Filamentous hemagglutinin of Bordetella pertussis: a key adhesin with immunomodulatory properties?

Review For reprint orders, please contact: [email protected]

Filamentous hemagglutinin of Bordetella pertussis: a key adhesin with immunomodulatory properties? Rodrigo Villarino Romero1, Radim Osicka1 & Peter Sebo*,1

Abstract The filamentous hemagglutinin of pathogenic Bordetellae is a prototype of a large two-partner-system-secreted and β-structure-rich bacterial adhesin. It exhibits several binding activities that may facilitate bacterial adherence to airway mucosa and host phagocytes in the initial phases of infection. Despite three decades of research on filamentous hemagglutinin, there remain many questions on its structure–function relationships, integrin interactions and possible immunomodulatory signaling capacity. Here we review the state of knowledge on this important virulence factor and acellular pertussis vaccine component. Specific emphasis is placed on outstanding questions that are yet to be answered.

The Gram-negative coccobacillus Bordetella pertussis, and occasionally also the closely related Bordetella parapertussis, cause a highly contagious respiratory infectious disease called pertussis or whooping cough. Pertussis affects children and adults of all age groups and used to be a major cause of infant mortality prior to global introduction of whole cell-based pertussis (wP) vaccines some six decades ago [1] . Unrecognized infections and/or mild disease due to B. pertussis continue to persist also in highly vaccinated populations. This makes whooping cough to be the least controlled vaccine-preventable infectious disease, accounting for more than 48 million cases and up to 300,000 pertussis-linked deaths annually worldwide [2–10] . Despite the efficacy of the wP vaccines, their acceptance by the general public in developed countries steeply decreased in the 1970s because of occasional adverse reactions due to the endotoxic component of the wP vaccine, the lipooligosaccharide (LOS) [7,11] . Research in the 1980s of the past century on antigens conferring protection against B. pertussis in the mouse challenge model then enabled development of less reactogenic acellular pertussis (aP) vaccines. These are made of purified antigens and progressively displaced the wP vaccines in the wealthiest countries. Recent epidemiological evidence, however, strongly suggests that the aP vaccines induce importantly shorter lasting immune protection in humans than the wP vaccines [12–18] . Despite high aP vaccine intake, whooping cough is on the rise in the most developed countries, with massive pertussis outbreaks occurring in countries such as Australia and the USA [19–22] . This situation calls for development of a next generation of pertussis vaccines that would not only confer protection against the severe (critical) pulmonary disease of infants, but would also prevent B. pertussis colonization and spread in highly vaccinated populations. Tackling of this challenge is complicated by the number of virulence factors that are produced by B. pertussis. The reader is referred to a recent comprehensive review for more detailed discussion of B. pertussis pathogenesis [23] . Briefly, several protein toxins, adhesins, autotransporters

Keywords

• adhesion • Bordetella • filamentous hemagglutinin • immunomudulation • integrins • pertussis • vaccine • virulence factors

1 Institute of Microbiology of the Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic *Author for correspondence: Tel.: +420 241 062 762; Fax: +420 241 062 152; [email protected]

10.2217/FMB.14.77 © 2014 Future Medicine Ltd

Future Microbiol. (2014) 9(12), 1339–1360

part of

ISSN 1746-0913

1339

Review Romero, Osicka & Sebo and complement resistance factors have been shown to play a role in B. pertussis virulence [1,24] . Among the key protein toxins are the adenylate cyclase toxin-hemolysin (ACT) and the more notoriously known pertussis toxin (PT) [25–27] . Although virulence of the related species B. bronchiseptica in mammals also relies on the cytotoxic and immunosuppressive effectors of the type III secretion system, the role of these virulence factors in B. pertussis infection remains unclear [28–32] . Other important virulence factors belong to the autotransporter and adhesin groups, which comprise critical cellular receptor-binding factors, including filamentous hemagglutinin (FHA), pertactin (PRN), tracheal colonization factor (Tcf), fimbriae (Fim) and complement resistance factors like BrkA and Vag8 [24,33] . These appear to contribute by either promoting bacterial adhesion and/or invasion into cells [34–47] . Moreover, some of them are recognized by the immune system as antigenic targets for antibodies and T cells [48–55] . Among such factors is the FHA, which is an important adhesin, present both in a secreted and surface-associated form [56,57] . In addition to its role as an adhesin, several studies over the past decade suggested that FHA also possesses immunomodulatory properties that may contribute to subversion of host innate and adaptive immunity [43,58–65] . FHA is highly immunogenic and is thus contained in all but one of the used aP vaccines. However, results obtained in mice as well as clinical studies in humans question the protective antigen potency of FHA and its contribution to the efficacy of pertussis vaccines [49,66–68] . Despite extensive research on FHA, particularly in the early 1990s, many questions on the role of FHA in Bordetella virulence and immunomodulatory capacities remain unanswered. Therefore, we review here the literature on FHA placing an emphasis on open questions on its structure–function relationships and biological role. fha locus organization FHA is encoded by the structural gene fhaB, the first gene of a polycistronic operon (Figure 1) in which fimB-D genes for fimbriae biogenesis are inserted downstream of fhaB [69–71] . More specifically, the fimbrial genes fimB and fimC are required for production of the fimbrial subunits, presumably participating in their folding and transport and anchorage to the outer membrane [70,71] while the fimD gene codes for the

1340

Future Microbiol. (2014) 9(12)

minor fimbrial subunit FimD [72] . As depicted in Figure 1, between the fhaB and fimB-D genes there is a pseudogene inserted designated fimA, which shows homology with the major fimbrial subunit genes [71] . Moreover, a large open reading frame designated fhaC and located downstream of fimD codes for a specific accessory protein FhaC, involved in the secretion of FHA across the outer membrane by the two partner system (TPS) secretion pathway [70,73–75] . The fimD and fhaC genes overlap, whereby their transcription and translation are coupled [70] . The fact that structural as well as accessory genes for both FHA and fimbriae are part of a polycystronic operon is noteworthy because site-directed deletions as well as transposon insertions may affect the production of both FHA and fimbriae [69,70] . For instance, it has been shown that insertion mutations in fimB-D affect FHA production because of polar effects on fhaC expression [70] . The fhaB gene is under the control of the central regulator of Bordetella virulence, the BvgAS two-component system [47,76–77] . It belongs to the first to be activated Bordetella virulence genes (vag) transcribed upon temperature upshift of Bordetella cultures from 25 to 37°C, or upon removal from culture media of BvgS sensor kinase activity modulators (i.e., sulfate or nicotinic acid) [47,78] . fhaB transcription is also rapidly resumed upon infection of mice by bacteria grown under modulating conditions in vitro, indicating that FHA is required early upon Bordetella entry into host airways [79] . Biogenesis of FHA The fhaB gene codes for a 367 kDa precursor protein (proFhaB, Figure 2A) that is exported in large quantities to the cell surface of exponentially growing B. pertussis [80–83] . Upon export to the cell surface, the proFhaB can undergo processing by proteases that yields the approximately 220 kDa mature FHA protein (Figure 2A) [80–83] . Mature FHA, composed of the N-terminal two thirds of proFhaB, can then remain on the cell surface or be released [45,84] . On its way across the Gram-negative bacterial envelope to the cell surface and extracellular milieu, the FhaB protein depends on the integral outer membrane protein FhaC, the second component of the TPS secretion pathway. FhaB is first exported across the cytoplasmic membrane via the signal peptide-dependent Sec pathway [88] , employing an unusually long N-terminal signal peptide comprising 71 amino

future science group

Filamentous hemagglutinin of Bordetella pertussis

bvgS bvgS bvgA bvgA

fhaB fhaB

fimA fimB fimA

fimC

fimD

Review

fhaC fhaC

Figure 1. fha locus organization. fhaB is the first gene of a polycistronic operon. Binding regions for the BvgA transcription factor are immediately upstream of the structural gene for filamentous hemagglutinin (fhaB). Genes implicated in fimbriae biogenesis (fimB–fimD), as well as the fhaC gene coding for the outer membrane protein FhaC, are located downstream of fhaB. Note that the pseudogene fimA is crossed over, indicating the absence of protein-coding ability. The genes fimD and fhaC are transcriptionally and translationally coupled and thus, no intergenic region is present between them.

acid residues (Figure 2A) . This has a 22-residuelong ‘N-terminal extension’ that is dispensable for FhaB production and secretion of the mature protein, but is thought to regulate the export rate of the protein across the inner membrane [88,89] . The unusual extension is followed by a positively charged region (residues 33–43) and a long hydrophobic core (residues 44–65) of the signal peptide, which is followed by a processing site at residue Ala-71. The latter fits the rule for Lep-type leader peptidase cleavage sites, having small amino acid residues at positions -1 and -3 relative to the processing site [86,90] . Employing FHA44, a mutant polypeptide corresponding to the first N-terminal 80 kDa of FhaB, it was shown that the signal peptide of FHA further contains three cysteines, two of which, Cys-24 and Cys-31, are necessary for post-translational cyclization of the N-terminal glutamine residue (Gln-72) of FHA to a cyclic pyroglutamyl residue [89] . The transit of FhaB through the cytoplasmic and outer membranes appears to be coupled. FhaB transits the periplasmic space in an extended and protease-sensitive conformation and its translocation through the outer membrane via FhaC occurs immediately upon export from the cytosol [91] . The mechanism of TPS action has been studied using FhaB as a prototype [75,80] . The FhaB protein carries between residues 72 and 316 (equivalent to residues 1–245 of the mature FHA protein) a characteristic N-proximal TPS domain (Figure 2A) [70] . This exhibits extensive homology to segments of the Serratia marcescens and Proteus mirabilis hemolysins and is recognized by the TpsB translocator, FhaC [92] . The structure of FhaC has recently been solved at a 3.15 Å resolution [87] . The C-terminus forms a transmembrane β barrel composed of


16 antiparallel β strands with globular periplasmic domains designated as polypeptidetransport-associated (POTRA) domains, which are likely involved in FHA recognition and are strictly required for the secretion process [87] . A large extracellular loop designated L6 is folded as a hairpin in the barrel interior, reaching the periplasm and thus occluding the barrel channel [87] . The N-terminal TPS of FHA was proposed to initially interact in an extended conformation with the POTRA 1 domain in the periplasm. This would bring in close contact the first repeats of the central β-helical domain of FHA with the tip of L6 loop, thus resulting in a conformational change that expels out loop L6 from the β channel, opening the channel for FHA translocation [87] . The FhaC channel appears to be rather narrow, with an estimated diameter of less than 10 Å, indicating that FHA transits the outer membrane in an unfolded state. This goes well with the observation that FhaC recognizes an unfolded conformation of the TPS domain of FhaB, further indicating that FHA does not fold inside the periplasm and acquires its native conformation only upon reaching the cell surface in a process of successive folding. This would progress as the protein emerges from the FhaC pore. The authors further proposed that the FhaB polypeptide might form a hairpin looping through the FhaC pore, comprising two extended chains, with the TPS domain remaining anchored to the periplasmic portion of FhaC. The formation of a rigid β-helix outside the cell would then provide the energy driving FhaB translocation through FhaC. The extracellular portion of the FhaB precursor is processed into mature FHA at some point after translocation to the cell surface. The processing is primarily accomplished by

www.futuremedicine.com

1341

Review Romero, Osicka & Sebo

367 kDa proFhaB SP TPS

PNT

Cytoplasm

N

C 1-71 72-316

2362-2492 2600-2602 3590 Proteolytic processing

Periplasm

1-71

3590

TPS N

Extracellular 72-316 220 kDa mature FHA N

Extracellular

FhaC C Periplasm

T

E

Intracellular P SP TPS

C

Extracellular

Periplasm

PNT N

Figure 2. Structural domains of filamentous hemagglutinin and proposed models of filamentous hemagglutinin biogenesis. (A) Schematic representation of the proFhaB precursor protein and of the processed mature form of FHA that results from proteolytic processing of proFhaB in two steps. Upon export of FhaB across the cytoplasmic membrane via the SP-dependent Sec pathway, the 71 residue-long N-terminal SP (light blue) is cleaved-off by signal peptidase. Following recognition of the N-proximal TPS domain (TPS, residues 72–332, in blue) by FhaC and translocation of its major portion through the outer membrane, the FhaB precursor is eventually processed by the outer bacterial membrane-anchored protease SphB1 and other unidentified proteases to the mature FHA of approximately 220 kDa.

1342




Review

Figure 2. Structural domains of filamentous hemagglutinin and proposed models of filamentous hemagglutinin biogenesis (cont.). Sequences downstream to the SphB1 processing site, indicated as PNT were implicated in intracellular retention of the C-terminal prodomain in the periplasm and/or outer bacterial membrane [85]. (B) Initially proposed model of FHA secretion according to Jacob Dubuisson and coworkers [86]. FHA export across the cytoplasmic membrane through T is initiated upon recognition of the N-terminal SP by P. During or after translocation, the signal peptide of FHA is processed and cyclization of the new N-terminal residue (Gln72) to a cyclic pyroglutamate (indicated by the yellow star) is catalyzed by E. The N-proximal TPS domain is recognized by the outer membrane protein FhaC and secretion of the FhaB protein across the outer membrane is initiated with the N-terminus crossing the outer membrane and emerging on cell surface in an extended conformation. Folding of the emerging protein portions into a β-helix on cell surface would drive directional translocation of FhaB by a Brownian ratchet mechanism. Upon processing by SphB1 on cell surface, the mature FHA protein is released into the extracellular milieu while the approximately 130 kDa C-terminal prodomain of FhaB (in red) is degraded in the periplasm. (C) Alternative model of FHA biogenesis proposed by Cotter and coworkers and later endorsed by others [45,62,85,87]. In this model, the N-terminal TPS domain binds FhaC but remains intracellular, while an unfolded FhaB protein hairpin loops across the FhaC channel to start translocation across the outer membrane. Folding proceeds and facilitates pulling of the rest of FhaB chain across the FhaC channel. The figure depicts the β-helix already formed while the C-terminus (blue) and the prodomain (in red) are still inside the periplasm. During the secretion process, the PNT adopts a conformation that halts prodomain transit through FhaC. In this model, the tension conferred by the translocation-impaired PNT determines the conformation that the C-terminus may adopt while folding. The FhaC is next primed for release of FhaB (indicated by the shift in FhaC color from gray to green). Upon processing by SphB1, the mature FHA can remain surface associated or is released into the supernatant. In this model, it is the C-terminus that is exposed on and protrudes from the cell surface, while the N-terminus is surface associated until FHA is released from cells. E: Unknown enzyme; FHA: Filamentous hemagglutinin; P: Cytoplasmic chaperone protein; PNT: Prodomain N-terminus; SP: Signal peptide; T: Sec translocase; TPS: Two-partner system. (B) Adapted with permission from [86]; (C) adapted with permission from [85].

the subtilisine-like domain of the self-processing autotransporter protease SphB1 [84] that remains anchored to the outer bacterial surface through an N-terminal lipid moiety [93] . Significant amounts of unprocessed FhaB accumulate, indeed, on the cell surface of sphB1deficient mutants [93] . These exhibit increased levels of in vitro attachment to epithelial and macrophage-like cells, presumably due to a tendency to self-aggregate [94] . In contrast, lack of an active SphB1 resulted in impaired colonization in a mouse model of infection, suggesting that the processing and release of FHA may be necessary for optimal colonization of the mouse lungs by B. pertussis [94] . However, it should be noted that it remains unclear whether SphB1 processes FHA only or other virulence factors as well. Hence, caution is needed in interpreting phenotypes observed with SphB1-deficient strains that must not necessarily be due to effects on FhaB processing, only. Moreover, processing by SphB1 does not seem to be truly essential for FHA release and shedding, as both B. pertussis


and B. bronchiseptica mutants deficient in SphB1 do still release a slightly larger (∼250 kDa) form of FHA into culture supernatants [45] . Processing of FhaB by SphB1, in turn, yields several cellassociated and released forms of mature FHA that range between approximately 210 and 230 kDa in size [45] . The processing sites recognized by SphB1 remain to be precisely identified and appear to be located between residues 2340 and 2485 of FhaB from B. bronchiseptica and residues 2231–2376 of B. pertussis FhaB, respectively [45,85] . Since SphB1-dependent cleavage does not appear to be essential for FHA release, its relevance for B. pertussis virulence remains to be demonstrated in an animal model better reflecting human infection than mice. The 130 kDa C-terminal prodomain portion of the FhaB precursor was recently proposed to act as an intra-molecular chaperone domain that enables acquisition of a functional conformation of the C-terminus of mature FHA, facilitating its folding prior to the SphB1-mediated processing [43,45,85] . Further characterization of


1343

Review Romero, Osicka & Sebo the prodomain revealed that sequences at its N-terminus (PNT) (Figure 2A) are required for the C-terminus of the prodomain to be retained intracellularly [85] . The topology of the FhaB molecule during secretion through the FhaC pore remains, however, a matter of debate. Secretion of FhaB was initially suggested to proceed directionally with the N-terminus emerging first and protruding from the cell surface as the movement of the protein through the FhaC pore proceeds (Figure 2B) [86] . In contrast, recent work would indicate that it is the processed C-terminus of the mature FHA protein that protrudes out of the bacterial cell surface and may play a role in adhesive and immunomodulatory activities of FHA (Figure 2C) [45,62,85,87] . FHA structure & properties The rod-like shape of the mature FHA molecule (Figure 3A) resembles that of a horseshoe nail of approximately 50 nm in length and approximately 4 nm in width [95] . Its approximately 9 nm long and 5 nm wide globular head is linked to an approximately 35-nm-long shaft, which is followed by a small flexible tail [95] . The shaft comprises two extended repetitive regions, R1 and R2 (Figure 3A) . The R1 region starts from residue 324 and comprises two copies of a 20-residue repeat followed by 37 copies of a 19-residue pseudorepeat [95,96] . The R2 region starts at residue 1431 and comprises 13 copies of 19–20 residue repeats of different consensus pattern from the R1 region [95,96] . In addition, over approximately 27 ‘covert’ and less conserved copies of these tandem repeats are dispersed along the FHA molecule (Figure 3A) [96] . These regions are predicted to comprise β-strands and turns [95,96] , having common features with the leucine-rich repeats present in many eukaryotic proteins [97–100] . Two models have been proposed for the overall structure of mature FHA, as depicted and compared in Figure 3B. The first proposed model predicted formation of a hairpin structure, in which the head of the ‘horseshoe nail’ would be composed of juxtaposed N- and C- terminal domains. The tail would then be composed of the intervening sequence and the shaft comprising most of the repeat stretches forming amphipathic hyperelongated β-sheets, with their hydrophobic faces apposed [95] . The second model was proposed by the same group and endorsed later by others [96] . It suggests that the shaft is a single β-helix containing three long parallel

1344


β-sheets that are arranged around the axis of the molecule. Unlike in the hairpin model, in the β-helix model the N- and C- termini would be located at opposite ends of the FHA molecule and the repeats would form successive coils of an elongated β helix that would correspond to the approximately 350-Å long rod in the central region of FHA. In 2004, Clantin and coworkers solved the structure of the 30-kDa N-terminal secretion domain that plays a central role in recognition and secretion of FHA by the TPS secretion pathway [73] . The TPS domain fragment also contained the first three repeats of the R1 domain that were found to form regular β-helical coils. The overall structure adopted by the protein was that of a right-handed β-helical fold with extra-helical motifs, a β-hairpin, a four-stranded β-sheet and an N-terminal capping (Figure 3C) . These results further indicated that mature FHA forms an elongated β-helix, rather than a hairpin. The adherence determinants would thus be located on loops or extrahelical motifs at different sites along the helix [73] . Adhesin activities of FHA The adhesin activities of FHA as well as its contribution to virulence of B. pertussis have been intensely studied early after its identification as a potential vaccine antigen, yielding somewhat controversial results [101–107] . Initially, adherence of the Tohama I strain to HeLa and Vero cells was found to be blocked by anti-FHA antibodies [103] , suggesting that FHA might be a virulence factor important for attachment to target cells. Nevertheless, the FHA preparations used to obtain the anti-FHA antibodies were later shown to be quite heterogeneous, containing PT and probably other contaminants such as fimbriae as well, and the anti-FHA antiserum was shown to react with additional distinct antigens present in the FHA preparation [102,103] . Furthermore, antibodies against fimbriae were also reported to inhibit adherence of B. pertussis to Vero cells [108] . This hindered the recognition of whether attachment is exclusively due to FHA, other components such as fimbriae, or both. Since then, several studies have supported a prominent role of FHA in the in vitro adherence of B. pertussis to epithelial cells [46,82,104,109] and phagocytic cells [39,41,46,109–110] of human and animal origin. Although a lot of evidence has been accumulated over the years implicating FHA as an important adherence factor, it is



B0 72

1065

HBD (442–863)

N

R1

232

B1

324

1680

CRD (1141–1279)

RGD (1097-1099)

R2

Review

C

B2

1431

1871 2165

220 kDa mature FHA

≈4 nm RGD

TPS N

CRD

RRARR

N

≈50 nm

R2

HBD R1

R1

RRARR RGD CRD

HBD

R2 TPS N

C

C

C

Figure 3. Schematic depiction of the domain structure of filamentous hemagglutinin. (A) Schematic representation of the processed mature form of FHA that results from proteolytic processing of proFhaB. Five regions are depicted that represent β-helical coils: the R1 and R2 extended regions, comprising coils characterized by 19-residue sequence repeats (blue); and three B regions composed of covert copies of similar motif but less conserved length (white). The three domains of FHA implicated in binding of host cell surface structures, in other words, identified as mediating binding to and/or invasion of eukaryotic cells by Bordetellapertussis, are also shown. These are the HBD located between residues 442 and 863 (HBD), the Arg-Gly-Asp (RGD) motif comprising a domain centered at the Gly residue 1098 and the CRD mapped between residues 1141 and 1279. (B) Schematic representation of the two proposed models of mature FHA structure, forming a hairpin (left) or a β-helix (right). The N- and C-termini as well as the R1 and R2 repeat regions are indicated together with the three known binding domains of mature FHA mentioned above. β-strands are depicted as dark grey bars. (C) Crystal structure of a secreted 30-kDa N-terminal fragment of FHA that includes the TPS domain. The structure comprises amino acid residues 1–301 of mature FHA (PDB ID code 1RWR). The N- and C-termini are also indicated. CRD: Carbohydrate recognition domain; FHA: Filamentous hemagglutinin; HBD: Heparin-binding domain; RRARR: Arg-Arg-Ala-Arg-Arg; TPS: Two-partner system. (A) Data taken from [96]; (B) data taken from [95,96]; (C) adapted from structure deposited in PBD under ID code 1RWR.

also important to recognize the caveats of these studies. Many experiments were performed with parental strains or derived mutants that due to the nature of the insertional mutations or deletions, yielding polar effects on the fim genes, were not only deficient in FHA but also in fimbriae. Therefore phenotypes cannot be unambiguously attributed to FHA. Other experiments relied on


fragments of FHA purified from Escherichia coli and used for experiments in vitro [46] . Given the complexity of the FHA export and folding (see structure and biogenesis) it is quite unlikely that these fragments were properly folded and maintained the native function in the absence of the rest of the FHA molecule. Other studies relied on rather artificial approaches, such


1345

Review Romero, Osicka & Sebo as antibody-blocking experiments that present problems of reproducibility. Furthermore, some studies showed that adhesion of B. pertussis to epithelial cells and monocytes involves cooperative contributions of several independent virulence factors such as FHA, fimbriae, pertactin or pertussis toxin [39,105,109,111] , but disagreed in which virulence factors are actually involved and in the cell types they act on. Some investigators reported that adherence of B. pertussis to human macrophages is not decreased by genetic alteration of either fimbriae, FHA or pertussis toxin alone and more than one of these virulence factors needs to be affected to yield decrease of adherence [109] . Cooperative interaction of these virulence factors on the cell surface during bacterial adhesion is indeed, plausible. For example, immunogold labeling revealed that FHA and PRN are distributed over the entire surface of the B. pertussis cells and often cluster together [37] . In one study, the expression of the virulence regulon was synchronized by shifting cultures of B. pertussis from 22 to 37°C and the results showed that PRN was also necessary for FHA-mediated adhesion to Chinese hamster ovary (CHO) cells [112] . In the absence of PRN, the FHA was poorly exposed on the external surface of the bacterium, while the binding activity was delayed and considerably impaired when compared with the wild-type strain [112] . Similarly, a direct physical association between FHA and ACT was reported, allowing for cell-surface retention of the AC toxin [113] , which has been shown to influence the FHA-mediated attachment of B. pertussis to epithelial cells [114] . Formation of complexes between different factors may thus be important in mediating bacterial attachment, adding a layer of complexity to the already tricky task of addressing the contribution of individual virulence factors to B. pertussis adherence to host cells. FHA was further shown to play a role in invasion of both epithelial and phagocytic cells [42,44,115–118] . Although major differences between B. pertussis strains or isolates were found and some discrepancy on the role of FHA appeared depending on the eukaryotic cells employed (i.e., nonrespiratory cells vs respiratory) [115,116] , generally the accumulated data led to the suggestion that FHA is required for invasion of eukaryotic cells by virulent bacteria. This process may represent a strategy by which B. pertussis forms intracellular reservoirs, avoiding host-immune

1346


responses [115–118] . However, all the virulence factors implicated in the invasion process (FHA, ACT and PRN) have also been shown to interact and mutually affect each other’s functionality [37,112–115] , participating in similar processes and/or sharing host receptors [41,44,109,119] . This makes it difficult to discriminate between the roles of these components. Furthermore, while some studies indicated that internalized bacteria do not multiply and that phagocytosis may be followed by killing of B. pertussis [115–117] , other studies suggest that B. pertussis can survive and replicate within epithelial and phagocytic cells, potentially contributing to B. pertussis evasion of the host immune response and persistence within hosts and populations [118,120–122] . Given the adhesive properties of FHA observed in in vitro experiments, the role of FHA in colonization of the respiratory tract has also been investigated [43,63,101,118,123] . One study showed that FHA may play a role in mouse lung colonization [63] , but the FHA-deficient strain was also defective in fimbriae expression. Other studies indicate that FHA does not play any essential role in B. pertussis colonization of the mouse lungs [43,101,123,124,101] , possibly due to redundancy of function of several virulence factors in this process [124] . FHA was found to be important in mediating bacterial colonization of the rabbit lungs [118] , although the methodology employed to inoculate the bacteria was rather far from reflecting the natural course of infection. In contrast, FHA has been shown to play a role during initial colonization of the mouse trachea [101] . All of these results should, however, be interpreted with caution as such experiments are flawed by the use of animals that are not naturally infected by B. pertussis, a strictly human-specific pathogen. The role that FHA may play in colonization of the human respiratory tract, hence, remains unknown and deserves to be addressed in the newly developed baboon animal model in which human pertussis pathophysiology appears to be reproduced quite truly [18,125–126] . Extensive research on the adhesive capacity of FHA has led to the recognition of its several binding activities/specificities for which different binding domains have been identified [37,41,46,82,104,127] . The first identified FHA domain involved in binding to ciliated respiratory cells and macrophages was a carbohydrate recognition domain (CRD) localized between residues 1141 and 1279 (Figure 3A) , which was


Filamentous hemagglutinin of Bordetella pertussis found to account for FHA binding to glycolipids of human cilia and to galactose-containing glycoconjugates [37,46,104,109] . It recognizes in particular the β1-4-linked galactose residues and galactose-N-acetylglucosamine moieties of glycolipids, such as lactosylceramide [46,104,109] . A second binding domain, located between residues 442 and 863 of the R1 repeat region (Figure 3A) , appears to mediate binding to sulfated carbohydrates and to account for the hemagglutinating activity of FHA [37,46] . Intriguingly, outside of this heparin-binding domain (HBD) an Arg-Arg-Ala-Arg-Arg (RRARR) sequence is found between residues 1069 and 1073 (Figure 3B) , which comprises the RRAR motif found in the consensus heparin-binding sites of fibronectins [128–130] . FHA would bind through this domain both sulfated glycolipids and proteo glycans, such as heparan and chondroitin sulfate [37,131,132,37] . This binding domain was shown to be responsible for FHA-mediated attachment to a variety of cultured epithelial cells [132] but its relevance, if any, in binding to target cells such as ciliated epithelial cells or macrophages awaits confirmation. Interestingly, in the study showing attachment of FHA to cultured cells via this second binding domain, the attachment of FHA to cells could not be inhibited by the sugars galactose and lactose [132] , unlike what was previously shown using ciliated respiratory cells or macrophages [46,104,109] . Furthermore, both the CRD and the HBD have not been conclusively shown to contribute to pathogenesis using in vivo models and their role during infection, hence, remains unknown. Finally, a subdomain harboring an Arg-GlyAsp (RGD) motif centered around the Gly residue 1098 (Figure 3A) was initially shown to participate together with the CRD in FHAdependent binding of bacteria to macrophages [109] . Interestingly, the sequence next to the RGD motif exhibits residue identity at seven of the nine positions with the sequence flanking the RGD motif of fibronectin [82] . The interaction with macrophages was proposed to occur through direct binding of the RGD motif of FHA to the α Mβ2 integrin, known also as CR3, or CD11b/CD18 [109] . Later studies, however, suggested that binding of FHA to CR3 may depend on initial RGD-mediated interaction of FHA with another eukaryotic integrin, the αvβ3 LRI, also known as CD51/CD61 and IAP (Figure 4) [41] . The authors proposed that crosslinking of the LRI/IAP complex of monocytes by


Review

FHA would initiate LRI/IAP-mediated intracellular signaling, in which PI3K activation seems to be involved, leading to upregulation of CR3 binding activity for FHA (Figure 4) [133] . The implications for this upregulation of CR3 are unknown but may indeed serve several purposes such as facilitating B. pertussis adherence, toxins delivery into cells, intracellular entry and/or survival of the bacteria [41] . The RGD motif of FHA has also been implicated in B. pertussis adhesion to CHO cells [112] and in B. pertussis invasion into and survival within primary human macrophages [117,118] . Similarly, it was suggested that following an initial attachment of bacteria to the surface of human respiratory epithelial cells, FHA may promote the invasion of these cells through interaction of the RGD motif with the very late antigen-5 (VLA-5), known also as the α5β1 integrin or CD49e/CD29 heterodimer [42] . However, and despite the large amount of accumulated data, it is worth noting that the conclusions from these in vitro studies are mostly based on peptide and antibody blocking experiments, and a direct interaction of FHA with integrin receptors remains to be demonstrated. Unlike with the CRD and the HBD, there have been some studies addressing the role of the RGD motif in vivo. Interestingly, peptides derived from the RGD region of FHA inhibited integrin-mediated adherence and transendothelial migration of neutrophils in vitro and prevented recruitment of leukocytes into the cerebrospinal fluid in an experimental model of meningitis in rabbits [134] . Another study showed that a B. pertussis mutant deficient in pertactin expression colonized the mouse lungs as efficiently as the parental strain, while a mutant lacking pertactin and having the RGD sequence of FHA mutated was able to multiply but was cleared faster than the parental strain, indicating a role of the RGD sequence of FHA in bacterial colonization [124] . In contrast to previous reports, however, a more recent study showed that antibodies against a fourth domain of FHA, the mature C-terminal domain (MCD), but not antibodies against the CRD, blocked FHA-mediated adherence of B. pertussis to epithelial and macrophage-like cells, suggesting a role for the MCD in the adhesion process [43] . Furthermore, several studies suggest that it is the MCD and not the RGD which mediates adherence of B. bronchiseptica to epithelial and macrophage-like cells and that the MCD is required for FHA


1347


C CR3-binding domain

N RGD

CR3

LRI

IAP

(CD47)

(β3)

(αM)

(αv)

(β2)

p85 Tyr

P ?

p110

Figure 4. Binding of surface-bound filamentous hemagglutinin to leukocyte response integrin/integrin-associated CD47 protein. As proposed by Ishibashi and coworkers [41,133], recognition of monocyte LRI/IAP by RGD domain of Bordetella pertussis FHA would lead to LRI/IAP cross-linking and triggering of intracellular signaling. This would activate CR3 to enhance its availability for interaction with another as yet unknown domain of FHA. Crosslinking of LRI/IAP was found to result in tyrosine phosphorylation of an as yet unidentified 60 kDa protein, which would recruit PI3K that activates CR3 for interaction with FHA. FHA: Filamentous hemagglutinin; P: Phosphate; RGD: Arg-Gly-Asp; Tyr: Tyrosine.

function during B. bronchiseptica infection in vivo [43,45,62] . A segment of the C-terminal prodomain of FhaB, located between residues 103 and 886 downstream to the presumed SphB1 processing site, was then suggested to play a role in acquisition of an adherence-competent conformation of the C-terminal portion of mature B. bronchiseptica FHA [43,45,62] . The structural integrity of the prodomain of the FhaB precursor appears, thus, to impact on functionality of the mature FHA. Noteworthy, equal virulence in the mouse challenge model was observed for wild-type B. pertussis and its ΔfhaB mutants in most of the studies [43,101,123,124,101] , thus making difficult the investigation of the role of specific segments of FHA in B. pertussis infection. The FHA molecules from B. bronchiseptica and

1348


B. pertussis were, however, found to be functionally interchangeable in mice challenge by B. bronchiseptica, indicating that results obtained in rodent models available for B. bronchiseptica may reflect the function of the FHA molecules from both species [43] . Due to different host specificity and differences in host respiratory tract colonization between the two Bordetella species, however, the data obtained with B. bronchiseptica should be interpreted with caution when extrapolated to FHA role and function in B. pertussis infection of man. In this context, it is important to note that it remains unclear what is the physiological role of processing and shedding of FHA by the bacteria during infection. A kinetic experiment of vag gene upregulation showed that it takes


Filamentous hemagglutinin of Bordetella pertussis several hours from induction of FhaB production until detectable amounts of processed mature FHA are released by the bacteria into the extracellular milieu [112] . This suggests that the full-length and cell membrane anchored FhaB and the processed soluble mature FHA form may play different roles in the course of infection. Interestingly, there is a second RGD motif located at amino acid position 2599 within the C-terminal prodomain of FhaB [82] . However, it remains unexplored whether this motif plays any role in B. pertussis adhesion or in any of the potential immunomodulatory activities of FHA. For example, FHA is also responsible for homotypic interactions that result in formation of bacterial aggregates [135] . It is believed that addition of β-cyclodextrin to the growth medium decreases the bacterial aggregation by reducing the surface hydrophobicity of FHA. In the absence of β-cyclodextrin, FHA is found mostly attached to the bacterial surface and to mediate massive bacterial aggregation [135] . Release of FHA from the bacterial surface might then facilitate bacterial dispersion after an initial adherence phase [93,112] . Pre-incubation of B. pertussis with added FHA appeared to enhance its adherence, showing that the free protein can reassociate with bacterial surface [107] and suggesting that free FHA may act by bridging the bacteria and eukaryotic cells. Finally, several recent studies have shown that free mature FHA may also play an immunomodulatory role during infection, whose characteristics are described in detail below. Immunomodulatory properties of FHA Over the past decade, a number of studies pointed towards an immunomodulatory activity of FHA that would result presumably from triggering of signaling of its diverse cellular receptors. As some of these data are contradictory and confusing, the results obtained in the murine and the human cell systems are discussed separately below. A puzzling observation is the capacity of B. pertussis FHA to bind the human regulator of the classical complement activation pathway, the C4BP, in a way that preserves its activity [136,137] . This is, however, unlikely to foster serum resistance of B. pertussis, since mutants not producing FHA remain serum resistant [138] . It remains to be explored, nevertheless, if by analogy to interaction with the long fibrillar M protein of Streptococcus pyogenes [139,140] , the binding of C4BP to FHA may also


Review

downregulate complement opsonization and enable phagocytosis evasion of B. pertussis. FHA has been shown to trigger the NF-κB pathway in a complex manner. Initial in vitro studies suggested that RGD-dependent engagement of VLA-5 on human bronchial epithelial cells by bacterial surface-bound FHA triggers NF-κB activation, followed by upregulation of expression of ICAM-1 (Figure 5) [127,141] that contributes to recruitment of inflammatory cells to respiratory epithelia [142–144] . This contrasts with the inhibition observed when epithelial cells were treated with soluble FHA [59] . In the latter study, exposure to soluble FHA did not result in NF-κB activation but resulted in attenuated proteasome function and inhibition of IκBα degradation and block of RelA translocation into the cell nucleus in response to other pro-inflammatory stimuli [59] . It is noteworthy that the RGD-dependent activation of the NF-κB pathway was observed in epithelial cells seeded into FHA-coated wells [127,141] . It is possible, hence, that these discrepancies were due to differences in presentation and mode of interaction of the FHA molecules (surface-bound vs soluble). Both modes may nevertheless differ from the positioning and mode of interaction of FHA when attached to and protruding from the bacterial surface. As mentioned above in the section on ‘adhesin properties of FHA,’ recent data would suggest that it may be the C-terminal domain of the mature FHA (MCD) and not the RGD motif that accounts for bacterial–host interactions [43] . In contrast to observations on epithelial cells, however, a rapid degradation of IκBα was observed within 30 min of treatment of monocytes and macrophage-like cells with soluble FHA [59] that yielded increased binding of RelA/p50 to κB-DNA binding sites in 2–4 h. Following the initial rapid activation, however, prolonged exposure of macrophages to FHA resulted in inhibition of the NF-κB pathway response to other inflammatory signals [59] . Similar to what was observed in epithelial cells, however, longterm exposure to FHA resulted in inhibition of proteasome activity in macrophages, which may explain the limited degradation of IκBα [59] . In line with previously observed effects of proteasome inhibition in increasing IL-10 secretion by monocytic cell lines in response to LPS [145] , an increase of IL-10 secretion by soluble FHAtreated cultured macrophage-like cells and fresh monocytes was observed [59] . Moreover, the anti-inflammatory cytokine IL-10 is known to


1349

Review Romero, Osicka & Sebo FHA

PT A

RGD

B ICAM-1

VLA-5 (β1)

(α5)

A

IκB Ub

Ub Ub Ub IκB

Transcription

Figure 5. Binding of surface-bound filamentous hemagglutinin to the integrin VLA-5. Engagement of VLA-5 on epithelial cells by surface-bound FHA that harbors an RGD motif would result in activation of NF-κB and upregulation of ICAM-1 expression [127,141]. FHA binding would activate the VLA-5 receptor-associated trimeric G protein that signals for ubiquitination and degradation of IκB. NF-κB can then translocate into the nucleus and initiate transcription of genes upregulating expression of ICAM-1 on the epithelial cell surface. The G protein involved in signaling appears to be PT-sensitive [141]. A and B: Pertussis toxin enzyme (A) and transport (B) subunits; FHA: Filamentous hemagglutinin; PT: Pertussis toxin; RGD: Arg-Gly-Asp; Ub: Ubiquitin.

disrupt NF-κB activation by interfering with IκBα degradation and Iκκβ-induced phosphorylation [146] , suggesting an autocrine positivefeedback loop amplification of IL-10 secretion by FHA. It remains however, unclear if the FHA-induced secretion of IL-10 alone mediates the inhibitory effects of FHA on macrophages. Proteasome inhibition might also explain the reported FHA-induced apoptosis [58] , since accumulation of IκBα has been shown to lead to apoptosis in other systems [147] . On the other hand, secreted and cell-associated FHA was shown to elicit pro-inflammatory and pro-apoptotic responses in human bronchial epithelial and monocyte-like cells [58,59] . Together with other pro-inflammatory

1350


cytokines, such as IL-8, a dose-dependent release of TNF-α was observed when macrophage-like cells and fresh monocytes were treated with soluble FHA [58,59] , suggesting that FHA bears also an intrinsic pro-inflammatory activity, or can at least act synergistically with LOS, or both [58] . When FHA-induced changes in genome-wide transcript abundance were studied on human peripheral blood mononuclear cells (PBMCs) stimulated with purified FHA [61] , the used FHA preparation was not only found to be a strong pro-inflammatory stimulus, but genes involved in the negative regulation of the inflammatory response were also induced by FHA. The results showed that FHA was a strong inducer of the IFN type I response and triggered the interferon


Filamentous hemagglutinin of Bordetella pertussis stimulated gene (ISGylation) pathway, including the ISG15 component that becomes conjugated to cellular target proteins and is associated with cytokine-like immunomodulatory activities [148–151] . FHA treatment of PBMCs resulted in larger quantities of ISGylated protein and free ISG15 as well as an additional ISGylated protein, of about 130 kDa, not detected in cells treated with LPS or IFN-α [61] . Monocytes and NK cells were shown to be the major producers of ISG15 upon exposure of PBMCs to FHA [61] . The important contribution of cell-free FHA to the inflammatory process triggering was also highlighted by the observation that most of the genes involved in mediating inflammation as part of a ‘common host-transcriptional response’ to bacteria were also upregulated upon exposure of cells to FHA [61] . Furthermore, some of the cytokines induced by FHA (all induced at least at the transcriptional level) are known to promote expression of CR3, and therefore the authors suggested that FHA might also indirectly promote B. pertussis binding to host cells, where CR3 may be exploited as a receptor for FHA at the surface of neutrophils and monocytes [41,109] . FHA might also influence host cell–cell interactions by upregulating ICAM-1 at the surface of PBMCs [61] . Activation of type I IFN signaling has been shown to sensitize cells of the immune system to death [152] . Since FHAinduced apoptosis in human myeloid cells is only partly dependent on the production of TNF-α [58] , it is tempting to speculate that the FHAinduced type I IFN response might contribute to induction of phagocyte death, thus representing an additional mechanism to render the host more susceptible to infection. Having said that, important caveats of these studies [41,61,109] need to be mentioned as well. In particular, the FHA preparations used in these studies still contained considerable amounts of contaminating LOS and therefore a contributory role of Bordetella LOS and/or other minor contaminants that may copurify with FHA cannot be ruled out. Important controls providing evidence that the employed FHA maintained its native conformation and was active (i.e, hemagglutinating activity) were missing in some of the studies [41,109] . Moreover, none of these studies provided controls indicating that the observed properties of the FHA preparation required a native and active FHA molecule. This could have been shown by including a heat/denatured or mutant form of FHA or a ‘mock’ control sample purified under


Review

the same conditions from cultures of bacteria not producing FHA or by including experiments where binding of FHA to cells is blocked. Thus, robust evidence indicating that the responses observed are due to a specific signaling activity of FHA is still missing. Finally, another group showed that PBMCs isolated from 3- and 6-month-old aP vaccinated infants secreted IFN-γ in response to in vitro stimulation with FHA [153] , supporting the finding that FHA acts as a pro-inflammatory stimulus. This IFN-γ response to FHA was highly variable because the monocytes of a substantial proportion of vaccinated infants appeared to spontaneously secrete IL-10, which seemed to have an inhibitory role on the B. pertussis antigen-specific IFN-γ secretion. The reason for this spontaneous IL-10 secretion remains unknown and it was only shown that it did not seem to depend on certain polymorphisms of the IL-10 gene promoter [153] . Similar to other studies mentioned above, a specification of the purity of the used FHA preparation was not provided, making interpretation of the obtained data difficult. In murine cell systems, anti-inflammatory activities of FHA have been studied in some detail and are summarized in the model proposed in Figure 6. Several types of immune cells were found to release IL-10 in response to soluble FHA [63,64] . At the same time, production of IL-12 by mouse macrophages and immature bone marrow dendritic cells in response to Escherichia coli LPS/IFN-γ was found to be significantly inhibited upon pre-incubation with soluble FHA. This suppressive effect seems to be due to receptor ligation by FHA, yielding induction of IL-10 that would downregulate IL-12 production [63,64] . These observations led to the suggestion that FHA may be capable of subverting immunity in the lungs during B. pertussis infection. Further research indicated that in the mouse model of B. pertussis infection, the exposure to FHA drives dendritic cells into a distinct subtype that induces pathogen-specific T regulatory (Tr1) cells expansion [63] . This led to the hypothesis that suppression of local Th1 responses during acute infection with B. pertussis could be due to FHA-dependent induction of Tr1 cells. The net effect of FHA would, hence, consist in inhibition of induction, activation and recruitment of Th1 cells, thus representing an immune subversion strategy employed by B. pertussis toward enabling of prolonged survival and


1351


Th1 Th1

IL-10, TGF-β IL-10, TGF-β

Bordetella Bordetella pertussis pertussis Phagocytosis Phagocytosis

Th17 Th17

Monocyte-

IL-10 IL-10 andandothers others

Treg Treg

Monocytederived derived cell

cell

CR3

CR3

Free soluble FHA FHA Free soluble

Monocytederived cell Transendothelial Transendothelial migration of migration of neutrophils

CR3 CR3

Surface-bound FHA

Surface-bound FHA

ICAM-1

ICAM-1

neutrophils

Bordetella pertussis Bordetella pertussis

Epithelial cell Epithelial cell

Figure 6. Immunomodulatory effects of filamentous hemagglutinin on airway mucosa. Binding of FHA via the RGD motif to LRI/IAP of macrophages would result in crosslinking of the receptors and subsequent increase in the binding activity of CR3, promoting the uptake of the bacteria [41,133]. Binding of FHA to the surface of human respiratory epithelial cells via the heparin-binding domain would be followed by interaction of the RGD sequence of FHA with VLA-5, resulting in signaling promoting internalization of bacteria into epithelial cells [42]. Finally, binding of free-soluble FHA to CR3 and/or other receptors on the surface of macrophages and dendritic cells would trigger signaling cascades leading to IL-10 secretion and expansion of Tregs and suppression of Th1- and Th17-polarized immune responses by secreted IL-10 and TGF-β [63,64,154]. FHA: Filamentous hemagglutinin.

persistence within the host. In support of this hypothesis, parenteral administration of FHA has been shown to reduce intestinal inflammation and other symptoms in a mouse model of T-cell-mediated colitis [60] . Treatment with FHA was associated with a reduction in proinflammatory Th1 cytokines and with a concomitant increase in anti-inflammatory cytokine production by innate cells and T cells, such as IL-10 and TGF-β. However, the FHA-induced protection against colitis did not appear to be dependent on IL-10 production by T cells [60] . Here again a description of how and to which extent the used antigen was purified (i.e., LOS levels) is missing in the study and use of a control, such as use of an FHA mutant, would have very much improved the robustness of the data. Nevertheless, in contrast to treatment with a one component vaccine containing only detoxified PT, another study showed that administration of a three-component FHA-containing acellular

1352


pertussis vaccine also increased the production of IL-10, induced apoptosis of activated Th1 cells and attenuated colitis in a mouse model of inflammatory bowel disease resembling ulcerative colitis [65] . FHA alone was further found to induce apoptosis of activated T lymphocytes in vivo and in vitro and Th1 cells appeared to be particularly sensitive to such FHA-induced apoptosis [65] . However, treatment with FHA alone, without any adjuvant did not abrogate colitis development. It remains, hence, possible that if FHA had been treated with formaldehyde and/or formulated with alum, as in the case of the three-component B. pertussis vaccine, it might have been more efficient in exerting an immunomodulatory activity in vivo. The contribution of FHA to B. pertussis virulence in vivo remains somewhat controversial, as most studies did not reveal any difference between virulence and colonization properties of wild-type and FHA-deficient B. pertussis bacteria


Filamentous hemagglutinin of Bordetella pertussis in the mouse model of infection [101,123,124,155– 157] . The lack of a clear phenotype for fhaB mutants in most of the studies may, hence, be due to the fact that mice are not a natural host for B. pertussis. Keeping in mind that B. pertussis produces pertussis toxin and presumably does not secrete the T3SS effectors during human infection, and hence may colonize its human host in a different mode than B. bronchiseptica does colonize its natural animal hosts, it is still of interest to indicate the results obtained for B. bronchiseptica FHA. The B. bronchiseptica bacteria persistently colonize both the nasal cavity and the trachea of rats and mice, whereas FHA-deficient B. bronchiseptica mutants are only able to colonize the nasal cavity, and often with decreased efficiency, but do not colonize lower airways such as trachea [43,158–159] . FHA was found to play a role in B. bronchiseptica colonization of the rat respiratory tract and to modulate the inflammatory response in mouse lungs [43] . FHA further appears to function by modulating the robustness of the inflammatory response. In the absence of functional FHA, a bimodal response to B. bronchiseptica challenge was observed, where a hyper-inflammatory response can develop, allowing rapid clearance of the bacteria, or alternatively, a local tissue damage can also occur that in turn promotes increased growth of FHA-deficient B. bronchiseptica bacteria, resulting in more inflammation, more damage and ultimate death of the animals [43] . The robust inflammatory response to FHA-deficient B. bronchiseptica is characterized by an early and sustained influx of IL-17-positive neutrophils and macrophages and later on by IL-17-positive CD4 + T cells. This suggests that FHA enables the bacteria to suppress recruitment of neutrophils and the development of an IL-17-mediated inflammatory response [62] . However, since FHA mediates attachment to cells and appears to form complexes with other virulence factors such as ACT and PRN [114] , it is also possible that attachment through FHA facilitates delivery of potent immunomodulatory toxins, such as PT or ACT, into cells. The phenotypes observed with FHA-deficient mutants may, hence, also reflect impaired delivery of these toxins, rather than a specific immunomodulatory role of FHA itself. Surprisingly, while several in vitro studies pointed to a role of the RGD motif of FHA in binding to target cells, upregulation of ICAM-1 and invasion of epithelial cells [41,42,109,127,133,141] , this motive was not found to play any role in the


Review

ability of B. bronchiseptica to cause respiratory infection in rats and mice, in induction or suppression of an inflammatory response in mice or in resistance of B. bronchiseptica to inflammation-mediated clearance in mice [43] . In turn, it was suggested that it is the C-terminal domain of mature FHA which has to be properly folded in order to allow adherence to epithelial and macrophage-like cells, upregulation of ICAM-1 expression in epithelial cells and modulation of the inflammatory response in mouse lungs [43] . Since, however, the B. pertussis and B. bronchiseptica species use different hosts, colonizing their respiratory tract in a different manner and causing different pathologies, it is difficult to compare the results obtained in mice in vivo and on human cells in vitro, where in the latter case a role of the RGD motif was reported [41,42,109,127,133,141] . It remains hence important to corroborate the analysis on a molecular level and reveal which of the described binding domains of FHA is involved in the attachment and other possible activities of B. pertussis FHA. Finally, FHA has been shown to possess an adjuvant activity [160] . Coadministration of FHA results in increased antigen-specific immunoglobulin titers against unrelated antigens delivered via intranasal, oral or subcutaneous route to mice. This indicates that the adjuvant effect is not restricted to binding to mucosal surfaces. This adjuvant effect does not seem to be due to crosslinking or direct interaction between FHA and the antigens tested. The domains implicated in this mechanism remain, however, unknown. Conclusion FHA was reported to harbor at least three domains that interact with ligands on host cells: a domain recognizing heparan sulfatecontaining glycosamynoglycans and potentially also proteoglycans on host cells; a CRD responsible for binding of FHA to glycolipids of human cilia and galactose-containing glycoconjugates; and a domain harboring an RGD motif that appears to mediate binding of FHA to at least two different cellular integrin receptors, the αvβ3 /IAP complex and the α5β1 integrin, the latter mediating bacterial invasion into epithelial and macrophage cells. Recent results indicate that FHA may have a fourth, previously unrecognized functional domain involved in receptor binding and signaling activities of the protein, the so-called mature C-terminal domain.


1353

Review Romero, Osicka & Sebo The massive amounts of processed approximately 220 kDa form of FHA that are secreted and shed by the bacteria under in vitro culture conditions remain perplexing. The vigorous antibody response in natural B. pertussis infections, however, indicates a massive FHA production also in vivo. Only speculations about the function of the released FHA form are available. The approximately 130 kDa large and rapidly degraded C-terminal prodomain portion of the FHA precursor is cleaved-off during FHA release. It remains cell associated and contains another RGD motif, which might eventually also participate in interaction of the precursor with integrins of target cells. It appears plausible that it is the unprocessed and cell-associated approximately 370 kDa form of FHA that truly mediates adherence of bacteria to eukaryotic cells under in vivo infection conditions. It also remains unclear whether cellassociated FHA forms complexes with other bacterial components that may be determining the binding properties of FHA. The host cell signaling pathways triggered by bacterial surface-associated FHA in the context of bacterial infection and by purified soluble FHA molecules may differ substantially. Besides serving in regulation of homotypic bacterial cell–cell interactions and biofilm formation, the shed FHA has a plausible potential to serve as an immunosuppressive modulin, a sort of protein toxin as well as a massively released decoy target of host antibodies. Several studies indicated that FHA may exert anti-inflammatory and immunosuppressive signaling activities through provoking IL-10 secretion by host monocytes, at least in the mouse model of infection. However, strong pro-inflammatory signaling of FHA, yielding IFN-γ and IL-12 induction in circulating mononuclear cells from B. pertussis-infected or pertussis-vaccinated humans, has also been reported. FHA interaction with primary human monocytic cells was then shown to trigger alteration of expression of hundreds of genes. The immunosignaling patterns of FHA appear, hence, to be rather complex, and may depend on the presence of other bacterial components. The authors of this review think that the above quoted results should be confirmed in experiments employing FHA preparations whose purity may raise fewer concerns about the effect of potential contaminants and containing controls that can provide strong evidence of the specificity of the effects

1354


observed and establishing the FHA role in the observed signaling in a more robust manner. Many basic questions on the structure and receptor interactions, glycan-binding mechanisms and immunomodulatory activities of FHA still remain to be addressed. Several concepts about FHA activity that are considered as previously established would to our opinion need to be reassessed using contemporary technologies and investigated further in detail. It has been established that FHA acts as a major adhesin of B. pertussis to cultured cells in vitro, mediating binding to both human ciliated respiratory epithelial cells and macrophages and enabling bacterial invasion of both cell types. Given the limitations imposed by in vitro studies and observing that B. pertussis is a human pathogen and mutations in fhaB gene often affect fimbriae expression, many of the earlier studies need to be interpreted with caution. The authors of this review also believe that the reported interaction of the different binding domains of FHA with the proposed cellular receptors and the nature thereof, remain to be proven in biochemical and molecular detail. It should be systematically reevaluated, because many results were obtained using rather indirect methods and a direct binding between FHA and its proposed integrin receptors has not been conclusively documented yet. In addition, contradictory and conflicting results are abundant in the available literature on FHA. Moreover, it is still unclear whether binding through FHA really mediates invasion of B. pertussis into target cells and whether this invasion process results in an intracellular reservoir of the bacteria enabling persistent infections in humans. It remains further unclear whether FHA is absolutely required for colonization and persistent infection of humans by B. pertussis. The role of B. pertussis FHA during in vivo infection, hence, urgently needs to be addressed experimentally in the recently developed baboon weanling model of human pertussis pathology. Future perspective Solving of the tertiary structure of the MCD and structural studies on the entire FHA molecule employing high-resolution cryo-electron microscopy are both likely to importantly advance our understanding of the structure–function relationships of the mature FHA molecule. As outlined above, a formal demonstration of the invoked direct physical-binding interactions of the mature FHA molecule with its several


Filamentous hemagglutinin of Bordetella pertussis proposed integrin receptors remains outstanding. Specific crosslinking of FHA to, or coimmunoprecipitation with, any of the potential integrin receptors appears, indeed, rather challenging in the light of the simultaneous presence of multiple cellular structures recognized by FHA. In particular, sulfated glucose aminoglycans recognized by the HBD, the glycans recognized by the CRD and some or even all of the three known integrin complexes possibly engaged by the RGD and MCD domains of FHA, may all be simultaneously present on the same primary monocyte-derived cell. Deciphering of the individual contributions of potentially weak interactions with integrin receptors to the overall binding and signaling activities of FHA represents a technical challenge. Its

Review

tackling by crosslinking and immunoprecipitation of FHA–receptor complexes and followed by high-resolution top-down MS analysis, complemented by site-directed mutagenesis and functional assays, will be required in order to sort out the individual versus multiple signaling interactions of FHA. Such knowledge, however, will be the key to deciphering the signaling mechanisms that underlie the reported immunomodulatory activity of this sophisticated adhesin. Important progress has recently been achieved in analyzing the biological activities of FHA of B. bronchiseptica in the permissive mice and rat models of infection. The use of the recently developed baboon weanling model, which reproduces well the human whooping cough pathophysiology, will now also enable a significantly

Executive summary Structure & biogenesis ●●

The fhaB gene codes for an approximately 367 kDa FhaB protein.

●●

This is exported across the cytoplasmic and outer bacterial membranes by a two-partner secretion system.

●●

Folding of β-helix stacks of FhaB occurs on bacterial cell surface.

●●

Processing of FhaB results in shedding of an approximately 220 kDa mature filamentous hemagglutinin (FHA) form into extracellular milieu.

Adhesin activities ●●

FhaB as well as mature FHA support in vitro adherence of Bordetella pertussis to a variety of nonciliated epithelial and phagocytic cells.

●●

Four different domains within the FHA molecule were implicated in interaction of FHA with eukaryotic cells in vitro. Their respective roles in B. pertussis pathogenesis in vivo remain, however, controversial.

Immunomodulatory properties ●●

FHA was found to act as a potent pro-inflammatory stimulus on human cells.

●●

In the mouse model, FHA was reported to trigger pathogen-specific T regulatory (Tr1) cell expansion and to inhibit induction, activation and recruitment of Th1 and Th17 cells.

●●

Contradictory results obtained with murine and human cells call for re-examination of the immunomodulatory

properties of FHA. Specific emphasis needs to be placed on purity of the used FHA preparations and on use of robust controls in future experiments. Conclusion & future perspective ●●

Direct physical interaction of mature FHA with the proposed integrin receptors remains to be demonstrated.

●●

Cross-linking with receptors and immunoprecipitation, followed by high-resolution top-down MS protein analytics, will need to be combined with site-directed mutagenesis, in order to finally map the functional binding domains of FHA.

●●

It remains unclear what are the respective biological roles of the surface-associated and of the soluble shed FHA.

●●

It remains to be established if FHA is required for colonization and persistent infection of humans by B. pertussis. This

can now be tested in the olive baboon weanling model that reproduces well the pathophysiology of human whooping cough illness.



1355

Review Romero, Osicka & Sebo closer-to-biological-reality analysis and verification of the immunosubversive role of B. pertussis FHA in human infection. Financial & competing interests disclosure Preparation of this review was supported by grant numbers P302/11/0580 (R Osicka) and GA13-14547S (P Sebo) of the Czech Science Foundation, Crucell Holland BV

References 1

2

3

(R Villarino) and the institutional support RVO61388971. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

12 Misegades LK, Winter K, Harriman K et al.

Mattoo S, Cherry JD. Molecular patho genesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev. 18(2), 326–382 (2005). Hodder SL, Cherry JD, Mortimer EA Jr, Ford AB, Gornbein J, Papp K. Antibody responses to Bordetella pertussis antigens and clinical correlations in elderly community residents. Clin. Infect. Dis. 31(1), 7–14 (2000). Cherry JD. The role of Bordetella pertussis infections in adults in the epidemiology of pertussis. Dev. Biol. Stand. 89, 181–186 (1997).

Association of childhood pertussis with receipt of 5 doses of pertussis vaccine by time since last vaccine dose, California, 2010. JAMA 308(20), 2126–2132 (2012). 13 Tartof SY, Lewis M, Kenyon C et al. Waning

immunity to pertussis following 5 doses of DTaP. Pediatrics 131(4), e1047–e1052 (2013). 14 Witt MA, Arias L, Katz PH, Truong ET,

Witt DJ. Reduced risk of pertussis among persons ever vaccinated with whole cell pertussis vaccine compared with recipients of acellular pertussis vaccines in a large US cohort. Clin. Infect. Dis. 56(9), 1248–1254 (2013). 15 Witt MA, Katz PH, Witt DJ. Unexpectedly

limited durability of immunity following acellular pertussis vaccination in pre adolescents in a North American outbreak. Clin. Infect. Dis. 54(12), 1730–1735 (2012).

Cherry JD. Pertussis in the preantibiotic and prevaccine era, with emphasis on adult pertussis. Clin. Infect. Dis. 28(Suppl. 2), S107–S111 (1999).

16 Cherry JD. Why do pertussis vaccines

5

Cherry JD. Epidemiology of pertussis. Pediatr. Infect. Dis. J. 25(4), 361–362 (2006).

17 Cherry JD, Olin P. The science and fiction of

6

Pittman M. History of the development of pertussis vaccine. Dev. Biol. Stand. 73, 13–29 (1991).

4

7

8

9

Sato Y, Sato H. Development of acellular pertussis vaccines. Biologicals 27(2), 61–69 (1999). Crowcroft NS, Stein C, Duclos P, Birmingham M. How best to estimate the global burden of pertussis? Lancet Infect. Dis. 3(7), 413–418 (2003). Cherry JD. The present and future control of pertussis. Clin. Infect. Dis. 51(6), 663–667 (2010).

10 Cherry JD. Pertussis: challenges today and for

the future. PLoS Pathog. 9(7), e1003418 (2013). 11 Baraff LJ, Manclark CR, Cherry JD,

Christenson P, Marcy SM. Analyses of adverse reactions to diphtheria and tetanus toxoids and pertussis vaccine by vaccine lot, endotoxin content, pertussis vaccine potency and percentage of mouse weight gain. Pediatr. Infect. Dis. J. 8(8), 502–507 (1989).

1356

fail? Pediatrics 129(5), 968–970 (2012). pertussis vaccines. Pediatrics 104(6), 1381–1383 (1999). 18 Warfel JM, Zimmerman LI, Merkel TJ.

Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc. Natl Acad. Sci. USA 111(2), 787–792 (2014). 19 Australian Government Department of

Health,National Notifiable Diseases Surveillance System: number of notifications of Pertussis in Australia in the period of 1991 to 2014. www9.health.gov.au/cda/source/rpt_3.cfm 20 Reported pertussis cases to the National

Notifiable Disease Surveillance System of CDC of the USA from 1922 to 2013. www.cdc.gov/pertussis/images/incidence 21 Octavia S, Sintchenko V, Gilbert GL et al.

Newly emerging clones of Bordetella pertussis carrying prn2 and ptxP3 alleles implicated in Australian pertussis epidemic in 2008–2010. J. Infect. Dis. 205(8), 1220–1224 (2012).


22 Winter K, Harriman K, Zipprich J et al.

California pertussis epidemic, 2010. J. Pediatr. 161(6), 1091–1096 (2012). 23 Melvin JA, Scheller EV, Miller JF, Cotter PA.

Bordetella pertussis pathogenesis: current and future challenges. Nature Rev. Microbiol. 12(4), 274–288 (2014). 24 Shrivastava R, Miller JF. Virulence factor

secretion and translocation by Bordetella species. Curr. Opin. Microbiol. 12(1), 88–93 (2009). 25 Carbonetti NH. Pertussis toxin and adenylate

cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiol. 5(3), 455–469 (2010). 26 Locht C, Coutte L, Mielcarek N. The ins and

outs of pertussis toxin. FEBS J. 278(23), 4668–4682 (2011). 27 Vojtova J, Kamanova J, Sebo P. Bordetella

adenylate cyclase toxin: a swift saboteur of host defense. Curr. Opin. Microbiol. 9(1), 69–75 (2006). 28 Fennelly NK, Sisti F, Higgins SC et al.

Bordetella pertussis expresses a functional type III secretion system that subverts protective innate and adaptive immune responses. Infect. Immun. 76(3), 1257–1266 (2008). 29 Gaillard ME, Bottero D, Castuma CE,

Basile LA, Hozbor D. Laboratory adaptation of Bordetella pertussis is associated with the loss of type three secretion system functionality. Infect. Immun. 79(9), 3677–3682 (2011). 30 Han Hj, Kuwae A, Abe A, Arakawa Y,

Kamachi K. Differential expression of type III effector BteA protein due to IS481 insertion in Bordetella pertussis. PLoS ONE 6(3), e17797 (2011). 31 Hegerle N, Rayat L, Dore G, Zidane N,

Bedouelle H, Guiso N. In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Microbes Infect. 15(5), 399–408 (2013). 32 Villarino Romero R, Bibova I, Cerny O et al.

The Bordetella pertussis type III secretion


Filamentous hemagglutinin of Bordetella pertussis system tip complex protein Bsp22 is not a protective antigen and fails to elicit serum antibody responses during infection of humans and mice. Infect. Immun. 81(8), 2761–2767 (2013). 33 Marr N, Shah NR, Lee R, Kim EJ,

Fernandez RC. Bordetella pertussis autotransporter Vag8 binds human C1 esterase inhibitor and confers serum resistance. PLoS ONE 6(6), e20585 (2011). 34 Everest P, Li J, Douce G et al. Role of the

Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells. Microbiology 142(Pt 11), 3261–3268 (1996). 35 Geuijen CA, Willems RJ, Bongaerts M,

Top J, Gielen H, Mooi FR. Role of the Bordetella pertussis minor fimbrial subunit, FimD, in colonization of the mouse respiratory tract. Infect. Immun. 65(10), 4222–4228 (1997). 36 Geuijen CA, Willems RJ, Mooi FR. The

major fimbrial subunit of Bordetella pertussis binds to sulfated sugars. Infect. Immun. 64(7), 2657–2665 (1996). 37 Hannah JH, Menozzi FD, Renauld G,

Locht C, Brennan Mj. Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA. Infect. Immun. 62(11), 5010–5019 (1994). 38 Hazenbos WL, Geuijen CA,

Van Den Berg BM, Mooi FR, Van Furth R. Bordetella pertussis fimbriae bind to human monocytes via the minor fimbrial subunit FimD. J. Infect. Dis. 171(4), 924–929 (1995). 39 Hazenbos WL, Van Den Berg BM,

Van’t Wout JW, Mooi FR, Van Furth R. Virulence factors determine attachment and ingestion of nonopsonized and opsonized Bordetella pertussis by human monocytes. Infect. Immun. 62(11), 4818–4824 (1994). 40 Charles IG, Dougan G, Pickard D et al.

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc. Natl Acad. Sci. USA 86(10), 3554–3558 (1989). 41 Ishibashi Y, Claus S, Relman DA. Bordetella

pertussis filamentous hemagglutinin interacts with a leukocyte signal transduction complex and stimulates bacterial adherence to monocyte CR3 (CD11b/CD18). J. Exp. Med. 180(4), 1225–1233 (1994). 42 Ishibashi Y, Relman DA, Nishikawa A.

Invasion of human respiratory epithelial cells by Bordetella pertussis: possible role for a filamentous hemagglutinin Arg-Gly-Asp


sequence and alpha5beta1 integrin. Microb. Pathog. 30(5), 279–288 (2001). 43 Julio SM, Inatsuka CS, Mazar J, Dieterich C,

Relman DA, Cotter PA. Natural-host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C-terminal domain, but not the RGD motif, during infection. Mol. Microbiol. 71(6), 1574–1590 (2009). 44 Leininger E, Ewanowich CA, Bhargava A,

Peppler MS, Kenimer JG, Brennan MJ. Comparative roles of the Arg-Gly-Asp sequence present in the Bordetella pertussis adhesins pertactin and filamentous hemagglutinin. Infect. Immun. 60(6), 2380–2385 (1992). 45 Mazar J, Cotter PA. Topology and maturation

of filamentous haemagglutinin suggest a new model for two-partner secretion. Mol. Microbiol. 62(3), 641–654 (2006). 46 Prasad SM, Yin Y, Rodzinski E,

Tuomanen EI, Masure HR. Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis. Infect. Immun. 61(7), 2780–2785 (1993). 47 Scarlato V, Arico B, Prugnola A, Rappuoli R.

Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. EMBO J. 10(12), 3971–3975 (1991). 48 Hafler JP, Pohl-Koppe A. The cellular

immune response to Bordetella pertussis in two children with whooping cough. Eur. J. Med Res. 3(11), 523–526 (1998). 49 Cherry JD, Gornbein J, Heininger U,

Stehr K. A search for serologic correlates of immunity to Bordetella pertussis cough illnesses. Vaccine 16(20), 1901–1906 (1998). 50 Mascart F, Verscheure V, Malfroot A et al.

Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses. J. Immunol. 170(3), 1504–1509 (2003). 51 Mills KH. Immunity to Bordetella pertussis.

Microbes Infect. 3(8), 655–677 (2001). 52 Novotny P, Chubb AP, Cownley K,

Charles IG. Biologic and protective properties of the 69-kDa outer membrane protein of Bordetella pertussis: a novel formulation for an acellular pertussis vaccine. J. Infect. Dis. 164(1), 114–122 (1991). 53 Olin P, Rasmussen F, Gustafsson L,

Hallander HO, Heijbel H. Randomised controlled trial of two-component, threecomponent, and five-component acellular pertussis vaccines compared with whole-cell pertussis vaccine. Ad Hoc Group for the

Review

Study of Pertussis Vaccines. Lancet 350(9091), 1569–1577 (1997). 54 Ryan M, Murphy G, Gothefors L, Nilsson L,

Storsaeter J, Mills KH. Bordetella pertussis respiratory infection in children is associated with preferential activation of type 1 T helper cells. J. Infect. Dis. 175(5), 1246–1250 (1997). 55 Storsaeter J, Hallander HO, Gustafsson L,

Olin P. Levels of anti-pertussis antibodies related to protection after household exposure to Bordetella pertussis. Vaccine 16(20), 1907–1916 (1998). 56 Locht C, Bertin P, Menozzi FD, Renauld G.

The filamentous haemagglutinin, a multifaceted adhesion produced by virulent Bordetella spp. Mol. Microbiol. 9(4), 653–660 (1993). 57 Weiss AA, Hewlett EL. Virulence factors of

Bordetella pertussis. Annu. Rev. Microbiol. 40, 661–686 (1986). 58 Abramson T, Kedem H, Relman DA.

Proinflammatory and proapoptotic activities associated with Bordetella pertussis filamentous hemagglutinin. Infect. Immun. 69(4), 2650–2658 (2001). 59 Abramson T, Kedem H, Relman DA.

Modulation of the NF-kappaB pathway by Bordetella pertussis filamentous hemagglutinin. PLoS ONE 3(11), e3825 (2008). 60 Braat H, Mcguirk P, Ten Kate FJ et al.

Prevention of experimental colitis by parenteral administration of a pathogenderived immunomodulatory molecule. Gut 56(3), 351–357 (2007). 61 Dieterich C, Relman DA. Modulation of the

host interferon response and ISGylation pathway by B. pertussis filamentous hemagglutinin. PLoS ONE 6(11), e27535 (2011). 62 Henderson MW, Inatsuka CS, Sheets AJ et al.

Contribution of Bordetella filamentous hemagglutinin and adenylate cyclase toxin to suppression and evasion of interleukin-17mediated inflammation. Infect. Immun. 80(6), 2061–2075 (2012). 63 Mcguirk P, McCann C, Mills KH. Pathogen-

specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195(2), 221–231 (2002). 64 Mcguirk P, Mills KH. Direct anti-

inflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous hemagglutinin


1357

Review Romero, Osicka & Sebo from Bordetella pertussis. Eur. J. Immunol. 30(2), 415–422 (2000). 65 Ohman L, Willen R, Hultgren OH,

Hultgren Hornquist E. Acellular Bordetella pertussis vaccine enhances mucosal interleukin-10 production, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2-deficient mice. Clin. Exp. Immunol. 141(1), 37–46 (2005). 66 Munoz JJ, Arai H, Cole RL. Mouse-

protecting and histamine-sensitizing activities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis. Infect. Immun. 32(1), 243–250 (1981). 67 Olin P, Hallander HO, Gustafsson L,

Reizenstein E, Storsaeter J. How to make sense of pertussis immunogenicity data. Clin. Infect. Dis. 33(Suppl. 4), S288–S291 (2001). 68 Watanabe M, Nakase Y. Mutant of Bordetella

pertussis which lacks ability to produce filamentous hemagglutinin. Infect. Immun. 35(3), 1018–1023 (1982). 69 Locht C, Geoffroy MC, Renauld G.

Common accessory genes for the Bordetella pertussis filamentous hemagglutinin and fimbriae share sequence similarities with the papC and papD gene families. EMBO J. 11(9), 3175–3183 (1992). 70 Willems RJ, Geuijen C, Van Der Heide HG

et al. Mutational analysis of the Bordetella pertussis fim/fha gene cluster: identification of a gene with sequence similarities to haemolysin accessory genes involved in export of FHA. Mol. Microbiol. 11(2), 337–347 (1994). 71 Willems RJ, Van Der Heide HG, Mooi FR.

Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene. Mol. Microbiol. 6(18), 2661–2671 (1992). 72 Willems RJ, Geuijen C, Van Der Heide HG

et al. Isolation of a putative fimbrial adhesin from Bordetella pertussis and the identification of its gene. Mol. Microbiol. 9(3), 623–634 (1993). 73 Clantin B, Hodak H, Willery E, Locht C,

Jacob-Dubuisson F, Villeret V. The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway. Proc. Natl Acad. Sci. USA 101(16), 6194–6199 (2004). 74 Jacob-Dubuisson F, El-Hamel C, Saint N

et al. Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin. J. Biol. Chem. 274(53), 37731–37735 (1999).

1358

75 Jacob-Dubuisson F, Locht C, Antoine R.

Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins. Mol. Microbiol. 40(2), 306–313 (2001). 76 Stibitz S. Mutations in the bvgA gene of

Bordetella pertussis that differentially affect regulation of virulence determinants. J. Bacteriol. 176(18), 5615–5621 (1994). 77 Stibitz S, Aaronson W, Monack D, Falkow S.

Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system. Nature 338(6212), 266–269 (1989). 78 Williams CL, Boucher PE, Stibitz S,

Cotter PA. BvgA functions as both an activator and a repressor to control Bvg phase expression of bipA in Bordetella pertussis. Mol. Microbiol. 56(1), 175–188 (2005). 79 Veal-Carr WL, Stibitz S. Demonstration of

86 Jacob-Dubuisson F, Buisine C, Mielcarek N,

Clement E, Menozzi FD, Locht C. Aminoterminal maturation of the Bordetella pertussis filamentous haemagglutinin. Mol. Microbiol. 19(1), 65–78 (1996). 87 Clantin B, Delattre As, Rucktooa P et al.

Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily. Science 317(5840), 957–961 (2007). 88 Chevalier N, Moser M, Koch Hg et al.

Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide. J. Mol. Microbiol. Biotechnol. 8(1), 7–18 (2004). 89 Lambert-Buisine C, Willery E, Locht C,

Jacob-Dubuisson F. N-terminal characterization of the Bordetella pertussis filamentous haemagglutinin. Mol. Microbiol. 28(6), 1283–1293 (1998).

differential virulence gene promoter activation in vivo in Bordetella pertussis using RIVET. Mol. Microbiol. 55(3), 788–798 (2005).

90 Von Heijne G. A new method for predicting

80 Delisse-Gathoye AM, Locht C, Jacob F et al.

Jacob-Dubuisson F. Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin. Mol. Microbiol. 29(3), 763–774 (1998).

Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis. Infect. Immun. 58(9), 2895–2905 (1990). 81 Domenighini M, Relman D, Capiau C et al.

Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor. Mol. Microbiol. 4(5), 787–800 (1990). 82 Relman DA, Domenighini M, Tuomanen E,

Rappuoli R, Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc. Natl Acad. Sci. USA 86(8), 2637–2641 (1989). 83 Watanabe H, Kanesaki S, Shukuda Y,

Mayahara H. The location of filamentous hemagglutinin and pertussis toxin antigens of Bordetella pertussis by immunoelectron microscopy. J. Electron. Microsc. 38(3), 172–181 (1989). 84 Coutte L, Antoine R, Drobecq H, Locht C,

Jacob-Dubuisson F. Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway. EMBO J. 20(18), 5040–5048 (2001). 85 Noel CR, Mazar J, Melvin JA, Sexton JA,

Cotter PA. The prodomain of the Bordetella two-partner secretion pathway protein FhaB remains intracellular yet affects the conformation of the mature C-terminal domain. Mol. Microbiol. 86(4), 988–1006 (2012).


signal sequence cleavage sites. Nucleic Acids Res. 14(11), 4683–4690 (1986). 91 Guedin S, Willery E, Locht C,

92 Renauld-Mongenie G, Cornette J,

Mielcarek N, Menozzi FD, Locht C. Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin. J. Bacteriol. 178(4), 1053–1060 (1996). 93 Coutte L, Willery E, Antoine R, Drobecq H,

Locht C, Jacob-Dubuisson F. Surface anchoring of bacterial subtilisin important for maturation function. Mol. Microbiol. 49(2), 529–539 (2003). 94 Coutte L, Alonso S, Reveneau N et al. Role of

adhesin release for mucosal colonization by a bacterial pathogen. J. Exp. Med. 197(6), 735–742 (2003). 95 Makhov AM, Hannah JH, Brennan Mj et al.

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns. J. Mol. Biol. 241(1), 110–124 (1994). 96 Kajava AV, Cheng N, Cleaver R et al.

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins. Mol. Microbiol. 42(2), 279–292 (2001). 97 Hashimoto C, Hudson KL, Anderson KV.

The Toll gene of Drosophila, required for


Filamentous hemagglutinin of Bordetella pertussis dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 52(2), 269–279 (1988). 98 Kobe B, Deisenhofer J. Crystal structure of

porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 366(6457), 751–756 (1993). 99 Lopez JA, Chung DW, Fujikawa K,

Hagen FS, Davie EW, Roth GJ. The alpha and beta chains of human platelet glyco protein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc. Natl Acad. Sci. USA 85(7), 2135–2139 (1988). 100 Reinke R, Krantz DE, Yen D, Zipursky SL.

Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell 52(2), 291–301 (1988). 101 Kimura A, Mountzouros KT, Relman DA,

Falkow S, Cowell JL. Bordetella pertussis filamentous hemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory infection model. Infect. Immun. 58(1), 7–16 (1990). 102 Redhead K. An assay of Bordetella pertussis

adhesion to tissue-culture cells. J. Med. Microbiol. 19(1), 99–108 (1985). 103 Sato Y, Izumiya K, Sato H, Cowell JL,

Manclark CR. Role of antibody to leukocytosis-promoting factor hemagglutinin and to filamentous hemagglutinin in immunity to pertussis. Infect. Immun. 31(3), 1223–1231 (1981). 104 Tuomanen E, Towbin H, Rosenfelder G et al.

Receptor analogs and monoclonal antibodies that inhibit adherence of Bordetella pertussis to human ciliated respiratory epithelial cells. J. Exp. Med. 168(1), 267–277 (1988). 105 Tuomanen E, Weiss A. Characterization of

two adhesins of Bordetella pertussis for human ciliated respiratory-epithelial cells. J. Infect. Dis. 152(1), 118–125 (1985). 106 Tuomanen E, Weiss A, Rich R, Zak F, Zak O.

Filamentous hemagglutinin and pertussis toxin promote adherence of Bordetella pertussis to cilia. Dev. Biol. Stand. 61, 197–204 (1985). 107 Urisu A, Cowell JL, Manclark CR.

Filamentous hemagglutinin has a major role in mediating adherence of Bordetella pertussis to human WiDr cells. Infect. Immun. 52(3), 695–701 (1986). 108 Gorringe AR, Ashworth LAE, Irons LI,

Robinson A. Effect of monoclonal-antibodies on the adherence of Bordetella-pertussis to vero cells. FEMS Microbiol. Lett. 26(1), 5–9 (1985).


109 Relman D, Tuomanen E, Falkow S,

Golenbock DT, Saukkonen K, Wright SD. Recognition of a bacterial adhesion by an integrin: macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 61(7), 1375–1382 (1990). 110 Mobberley-Schuman PS, Weiss AA. Influence

of CR3 (CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human neutrophils. Infect. Immun. 73(11), 7317–7323 (2005). 111 Van Den Berg BM, Beekhuizen H,

Willems RJ, Mooi FR, Van Furth R. Role of Bordetella pertussis virulence factors in adherence to epithelial cell lines derived from the human respiratory tract. Infect. Immun. 67(3), 1056–1062 (1999). 112 Arico B, Nuti S, Scarlato V, Rappuoli R.

Adhesion of Bordetella pertussis to eukaryotic cells requires a time-dependent export and maturation of filamentous hemagglutinin. Proc. Natl Acad. Sci. USA 90(19), 9204–9208 (1993). 113 Zaretzky FR, Gray MC, Hewlett EL.

Mechanism of association of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for toxin-filamentous haemagglutinin interaction. Mol. Microbiol. 45(6), 1589–1598 (2002). 114 Perez Vidakovics ML, Lamberti Y,

Van Der Pol WL, Yantorno O, Rodriguez ME. Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells. FEMS Immunol. Med. Microbiol. 48(1), 140–147 (2006). 115 Bassinet L, Gueirard P, Maitre B, Housset B,

Gounon P, Guiso N. Role of adhesins and toxins in invasion of human tracheal epithelial cells by Bordetella pertussis. Infect. Immun. 68(4), 1934–1941 (2000). 116 Ewanowich CA, Melton AR, Weiss AA,

Sherburne RK, Peppler MS. Invasion of HeLa 229 cells by virulent Bordetella pertussis. Infect. Immun. 57(9), 2698–2704 (1989). 117 Friedman RL, Nordensson K, Wilson L,

Akporiaye ET, Yocum DE. Uptake and intracellular survival of Bordetella pertussis in human macrophages. Infect. Immun. 60(11), 4578–4585 (1992). 118 Saukkonen K, Cabellos C, Burroughs M,

Prasad S, Tuomanen E. Integrin-mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization. J. Exp. Med. 173(5), 1143–1149 (1991). 119 Guermonprez P, Khelef N, Blouin E et al. The

adenylate cyclase toxin of Bordetella pertussis

Review

binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18). J. Exp. Med. 193(9), 1035–1044 (2001). 120 Lamberti Y, Gorgojo J, Massillo C,

Rodriguez ME. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival. Pathog. Dis. 69(3), 194–204 (2013). 121 Lamberti Y, Perez Vidakovics ML,

Van Der Pol LW, Rodriguez ME. Cholesterolrich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils. Microb. Pathog. 44(6), 501–511 (2008). 122 Lamberti YA, Hayes JA,

Perez Vidakovics ML, Harvill ET, Rodriguez ME. Intracellular trafficking of Bordetella pertussis in human macrophages. Infect. Immun. 78(3), 907–913 (2010). 123 Alonso S, Pethe K, Mielcarek N, Raze D,

Locht C. Role of ADP-ribosyltransferase activity of pertussis toxin in toxin-adhesin redundancy with filamentous hemagglutinin during Bordetella pertussis infection. Infect. Immun. 69(10), 6038–6043 (2001). 124 Khelef N, Bachelet CM, Vargaftig BB,

Guiso N. Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins. Infect. Immun. 62(7), 2893–2900 (1994). 125 Warfel JM, Beren J, Kelly VK, Lee G,

Merkel TJ. Nonhuman primate model of pertussis. Infect. Immun. 80(4), 1530–1536 (2012). 126 Warfel JM, Merkel TJ. Bordetella pertussis

infection induces a mucosal IL-17 response and long-lived Th17 and Th1 immune memory cells in nonhuman primates. Mucosal Immunol. 6(4), 787–796 (2013). 127 Ishibashi Y, Nishikawa A. Bordetella pertussis

infection of human respiratory epithelial cells up-regulates intercellular adhesion molecule-1 expression: role of filamentous hemagglutinin and pertussis toxin. Microb. Pathog. 33(3), 115–125 (2002). 128 Busby TF, Argraves WS, Brew SA, Pechik I,

Gilliland GL, Ingham KC. Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a cationic cradle. J. Biol. Chem. 270(31), 18558–18562 (1995). 129 Cardin AD, Demeter DA, Weintraub HJ,

Jackson RL. Molecular design and modeling of protein-heparin interactions. Methods Enzymol. 203, 556–583 (1991). 130 Lucena S, Arocha Pinango CL, Guerrero B.

Fibronectin. Structure and functions associated to hemostasis. Invest. Clin. 48(2), 249–262 (2007).


1359

Review Romero, Osicka & Sebo 131 Menozzi FD, Gantiez C, Locht C. Interaction

of the Bordetella pertussis filamentous hemagglutinin with heparin. FEMS Microbiol. Lett. 62(1), 59–64 (1991). 132 Menozzi FD, Mutombo R, Renauld G et al.

Heparin-inhibitable lectin activity of the filamentous hemagglutinin adhesin of Bordetella pertussis. Infect. Immun. 62(3), 769–778 (1994). 133 Ishibashi Y, Yoshimura K, Nishikawa A,

Claus S, Laudanna C, Relman DA. Role of phosphatidylinositol 3-kinase in the binding of Bordetella pertussis to human monocytes. Cell Microbiol. 4(12), 825–833 (2002). 134 Rozdzinski E, Spellerberg B, Van Der Flier M

et al. Peptide from a prokaryotic adhesin blocks leukocyte migration in vitro and in vivo. J. Infect. Dis. 172(3), 785–793 (1995). 135 Menozzi FD, Boucher PE, Riveau G,

Gantiez C, Locht C. Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis. Infect. Immun. 62(10), 4261–4269 (1994). 136 Berggard K, Johnsson E, Mooi FR,

Lindahl G. Bordetella pertussis binds the human complement regulator C4BP: role of filamentous hemagglutinin. Infect. Immun. 65(9), 3638–3643 (1997). 137 Berggard K, Lindahl G, Dahlback B,

Blom AM. Bordetella pertussis binds to human C4b-binding protein (C4BP) at a site similar to that used by the natural ligand C4b. Eur. J. Immunol. 31(9), 2771–2780 (2001). 138 Fernandez RC, Weiss AA. Serum resistance in

bvg-regulated mutants of Bordetella pertussis. FEMS Microbiol. Lett. 163(1), 57–63 (1998). 139 Berggard K, Johnsson E, Morfeldt E,

Persson J, Stalhammar-Carlemalm M, Lindahl G. Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes. Mol. Microbiol. 42(2), 539–551 (2001). 140 Carlsson F, Berggard K,

Stalhammar-Carlemalm M, Lindahl G. Evasion of phagocytosis through cooperation between two ligand-binding regions in Streptococcus pyogenes M protein. J. Exp. Med. 198(7), 1057–1068 (2003).

1360

141 Ishibashi Y, Nishikawa A. Role of nuclear

factor-kappa B in the regulation of intercellular adhesion molecule 1 after infection of human bronchial epithelial cells by Bordetella pertussis. Microb. Pathog. 35(4), 169–177 (2003). 142 Bloemen PG, Van Den Tweel MC,

Henricks PA et al. Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion. Am. J. Physiol. 270(1 Pt 1), L80–L87 (1996). 143 O’Brien AD, Standiford TJ, Bucknell KA,

Wilcoxen SE, Paine R 3rd. Role of alveolar epithelial cell intercellular adhesion molecule-1 in host defense against Klebsiella pneumoniae. Am. J. Physiol. 276(6 Pt 1), L961–L970 (1999). 144 Tosi MF, Stark JM, Smith CW, Hamedani A,

Gruenert DC, Infeld MD. Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion. Am. J. Respir. Cell Mol. Biol. 7(2), 214–221 (1992). 145 Cuschieri J, Gourlay D, Garcia I, Jelacic S,

Maier RV. Implications of proteasome inhibition: an enhanced macrophage phenotype. Cell Immunol. 227(2), 140–147 (2004). 146 Ward PA, Lentsch AB. Endogenous

regulation of the acute inflammatory response. Mol. Cell. Biochem. 234–235(1–2), 225–228 (2002). 147 Jana NR, Dikshit P, Goswami A, Nukina N.

Inhibition of proteasomal function by curcumin induces apoptosis through mitochondrial pathway. J. Biol. Chem. 279(12), 11680–11685 (2004). 148 D’cunha J, Knight E Jr, Haas AL, Truitt RL,

Borden EC. Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc. Natl Acad. Sci. USA 93(1), 211–215 (1996). 149 D’cunha J, Ramanujam S, Wagner RJ,

Witt PL, Knight E Jr, Borden EC. In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine. J. Immunol. 157(9), 4100–4108 (1996). 150 Giannakopoulos NV, Luo JK, Papov V et al.

Proteomic identification of proteins conjugated to ISG15 in mouse and human


cells. Biochem. Biophys. Res. 336(2), 496–506 (2005). 151 Recht M, Borden EC, Knight E Jr. A human

15-kDa IFN-induced protein induces the secretion of IFN-gamma. J. Immunol. 147(8), 2617–2623 (1991). 152 Decker T, Muller M, Stockinger S. The yin

and yang of type I interferon activity in bacterial infection. Nat. Rev. Immunol. 5(9), 675–687 (2005). 153 Dirix V, Verscheure V, Goetghebuer T et al.

Monocyte-derived interleukin-10 depresses the Bordetella pertussis- specific gamma interferon response in vaccinated infants. Clin. Vaccine Immunol. 16(12), 1816–1821 (2009). 154 Higgs R, Higgins SC, Ross PJ, Mills KH.

Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol. 5(5), 485–500 (2012). 155 Goodwin MS, Weiss AA. Adenylate cyclase

toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice. Infect. Immun. 58(10), 3445–3447 (1990). 156 Roberts M, Cropley I, Chatfield S,

Dougan G. Protection of mice against respiratory Bordetella pertussis infection by intranasal immunization with P.69 and FHA. Vaccine 11(8), 866–872 (1993). 157 Weiss AA, Goodwin MS. Lethal infection by

Bordetella pertussis mutants in the infant mouse model. Infect. Immun. 57(12), 3757–3764 (1989). 158 Cotter PA, Yuk MH, Mattoo S et al.

Filamentous hemagglutinin of Bordetella bronchiseptica is required for efficient establishment of tracheal colonization. Infect. Immun. 66(12), 5921–5929 (1998). 159 Mattoo S, Miller JF, Cotter PA. Role of

Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response. Infect. Immun. 68(4), 2024–2033 (2000). 160 Poulain-Godefroy O, Menozzi FD, Alonso S

et al. Adjuvant activity of free Bordetella pertussis filamentous haemagglutinin delivered by mucosal routes. Scand. J. Immunol. 58(5), 503–510 (2003).


Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain.

Mucosal immunization with filamentous hemagglutinin protects against Bordetella pertussis respiratory infection.

Expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli using a two cistron system.

Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses.

Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis.

Direct expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli and Salmonella typhimurium aroA.

Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction.

Bordetella pertussis filamentous hemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory infection model.

Immunoglobulin A antibodies to pertussis toxin and filamentous hemagglutinin in saliva from patients with pertussis.

Fimbrial hemagglutinin in stationary and shake cultures of Bordetella pertussis.

Purification and crystallization of fimbrial hemagglutinin from Bordetella pertussis.

Comparative roles of the Arg-Gly-Asp sequence present in the Bordetella pertussis adhesins pertactin and filamentous hemagglutinin.

The longevity of the immune response to filamentous hemagglutinin and pertussis toxin in patients with pertussis in a semiclosed community.

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

New Insight into Filamentous Hemagglutinin Secretion Reveals a Role for Full-Length FhaB in Bordetella Virulence.

Common accessory genes for the Bordetella pertussis filamentous hemagglutinin and fimbriae share sequence similarities with the papC and papD gene families.

CD18) binds filamentous hemagglutinin of Bordetella pertussis.

Murine antibody response to oral infection with live aroA recombinant Salmonella dublin vaccine strains expressing filamentous hemagglutinin antigen from Bordetella pertussis.

Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis.

Conversion of Bordetella pertussis to Bordetella parapertussis.

Cooperative roles for fimbria and filamentous hemagglutinin in Bordetella adherence and immune modulation.

Is Bordetella pertussis clonal?

Is Bordetella pertussis clonal?