genes in their flagellar loci (608). The Bvg– phase of B. pertussis is characterized by the expression of several outer membrane proteins of unknown function (287). Experiments with phase-locked and ectopic expression mutants have demonstrated that the Bvg+ phase is necessary and sufficient for respiratory tract colonization by B. pertussis and B. bronchiseptica (175, 496). These experiments also demonstrated that the Bvg– phase of B. bronchiseptica was necessary and sufficient for survival under nutrient-limiting conditions, suggesting the existence of an environmental reservoir (175). An environmental reservoir for B. pertussis and B. parapertussishu seems less plausible, as these strains are more fastidious and appear to be confined to transmission by the respiratory droplet route. A role for the Bvg– phase of these human-adapted bordetellae remains to be identified.
So, why is this BvgAS phosphorelay so complex? One possibility is that multiple steps allow multiple levels of control. The complexity of the system may also reflect the ability to respond to signal intensity in a graded manner. Indeed, it was recently demonstrated that instead of controlling a biphasic transition between the Bvg+ and Bvg– states, BvgAS controls expression of a spectrum of phenotypic phases in response to quantitative differences in environmental cues (176, 203, 204, 732). Wild-type bordetellae grown in the presence of submodulating conditions, such as concentrations of 0.4 to 2 mM nicotinic acid for B. bronchiseptica, express a phenotypic phase distinct from those described above. This phase is characterized by the absence of Bvg-repressed phenotypes, the presence of a subset of Bvg-activated virulence factors and the expression of several polypeptides that are expressed maximally or exclusively in this phase. Bordetellae growing in this phase display phenotypes intermediate between the Bvg+ and Bvg– phases; as such, this phase has been designated the Bvg-intermediate (Bvgi) phase and corresponds to Lacey's I mode (Fig. 2A) (176). A single nucleotide change in bvgS at position 733 resulting in a Thr-to-Met substitution mimics a Bvgi-phase phenotype. Bvgi phase bordetellae containing this mutation (designated bvgS-II) display increased resistance to nutrient limitation and a decreased ability to colonize the respiratory tract compared to wild-type Bvg+-phase bacteria (176). The Bvgi phase appears to be conserved between B. pertussis and B. bronchiseptica, and is predicted to play a role in the respiratory transmission of these strains (257). Recently, the Bvgi phase of B. bronchiseptica was shown to be associated with biofilm formation (383). Biofilms are bacterial communities that are attached to a solid surface and have characteristics different from free-living planktonic bacteria (173). Bacteria growing within biofilms appear to be more resistant to antibiotics and host immune defenses than are their planktonic counterparts (457). While the physiological relevance of Bvg-dependent biofilm formation in B. bronchiseptica remains to be determined, studying biofilm formation has potential implications in understanding the life-style of B. bronchiseptica (versus B. pertussis) as a chronically colonizing pathogen. Systematic analysis of gene expression in the Bvg+, Bvgi, and Bvg– phases of Bordetella reveals the existence of at least four classes of Bvg-regulated genes: (i) those that are expressed maximally only in the Bvg+ phase, (ii) those that are expressed maximally in both the Bvg+ and Bvgi phases, (iii) those that are expressed exclusively in the Bvgi phase, and (iv) those that are expressed only in the Bvg– phase (Fig. 2B). From a phylogenetic perspective, however, Bvg-regulated genes fall into two categories. Some loci are commonly expressed by B. pertussis, B. parapertussis (human and ovine), and B. bronchiseptica. Their products are highly similar and in some cases interchangeable between different subspecies. In contrast, other loci which are present in the genomes of all four subspecies appear to be differentially expressed. These genes provide important clues for understanding fundamental differences between Bordetella-host interactions.
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Although differences in Bvg+-phase phenotypes expressed by Bordetella subspecies are apparent, they exist in a background of overall similarity. In contrast, the Bvg– phases of these organisms are remarkably different. To date, there are few loci that are coexpressed in the Bvg– phases of all four subspecies (174). An interesting example involves the motility phenotype of B. bronchiseptica. Although B. pertussis and B. parapertussis contain the entire complement of motility genes, they are not expressed and these subspecies are therefore nonmotile (7, 8). In a similar vein, the B. pertussis vrg loci encode several outer membrane proteins that are specifically expressed in the Bvg– phase. The vrg genes in the B. bronchiseptica genome appear to be transcriptionally silent.
Understanding the role of Bvg-mediated signal transduction in the Bordetella life cycle is crucial in determining the pathogenic mechanisms and evolutionary trends involved in Bordetella-host interactions. It provides insightful details into the dynamics of virulence gene regulation and its implications for host adaptations.
Filamentous hemagglutinin. The virulence determinants of B. pertussis and B. bronchiseptica are discussed in Table 2. FHA is a highly immunogenic, hairpin-shaped molecule which serves as the dominant attachment factor for Bordetella in animal model systems (174, 655). It has been included as a component in most acellular pertussis vaccines (149). Protein structure and immunological analyses suggest that the FHA proteins from B. pertussis and B. bronchiseptica are similar in their molecular mass, structure dimensions, and hemagglutination properties and have a common set of immunogenic epitopes (529, 594, 683).
FHA is encoded by fhaB, one of the strongest BvgAS-activated genes. It is maximally expressed under both Bvg+- and Bvgi-phase conditions. The fhaB promoter contains a primary high-affinity BvgA-binding site consisting of two nearly perfect inverted heptanucleotide repeats [TTTC(C/G)TA] that are centered at position –88.5 relative to the start of transcription (677). Binding of a phosphorylated BvgA dimer to this site, followed by cooperative binding of two additional BvgP dimers 3' to the high-affinity site, leads to the activation of fhaB transcription. Binding of the first BvgAP dimer to the primary high-affinity binding site seems to be the critical first step for fhaB transcription, since binding of the second and third dimers was found to be entirely cooperative and independent of nucleotide sequence (69, 71).
FHA is synthesized as a 367-kDa precursor, FhaB, which undergoes extensive N- and C-terminal modifications to form the mature 220-kDa FHA protein. It is exported across the cytoplasmic membrane by a Sec signal peptide-dependent pathway. Its translocation and secretion across the outer membrane requires a specific accessory protein, FhaC. FhaC folds into a transmembrane ß-barrel that facilitates secretion by serving as an FHA-specific pore in the outer membrane (304, 392). FHA most probably crosses the outer membrane in an extended conformation and acquires its tertiary structure at the cell surface, following extensive N- and C-terminal proteolytic modifications which have recently been characterized in a series of elegant experiments (180, 181, 303, 304, 391-393). On translocation across the cytoplasmic membrane, the N terminus of FhaB undergoes cleavage of an additional 8 to 9 kDa at a site that corresponds to a Lep signal peptidase recognition sequence. This portion of the N terminus is predicted to be important for interacting with FhaC. Once at the cell surface, approximately 130 kDa of the C terminus of FhaB is proteolytically removed by a newly identified subtilisin-like autotransporter/protease, SphB1 (180, 181). FHA release depends on SphB1-mediated maturation. The C terminus of the FhaB precursor is predicted to serve as an intramolecular chaperone, preventing premature folding of the protein. Together, FHA and FhaC serve as prototypes for members of the two-partner secretion (TPS) system, which typically include secreted proteins with their cognate accessory proteins from several gram-negative bacteria. Although efficiently secreted via this process, a significant amount of FHA remains associated with the cell surface by an unknown mechanism.
In vitro studies using a variety of mammalian cell types suggest that FHA contains at least four separate binding domains that are involved in attachment. The Arg-Gly-Asp (RGD) triplet, situated in the middle of FHA and localized to one end of the proposed hairpin structure, stimulates adherence to monocytes/macrophages and possibly other leukocytes via the leukocyte response integrin/integrin-associated protein (LRI/IAP) complex and complement receptor type 3 (CR3) (384, 654, 690). Specifically, the RGD motif of FHA has been implicated in binding to very late antigen 5 (VLA-5; an 
5ß1-integrin) of bronchial epithelial cells (387). Ligation of VLA-5 by the FHA RGD domain induces activation of NF-
B, which then causes the up-regulation of epithelial intercellular adhesion molecule 1 (ICAM-1) (385, 386). ICAM-1 up-regulation is involved in leukocyte accumulation and activation at the site of bacterial infection (59, 593, 762). Interestingly, purified PT can abrogate NF-B activation by this mechanism, suggesting the involvement of a PT-sensitive G protein in the signaling process (the role of PT is discussed in detail later in this review) (386). The CR3 recognition domain in FHA has yet to be identified. FHA also possesses a carbohydrate recognition domain (CRD), which mediates attachment to ciliated respiratory epithelial cells as well as to macrophages in vitro (636). In addition, FHA displays a lectin-like activity for heparin and other sulfated carbohydrates, which can mediate adherence to nonciliated epithelial cell lines. This heparin-binding site is distinct from the CRD and RGD sites and is required for FHA-mediated hemagglutination (530). FHA is also required for biofilm formation in B. bronchiseptica (383).
A role for FHA in vivo has been more difficult to discern mainly due to the lack of a natural animal host (other than humans) for B. pertussis, as well as the complexity of this molecule and its associated biological activities. In a rabbit model of respiratory tract infection, fewer FHA mutants compared to wild-type B. pertussis were recovered from the lungs at 24 hs after intratracheal inoculation (690). A comparison of in vivo results with in vitro binding characteristics of the various mutant strains used in the above study suggested that wild-type B. bronchiseptica was capable of adhering to both ciliated epithelial cells and macrophages. Further, competition experiments with lactose and anti-CR3 antibody suggested that both CRD- and RGD-dependent binding was involved (690). Using mouse models, however, others have found FHA mutants to be indistinguishable from wild-type B. pertussis in their ability to persist in the lungs but defective for tracheal colonization (421, 557). Still others, also using mouse models, have observed no difference between FHA mutants and wild-type B. pertussis (284, 419, 663, 810).
Construction and analysis of two types of FHA mutant derivatives of B. bronchiseptica, one containing an in-frame deletion in the structural gene fhaB and one in which FHA is expressed ectopically in the Bvg– phase, in the absence of the array of Bvg+-phase virulence factors with which it is normally expressed, proved invaluable in determining a role for FHA (7, 177). Comparison of these mutants with wild-type B. bronchiseptica showed that FHA is both necessary and sufficient to mediate adherence to rat lung epithelial cells in vitro. Using a rat model of respiratory infection, FHA was shown to be absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals (177). FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance activity of the mucociliary escalator (177). While all the in vitro and in vivo studies so far demonstrate a predominant role for FHA as an adhesin, the release of copious amounts of FHA from the cell surface seems counterintuitive since adhesins typically remain associated with the bacterial surface to promote maximum attachment. The significance of FHA release during bacterial infection was investigated using a B. pertussis SphB1-deficient mutant in a mouse model of respiratory tract infection (179). SphB1 mutants are incapable of secreting FHA, and mature FHA remains surface associated in these strains. These mutants were found to be defective in their ability to multiply and persist in the lungs of mice, despite their increased adhesiveness in vitro. Since surface-associated FHA also causes autoagglutination of bordetellae, a secondary role for FHA may be to facilitate the dispersal of bacteria from microcolonies and their detachment from epithelial surfaces to promote bacterial spread.
B. pertussis inhibits T-cell proliferation to exogenous antigens in vitro in an FHA-dependent manner (67). Further, McGuirk and Mills have demonstrated that interaction of FHA with receptors on macrophages results in suppression of the proinflammatory cytokine, interleukin-12 (IL-12), via an IL-10 dependent mechanism (513, 515). These data reveal a role for FHA in facilitating persistence by curbing protective Th1 immune responses. In contrast, a subsequent study suggests that FHA can elicit proinflammatory and proapoptotic responses in human monocyte-like cells and bronchial epithelial cells (2). As mentioned earlier, binding of FHA to the VLA-5 integrin induces the expression of proinflammatory genes, such as ICAM-1, in an NF-B-dependent manner in human bronchial epithelial cells (386). FHA-specific antibodies are also necessary to protect against reinfection by B. bronchiseptica in the rat model (503). Specifically, animals were challenged with marked B. bronchiseptica strains 30 days after receiving a primary inoculation of wild-type or mutant B. bronchiseptica. The animals developed a robust anti-Bordetella serum antibody response by the 30-day time point, which was monitored both qualitatively and quantitatively by enzyme-linked immunosorbent assay (ELISA). The presence of anti-FHA serum titers was correlated with the ability of the animal to resist further infection with the marked B. bronchiseptica challenge strains. However, antibodies to FHA also inhibit the phagocytosis of B. pertussis by neutrophils (554). Taken together, these data suggest FHA performs several immunomodulatory functions in vivo.
Data regarding the role played by FHA in the pathogenesis of B. pertussis infections in humans can be gleaned from recent pertussis vaccine studies. Vaccinees who received FHA containing pertussis vaccines mounted a substantial antibody response to this protein (217, 218, 332). In general, acellular vaccines which contain FHA as well as PT toxoid have slightly greater efficacy than monocomponent PT toxoids (3, 131, 149, 734, 735, 737). However, one whole-cell component DTP vaccine in which vaccinees did not mount an antibody response to FHA was nevertheless highly efficacious (218, 332, 334, 719). Most importantly, in two studies in which serologic correlates of immunity were determined, it was found that FHA made no contribution to protection (148, 736).
Analysis of the B. pertussis, B. bronchispetica, and B. parapertussishu genomes revealed the existence of two additional genes, fhaS and fhaL, which encode FHA-like proteins (608). While orthologs of these genes are conserved among the Bordetella subspecies, differences exist in their internal sequences. Bordetella subspecies display differential binding to ciliated cells derived from different hosts, suggesting that host specificity may in part be dependent on the interaction of bacterial adhesins to their host receptors (770). Analysis of fhaS and fhaL gene products may be of interest in this regard. It may also explain the exact contribution of FHA in modulating the host immune response.
Agglutinogens. Agglutinogens (AGGs) are surface proteins that, with infection, elicit the production of antibodies that cause the agglutination of Bordetella organisms in vitro (10, 219, 489, 666, 667, 803). Early studies determined 14 antigenic types of AGGs, 6 of which were specific for B. pertussis (666). A serotyping scheme was developed from the results of the agglutination studies using antisera raised against Bordetella in rabbits following multiple injections of killed organisms. The antisera were made "monospecific" by adsorption with heterologous strains.
Of the six AGGs specific for B. pertussis, AGG1 was common to all strains while AGG2 to AGG6 were found in various combinations in different isolated strains (666). Three AGGs (AGG1, AGG2, and AGG3) have subsequently been determined to be the main agglutinating antigens, while AGG4, AGG5, and AGG6 have been classified as minor antigens that apparently associate with either AGG2 or AGG3. AGG2 and AGG3 have since been determined to be fimbrial in nature (fimbriae are discussed in detail later in this review).
The nature of AGG1 is not known (666). Since both PRN and LPS can function as AGGs, either could be AGG1 (75, 462, 551). It must be noted, however, that the original serotyping scheme was based on heat labile antigens, thereby discounting LPS as AGG1.
Studies done over 50 years ago found that protection from pertussis in exposed children correlated with high titers of serum agglutinating antibody (agglutinins) (546, 682). In the early 1960s it was noted that the apparent efficacy of British pertussis vaccines had decreased. Preston suggested that this decline in vaccine efficacy was because the vaccine used in England at the time did not contain AGG3 and the most prevalent circulating B. pertussis strains were serotype 1.3 (640, 643). Efficacy in England apparently increased following the addition of serotype 3-containing strains to the vaccine. This seemed to support Preston's opinion (641, 642, 644). However, the protective unitage of the British vaccine was also increased at the time, so that the increased efficacy may not have been due to the inclusion of serotype 3 strains in the vaccine (667).
Since pertussis seems to have been well controlled in Japan since 1981 and since none of the DTaP vaccines used in Japan contain AGG3, it seems that antibody to this antigen is of minor importance in protection against disease (422). However, in a small study of B. pertussis isolates in Japan it was found that six of seven collected between 1992 and 1996 were serotype AGG 3 (306).
Fimbriae. Like many gram-negative pathogenic bacteria, bordetellae express filamentous, polymeric protein cell surface structures called fimbriae (FIM). The major fimbrial subunits that form the two predominant Bordetella fimbrial serotypes, Fim2 and Fim3 (AGG2 and AGG3), are encoded by unlinked chromosomal loci fim2 and fim3, respectively (470, 558). A third unlinked locus, fimX, is expressed only at very low levels if at all (660), and recently a fourth fimbrial locus, fimN, was identified in B. bronchiseptica (401). B. bronchiseptica and B. parapertussis contain a fifth gene, fimA, located immediately upstream of the fimbrial biogenesis operon fimBCD and 3' of fhaB, which is expressed and capable of encoding a fimbrial subunit type, FimA (68). Genome sequence analysis of B. pertussis, B. parapertussishu and B. bronchiseptica reveals that all three subspecies contain fim2 and fim3, although the predicted C terminus of Fim2 is variable in B. pertussis (638). FimX is intact in B. pertussis and B. bronchiseptica but frameshifted in B. parapertussishu. While fimA is truncated, fimN is deleted in B. pertussis. Further, variations are seen in the FimN C termini of B. bronchiseptica and B. parapertussishu. There is also a novel putative fimbrial subunit upstream of fimN in B. bronchiseptica and B. parapertussishu that is missing in B. pertussis (638).
In addition to positive regulation by BvgAS, the fim genes are subject to fimbrial phase variation by slip-strand mispairing within a stretch of cytosine residues located between the –10 and –35 elements of the fim2, fim3, fimX, and fimN promoters (401, 817). The putative promoter region of fimA does not contain a "C stretch" and therefore is predicted not to undergo phase variation where expressed. Since slip-strand mispairing affects transcription of the individual fimbrial genes independently of each other, bacteria may express Fim2, Fim3, FimX, FimN, FimA, or any combination at any given time. However, all fimbrial serotypes have a common minor fimbrial subunit, FimD, which forms the tip adhesin (265). The fimD gene is located within the fimbrial biogenesis operon downstream of fimB and fimC (472, 819). Interestingly, this operon is positioned between fhaB and fhaC, genes required for synthesis and processing of FHA. Based on the predicted amino acid sequence similarity to the E. coli PapD and PapC proteins, FimB and FimC have been proposed to function as a chaperone and usher, respectively (471, 819). Mutation of any one of the genes in the fimBCD locus results in a complete lack of fimbriae on the bacterial cell surface, suggesting that fimBCD is the only functional fimbrial biogenesis locus on the Bordetella chromosome (818).
Attachment to host epithelia is often a crtical, early step in bacterial pathogenesis. Although fimbriae are implicated in this process, it has been difficult to establish a definitive role for Bordetella fimbriae as adhesins for several reasons. First, the multiple, unlinked major fimbrial subunit genes, as well as the transcriptional and translational coupling of the fimbrial biogenesis operon with the fha operon, have impeded the ability to construct strains completely devoid of fimbriae. Second, the presence of several other putative adhesins with potentially redundant functions has obscured the detection of clear phenotypes for Fim– mutants. Finally, since the interactions between bacterial adhesins and host receptor molecules are expected to be highly specific, the use of heterologous hosts for studies with B. pertussis has severely limited the ability to detect in vivo roles for putative adhesins. Nonetheless, several studies suggest that fimbriae may mediate the binding of Bordetella to the respiratory epithelium via the major fimbrial subunits and to monocytes via FimD (328, 329, 557). Geuijen et al. have shown that purified B. pertussis fimbriae, with or without FimD, were able to bind to heparan sulfate, chondroitin sulfate, and dextran sulfate, sugars that are ubiquitously present in the mammalian respiratory tract (266). Heparin-binding domains within the Fim2 subunit were identified and found to be similar to those of the eukaryotic extracellular matrix protein, fibronectin. Studies by Hazenbos et al. suggest that FimD mediates the binding of nonopsonized B. pertussis to VLA-5 on the surface of monocytes, which then causes activation of CR3, thereby enhancing its ability to bind FHA (328, 329). Fimbriae have also been suggested to play a minor role in biofilm formation (383).
In vivo studies have shown that Fim– B. pertussis strains are defective in their ability to multiply in the nasopharynx and trachea of mice (265, 557). By using a B. bronchiseptica strain devoid of fimbriae but unaltered in its expression of FHA and other putative adhesins, fimbriae have been shown to contribute to the efficiency of establishment of tracheal colonization and are absolutely required for persistence in the trachea using both rat and mouse models (506). Moreover, the serum antibody profiles of animals infected with Fim– bacteria differ qualitatively and quantitatively from those of animals infected with wild-type B. bronchiseptica (506). Specifically, fimbriae play an immunomodulatory role by (i) acting as T-independent antigens for an early immunoglobulin M IgM response and (ii) inducing a Th2-mediated component of the host immune response to Bordetella infection (503). Challenge experiments suggest that the presence of fimbriae is important for eliciting an immune response that is protective against superinfection (S. Mattoo et al., unpublished data). Fimbriae are also important for exerting an anti-inflammatory function and inhibiting killing by lung macrophages in mice (784).
Data from the two trials in which serologic correlates of immunity in children were determined also suggest that antibody to FIM contributes to protection (148, 736). In addition, when a vaccine containing PT, FHA, and PRN was compared to one which contained FIM 2/3 as well as PT, FHA, and PRN, the latter vaccine displayed significantly greater efficacy (597) (see the section on DTaP vaccine efficacy, below).
Taken together, all the above results suggest FIM-mediated interactions with epithelial cells and/or monocytes/macrophages may play important roles not only in adherence but also in the nature and magnitude of the host immune response to Bordetella infection.
Pertactin and other autotransporters. Bordetella strains express a number of related surface-associated proteins belonging to the autotransporter secretion system, that are positively regulated by BvgAS. The autotransporter family includes functionally diverse proteins, such as proteases, adhesins, toxins, invasins, and lipases, that appear to direct their own export to the outer membrane (344). Autotransporters typically consist of an N-terminal region called the passenger domain, which confers the effector functions, and a conserved C-terminal region called the ß-barrel, which is required for the secretion of the passenger proteins across the membrane. The N-terminal signal sequence facilitates translocation of the preproprotein across the inner membrane via the Sec pathway. On cleavage of the N-terminal signal in the periplasm, the C terminus folds into a ß-barrel in the outer membrane, forming an aqueous channel. The linker region between the N and C termini directs the translocation of the passenger through the ß-barrel channel. On the surface, passenger domains may be cleaved from the translocation unit and remain noncovalently associated with the bacterial surface or may be released into the extracellular milieu following an autoproteolytic event (for example, when the passenger domain is a protease) or cleavage by an endogenous outer membrane protease.
The first member of autotransporter family to be identified and characterized in Bordetella is PRN. Mature PRN is a 68-kDa protein in B. bronchiseptica (556), a 69-kDa protein in B. pertussis (128), and a 70-kDa protein in B. parapertussis (human) (461). It has been proposed to play a role in attachment since all three PRN proteins contain an Arg-Gly-Asp (RGD) tripeptide motif as well as several proline-rich regions and leucine-rich repeats, motifs commonly present in molecules that form protein-protein interactions involved in eukaryotic cell binding (226). The B. pertussis, B. bronchiseptica, and B. parapertussis PRNs differ primarily in the number of proline-rich regions they contain (460). The X-ray crystal structure of B. pertussis PRN suggests that it consists of a 16-strand parallel ß-helix with a V-shaped cross section and is the largest ß-helix known to date (225, 226). In support of the autotransporter secretion model, Charles et al. have shown that deletion of the 3' region of prnBp prevents surface exposure of the molecule (127).
Other Bordetella proteins with predicted autotransport ability include TcfA (originally classified as a tracheal colonizations factor) (248), BrkA (238), SphB1 (180), and Vag8 (247). All of these proteins show significant amino acid sequence similarity in their C termini and contain one or more RGD tripeptide motifs. Unlike PRN, BrkA, SphB1, and Vag8, TcfA is expressed exclusively in B. pertussis. Based on predicted amino acid sequence similarity to all of these proteins, the B. pertussis genome appears to encode at least three additional members of this autotransporter family. A lot has been learned about Bordetella autotransporters in recent years. As mentioned earlier, SphB1 has been characterized as a subtilisin-like Ser protease/lipoprotein that is essential for cleavage and C-terminal maturation of FHA (180). SphB1 is the first reported autotransporter whose passenger protein serves as a maturation factor for another protein secreted by the same organism. BrkA is expressed as a 103-kDa preproprotein that is processed to yield a 73-kDa (passenger)-domain and a 30-kDa ß-domain that facilitates transport by functioning dually as a secretion pore and an intramolecular chaperone that effects folding of the passenger concurrent with or following translocation across the outer membrane (598, 599). Like PRN and SphB1, BrkA remains tightly associated with the bacterial surface. Vag8 is a 95-kDa outer membrane protein that is expressed in B. pertussis, B. bronchiseptica, and B. parapertussishu (247). The B. pertussis and B. bronchiseptica Vag8 homologs are highly similar, and their C termini show significant homology to the C termini of PRN, BrkA, and TcfA, suggesting that Vag8, too, may function as an autotransporter. However, cleavage of the -domain from the C terminus may not occur in Vag8, since the predicted size of the entire protein encoded by vag8 corresponds to the size seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (247). In contrast, TcfA is produced as a 90-kDa cell-associated precursor form that is processed to release a mature 60-kDa protein (248). It is interesting that TcfA, the only known B. pertussis-specific autotransporter, is also the only Bordetella autotransporter that is not surface associated.
The ability of PRN and the other autotransporters to function as adhesins has been tested both in vitro and in vivo. In vitro studies demonstrated that purified PRN could promote the binding of CHO cells to tissue culture wells and that expression of prn in Salmonella or E. coli could increase the adherence and/or invasiveness of these bacteria to various mammalian cell lines (228). In contrast, a PRN– strain of B. pertussis did not differ from its wild-type parent in its ability to adhere to or invade HEp2 cells in vitro or to colonize the respiratory tracts of mice in vivo (664). Similarly, a B. bronchiseptica strain with an in-frame deletion mutation in prn was indistinguishable from wild-type B. bronchiseptica in its ability to establish a persistent respiratory tract infection in rats (P. A. Cotter and J. F. Miller, unpublished data). In contrast to the animal model studies discussed above, several pieces of data derived from vaccine trials and household contact study suggest that PRN may be the most important adhesin of B. pertussis (148, 203, 736). Of the seven vaccine efficacy trials conducted in the early 1990s, two were performed in a manner in which antibody values at the time of exposure were known (148, 736). In both of these trials it was noted that antibody to PRN was most important in protection. In addition to these observations, it is clear that DTaP vaccines which contain PRN in addition to PT and FHA are significantly more effective in preventing B. pertussis illness (131, 149, 310) (this is discussed in detail in the section on DTaP vaccine efficacy studies below).
With regard to the above studies, we predict that protection may be afforded by blocking PRN-mediated attachment of B. pertussis to host cells. More recently, Hellwig et al. have presented evidence that anti-pertactin antibodies are required for efficient phagocytosis of B. pertussis by the host immune cells (343).
Potential adhesive functions for TcfA, BrkA, and Vag8 have not been investigated directly, although TcfA– B. pertussis strains show a decreased ability to colonize the murine trachea compared to wild-type B. pertussis (248). BrkA has been proposed to play a role in serum resistance and contribute to the adherence of B. pertussis to host cells in vitro and in vivo. It also protects against lysis by certain classes of antimicrobial peptides (239). Interestingly, BrkA does not appear to be required for serum resistance of B. bronchiseptica (647). Most recently, Vag8 has been proposed to facilitate the secretion of type III proteins in B. bronchiseptica (507). This is the first reported example of an autotransporter involved in regulating type III secretion.
Adenylate cyclase.
All of the Bordetella species that infect mammals secrete CyaA, a bifunctional calmodulin-sensitive adenylate cyclase/hemolysin. CyaA is expressed maximally in the Bvg+ phase. Unlike the promoter for fhaB, cyaA does not contain any high-affinity BvgA-binding sites in its promoter region. Instead, it contains several heptameric variants of the BvgA-binding consensus 5'-TTTCCTA-3' which extend between nucleotides –137 and –51 from the transcriptional start site. Phosphorylation of BvgA is absolutely required for binding at these sites. The main target sequence for the BvgAP and DNA interaction is located between positions –100 and –80; binding to this centrally located site is predicted to trigger cooperative interactions of BvgAP with the neighboring low-affinity sites.
CyaA is synthesized as a protoxin monomer of 1,706 amino acids. Its adenylate cyclase catalytic activity is located within the N-terminal 400 amino acids (277, 349). The 1,300-amino-acid C-terminal domain mediates delivery of the catalytic domain into the cytoplasm of eukaryotic cells and possesses low but detectable hemolytic activity for sheep red blood cells (46, 349, 668). Amino acid sequence similarity between the C-terminal domain of CyaA, the hemolysins of E. coli (HlyA) and Actinobacillus pleuropneumoniae (HppA), and the leukotoxins of Pasteurella hemolytica (LktA) and Actinobacillus actinomycetemcomitans (AaLtA) places CyaA within a family of calcium-dependent, pore-forming cytotoxins known as RTX (repeats-in-toxin) toxins (659, 672, 676, 813). Each of these toxins contains a tandem array of a nine amino acid repeat [LXGGXG(N/D)DX] that is thought to be involved in calcium binding (813). Before the CyaA protoxin can intoxicate host cells, it must be activated by the product of the cyaC gene, which is located adjacent to, and transcribed divergently from, the cyaABDE operon (36). CyaC activates the CyaA protoxin by catalyzing the palmitoylation of an internal lysine residue (Lys-983) (37, 311). The E. coli HlyA protoxin is also activated by fatty acyl group modification (322, 375, 388). Whereas E. coli hemloysin is released in the extracellular medium, the majority of the Bordetella CyaA remains surface associated, with only a small portion being released in the supernatant. It was recently suggested that FHA may play a role in retaining CyaA toxin on the bacterial cell surface; B. pertussis mutants lacking FHA released significantly more CyaA into the medium, and CyaA toxin association with the bacterial surface could be restored by expressing FHA from a plasmid in trans (844). CyaA also inhibits biofilm formation in B. bronchiseptica, possibly via its interaction with FHA and subsequent interference with FHA function (383).
The eukaryotic surface glycoprotein CD11b serves as the receptor for mature CyaA toxin. Interestingly, surface-bound CyaA does not appear to be responsible for host cell intoxication; a recent report demonstrates that intoxication requires close contact of live bacteria with target cells and active secretion of CyaA (292). CyaA can enter a variety of eukaryotic cell types (350). Once inside, CyaA is activated by calmodulin (830) and catalyzes the production of supraphysiologic amounts of cyclic AMP (cAMP) from ATP (89, 163, 164, 321). Purified CyaA inhibits chemiluminescence, chemotaxis and superoxide anion generation by peripheral blood monocytes and polymorphonuclear neutrophils in vitro (611). CyaA also induces apoptosis in cultured murine macrophages (419) and inhibits the phagocytosis of B. pertussis by human neutrophils (808, 809). Recently, CyaA was shown to inhibit the surface expression of costimulatory molecule CD40 and IL-12 production in bone marrow-derived dendritic cells from C57BL/6 mice infected with B. bronchiseptica (709). It was further shown to be required for p38 phosphorylation, suggesting that it plays a role in inhibiting the p38 mitogen-activated protein kinase pathway (709). In vivo studies have shown that, compared to wild-type B. pertussis<
