The Virulence Factors of Bordetella Pertussis

FEMS Microbiology Reviews 25 (2001) 309^333
www.fems-microbiology.org
The virulence factors of Bordetella pertussis: a matter of control

Adam M. Smith
b
a;1
, Carlos A. Guzman b , Mark J. Walker
a;
a
Department of Biological Sciences, University of Wollongong, Wollongong, N.S.W. 2522, Australia
Department of Microbial Pathogenicity and Vaccine Research, Division of Microbiology, GBF-National Research Centre for Biotechnology,
Braunschweig, Germany
Received 3 April 2000; received in revised form 8 November 2000 ; accepted 19 December 2000
Abstract
Bordetella pertussis is the causative agent of whooping cough, a contagious childhood respiratory disease. Increasing public concern over
the safety of whole-cell vaccines led to decreased immunisation rates and a subsequent increase in the incidence of the disease. Research into
the development of safer, more efficacious, less reactogenic vaccine preparations was concentrated on the production and purification of
detoxified B. pertussis virulence factors. These virulence factors include adhesins such as filamentous haemagglutinin, fimbriae and
pertactin, which allow B. pertussis to bind to ciliated epithelial cells in the upper respiratory tract. Once attachment is initiated, toxins
produced by the bacterium enable colonisation to proceed by interfering with host clearance mechanisms. B. pertussis co-ordinately
regulates the expression of virulence factors via the Bordetella virulence gene (bvg) locus, which encodes a response regulator responsible for
signal-mediated activation and repression. This strict regulation mechanism allows the bacterium to express different gene subsets in
different environmental niches within the host, according to the stage of disease progression. 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Virulence factor expression ; Pertussis toxin; Adenylate cyclase; Fimbria ; Filamentous hemagglutinin; Pertactin ; Bordetella; bvg
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
The genus Bordetella . . . . . . . . . . . . . . . . . . . . .
Genetic control of virulence . . . . . . . . . . . . . . . .
3.1. Bordetella virulence gene regulation system
3.2. Transcriptional regulation by BvgA . . . . . .
3.3. Involvement of RNA polymerase . . . . . . . .
3.4. Dierential activation of virulence factors .
3.5. `Bvgi ' . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. The Ris system . . . . . . . . . . . . . . . . . . . . .
3.7. Bvg accessory factor . . . . . . . . . . . . . . . . .
Virulence factors of B. pertussis . . . . . . . . . . . . .
4.1. Filamentous haemagglutinin . . . . . . . . . . .
4.2. Serotype-specic mbriae . . . . . . . . . . . . . .
4.3. Pertactin . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Pertussis toxin . . . . . . . . . . . . . . . . . . . . . .
4.5. Adenylate cyclase toxin/haemolysin . . . . . .
4.6. Dermonecrotic toxin . . . . . . . . . . . . . . . . .
4.7. Tracheal cytotoxin . . . . . . . . . . . . . . . . . .
4.8. Lipopolysaccharide . . . . . . . . . . . . . . . . . .
4.9. Tracheal colonisation factor . . . . . . . . . . .
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310
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* Corresponding author. Tel. : +61 (2) 4221 3439; Fax: +61 (2) 4221 4135; E-mail : mwalker@uow.edu.au
1
Present address: GADI Research Centre, University of Canberra, Canberra, A.C.T., Australia.
0168-6445 / 01 / $20.00 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 0 1 ) 0 0 0 5 6 - 0
FEMSRE 716 1-5-01
310
A.M. Smith et al. / FEMS Microbiology Reviews 25 (2001) 309^333
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
326
5.
6.
4.10. Serum resistance locus . . . . . . . . . . .

Elicitation of protective immune responses
5.1. Cell-mediated immunity . . . . . . . . . .
5.2. Development of vaccine resistance . .
Concluding remarks . . . . . . . . . . . . . . . .
...............
against B. pertussis
...............
...............
...............
1. Introduction
Pertussis or whooping cough is a highly contagious disease of the human respiratory tract, which is particularly
severe in infants. The disease is characterised by bronchopneumonia, paroxysmal coughing and the distinctive
`whooping' intake of air. Pertussis is especially prevalent
in developing countries where appropriate medical assistance is often unavailable and disease progression is unimpeded due to the lack of suitable antimicrobials. The
readily transmissible nature and the severity of the disease
in these countries have seen worldwide deaths from
whooping cough increase to approximately 346 000 per
year [1]. Whooping cough is caused by the obligate human
pathogen, Bordetella pertussis, a Gram-negative coccobacillus originally isolated in 1906 by Bordet and Gengou [2].
The normal course of infection begins with the bacterium entering the host via the airways, contained within
airborne droplets derived from the cough of an infected
individual. The pathogen proceeds down the respiratory
tract, adhering to ciliated epithelial cells in the trachea
and nasopharynx. Once attachment is initiated, the bacterium begins to replicate and colonises adjacent areas. Toxins secreted by the micro-organism damage the epithelial
lining, resulting in the loss of ciliated cells which induces
the characteristic coughing. These toxins also allow the
bacterium to evade the host immune response by interfering with clearance mechanisms. Halting ciliary function,
short-circuiting host G protein signalling apparatus and
inhibiting immune cell function by upregulating cAMP
levels are all examples of the damage caused by the various toxins of B. pertussis.
The incidence of whooping cough has been greatly
reduced since the advent of pertussis vaccines in the
1940s. These `whole-cell' vaccines consist of chemically
or heat-killed B. pertussis cells. These vaccines, as part
of the diphtheria, tetanus, pertussis (DTP) immunisation
regime, proved extremely eective at preventing the symptoms of whooping cough. Unfortunately, a loose association with rare neurologic complications and deaths has
led to a decline in public condence, a subsequent reduction in immunisation rates, and an increase in the incidence of the disease. The other constituents of the DTP
vaccine are puried diphtheria and tetanus toxoids. These
vaccine components have attained a comparatively less
controversial reputation, and signicant research has
been carried out in an attempt to bring the pertussis com-
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ponent into line with the less reactogenic constituents of

the vaccine.
2. The genus Bordetella
Historically, the bacteria currently classied within this
genus have had a very dynamic taxonomic existence, not
always being classied together. It was Lopez who proposed that a new group, the genus Bordetella, be created
to house the then three species, pertussis, parapertussis and
bronchiseptica [3]. More recent molecular evidence, including DNA sequencing analysis, multilocus enzyme electrophoresis and restriction fragment length polymorphism
typing, has justied the grouping of these three species
into the same genus [4,5]. B. pertussis is exquisitely tuned
to a single environment and has only been found to infect
humans.
B. parapertussis causes a milder `pertussis-like' disease
state in humans and has also been found associated with
ovine respiratory infections [6]. Most other mammals can
be colonised by B. bronchiseptica with many commercially
important diseases caused by this micro-organism [7], the
most notable being an association with atrophic rhinitis in
swine which costs the meat industry considerable prots,
and kennel cough in dogs [8,9]. The bird pathogen Bordetella avium, a later addition to the genus (Table 1) [10],
causes turkey coryza and other respiratory diseases of fowl
[11].
More recently, a number of new members of the genus
have been proposed. Bordetella hinzii is the name assigned
to a novel species recently isolated from a bacteraemic
individual with HIV/AIDS [12]. Associated with human
septicaemia, another species, designated Bordetella holmesii, has also been identied [13]. The name Bordetella trematum has recently been proposed for a novel species
isolated from human wounds and ear infections [14].
Although none of these new species were associated with
respiratory tract infections, they are similar to other members of the genus based on phylogenetic analyses [12^14].
Since its initial discovery, B. hinzii has also been isolated
from the respiratory tract of infected poultry [15].
3. Genetic control of virulence
The goal of any pathogenic bacterium, including B. per-
FEMSRE 716 1-5-01
311
Table 1
Comparison of the gene products synthesised by the four most studied members of the genus Bordetellaa
B. pertussis
Pertussis toxin (PT)
Adenylate cyclase toxin/haemolysin
Filamentous haemagglutinin (FHA)
Fimbriae
Pertactin
Tracheal cytotoxinc
Lipopolysaccharide (LPS)
Dermonecrotic toxin
Flagella
Tracheal colonisation factor (Tcf)
BrkA
Urease
+
+
+
+
+
+
+
+
3
+
+
3
B. parapertussis
B. bronchiseptica
B. avium
3
+
+
+
+
+
+
+
3
3
+
+/3
3
+
+
+
+
+
+
+
+
3
+/3
+
3
3
3
+
+
+
+
+
+
3
3
3
Updated and modied from Gross et al. [10].

Abbreviations: +, gene product expressed ; +/3, gene expressed in some strains; 3, gene cryptic or absent.
c
Expression of tracheal cytotoxin is not regulated by the bvg locus.
b
tussis, is to colonise and multiply by exploiting its environment to the fullest extent. This is achieved by the bacterium controlling the production of the specic factors that
enable it to infect its host.
3.1. Bordetella virulence gene regulation system
It was realised many years ago that B. pertussis existed
in more than one phase [16]. `Phase variation' occurs when
spontaneous mutations during DNA replication give rise
to avirulent variants. This process occurs at a frequency of
one in a thousand to one in a million cells and produces
avirulent bacteria which do not have the ability to invade
and colonise the host. However, if, when culturing wildtype B. pertussis, the growth conditions are altered, the
bacteria undergo a totally reversible conversion to one
of principally two states, called X and C modes by Lacey
in 1960 [17]. Termed àntigenic modulation', this phenomenon may be important in the survival of this micro-organism. This landmark study also recognised a third state
(I mode), with growth characteristics intermediate between
X and C modes, and `àn innitude of other antigenic
states''.
More recent research has dealt almost entirely with the
two extreme modes of growth, virulent (X) and avirulent
(C). However, studies on the related species B. bronchiseptica have provided new insight into the intricacies of antigenic modulation. Due to the expression of a set of genes
under modulating growth conditions which are easily
monitored, B. bronchiseptica may be used as a model for
other members of the genus. An intermediate-phase B.
bronchiseptica mutant similar to Lacey's I mode has recently been isolated which expresses phenotypes characteristic of both virulent and avirulent states [18].
The members of the genus Bordetella share a genetic
locus which encodes a biological `switch' enabling these
species to oscillate between dierent phenotypic states depending on their environment. In the human respiratory
tract, a subset of B. pertussis genes are expressed which

allow the bacterium to colonise the host. The genes for a
number of adhesins, such as lamentous haemagglutinin
(FHA), serotype-specic mbriae, and a 69-kDa outer
membrane protein known as pertactin, are expressed, as
are the genes encoding products involved in evasion of the
host immune system: pertussis toxin (PT), adenylate cyclase toxin/haemolysin and dermonecrotic toxin.
In the laboratory, the virulence control switch can be
manipulated to bring Bordetella species into an avirulent
state by the addition of sulfate ions or nicotinic acid to the
culture medium, or by reducing the culture temperature
from 37C to 25C [19].
The expression of virulence factors in Bordetella species
is co-ordinately regulated by what was originally termed
the vir locus [20] and is now known as the Bordetella
virulence gene (bvg) locus. This gene locus encodes two
proteins which enable the bacterium to `sense' the prevailing environmental conditions, and then àct' accordingly
by controlling the expression of specic genes and gene
loci. The sensor protein is known as BvgS and the activator as BvgA. Together these gene products constitute a
two-component signal transduction system and make up
part of a signal transduction superfamily [21]. This type of
system is utilised to respond to many dierent environmental stimuli by many prokaryotes, and recently has
been described in some eukaryotes [21^24].
The genes encoding BvgA and BvgS are contained within an operon [25] (Fig. 1) [26]. The original sequencing
data unfortunately contained mistakes in and downstream
of the bvgS gene, confusing the exact arrangement of the
operon and obscuring the presence of the bvgR open reading frame (ORF) [25]. The discovery and analysis of a
third gene, bvgR, has extended both the size and scope
of the bvg locus [27]. While bvgA and bvgS are transcriptionally linked, bvgR is transcribed separately from the
opposing DNA strand (Fig. 1). Transcription of bvgAS
is controlled by three self-regulated promoters, P1 , P3
FEMSRE 716 1-5-01
312
Fig. 1. Schematic representation of the genetic region encompassing the bvg operon and the fhaB gene, highlighting the promoter organisation of the
bvg/fha intergenic region. The genes encoding bvgA, bvgS and bvgR (shaded boxes) and fhaB (open box) are shown, and the direction of transcription
from each promoter is indicated by arrows. The genes for bvgA and bvgS are transcriptionally linked. bvgR is transcribed separately in the opposite direction. Modied from Merkel and Stibitz [48] and Coote [26].
and P4 , and one bvg-independent promoter, P2 (Fig. 1)

[28,29]. Transcription from P2 is constitutive, thereby
maintaining low-level production of intracellular BvgAS
proteins. While P1 , P2 and P3 are all involved in the transcription of bvgAS, P4 produces an antisense RNA strand
complementary to transcripts expressed from the other
three promoters. The exact in vivo function of this promoter remains unclear ; however, it may be involved in
stabilisation of the sense transcripts via the prevention of
secondary structure formation. Hybridisation between
mRNA from P1 and P4 may lead to a more ecient interaction with ribosomes [29].
The gene encoding FHA is transcribed from a region
just upstream of these four promoters [29] at Pfha (Fig. 1).
There has since been discovered a bvg-independent FHA
promoter, Pfha , which has been shown to express low
levels of the FHA precursor protein, FhaB, in vitro
[30,31].
3.1.1. BvgA
The transcriptional activator, BvgA, which can directly
and indirectly aect the expression of a number of virulence-controlled genes, is a 23-kDa cytoplasmic protein.
The two-domain structure of BvgA (Fig. 2), consisting
of a receiver at the N-terminus and a C-terminal helixturn-helix (HTH) motif, is characteristic of response regulators [25,32]. It is this HTH module which facilitates
specic DNA sequence binding at the promoters of bvgactivated genes [33]. Although not part of the HTH domain, the last 20 amino acid residues are also required for
binding, probably by stabilising the HTH^DNA binding
interaction [33,34].
The binding of DNA by proteins is a common phenomenon within cells, and the C-terminal HTH domain of
BvgA shares sequence homology with a number of dier-
Fig. 2. Domain structure of the 209-amino acid transcriptional activator

BvgA. The receiver domain (open box) is shown, with the phosphorylation site (aspartic acid, D54 ) indicated. The C-terminal domain (shaded
box) incorporating the helix-turn-helix domain (lled box; amino acids
168^185) is also shown. Modied from Uhl and Miller [46].
ent protein families. As expected, this domain is homologous to corresponding areas of other response regulators
such as FixJ, UhpA and NarL [34]. Other DNA binding
proteins such as LuxR from Vibrio scheri [35] as well as
the C-termini of selected bacterial c factors [36] also share
homology with this evolutionarily conserved domain.
Direct control over the transcription of a number of
bvg-activated genes by BvgA is achieved via binding to
specic sequences at target promoter regions [37,38]. The
heptameric
sequence-specic
BvgA
binding
site
TTTCCTA rst proposed by Roy and Falkow, or homologous sequences, is present as either direct or inverted
repeats upstream of various bvg-regulated genes [38^40].
These homologous heptameric repeats are involved in the
binding of BvgA to virulence-activated promoters, a point
substantiated in a number of virulence-activated promoters by DNase I protection studies [39^42].
3.1.2. BvgS
In a process that is not yet fully understood, a histidine
kinase residue in the transmitter domain of BvgS is autophosphorylated, a state which can be altered if the environment changes. Following a series of intramolecular
phosphorylation events, the phosphoryl group is eventually transferred to BvgA [43]. If there is a change in the
environment to modulating conditions, the initial phosphorylation does not occur, preventing the activation of
bvgA.
The 135-kDa sensory component of the signal transduction system, BvgS, is often termed an unorthodox sensor
protein because of the number of extra functional domains
Fig. 3. Domain structure of the 1236-amino acid protein BvgS. The

514-aa sensor domain (single line), the 159-aa linker region (single line),
232-aa transmitter domain (open box), 131-aa receiver domain (lightly
shaded box) and the 126-aa C-terminal output (heavily shaded box) regions are shown. Phosphorylation sites (histidine, H729 ; aspartic acid,
D1023 ; and histidine, H1172 ) are indicated. The transmembrane domains
(TM; black boxes) are also depicted. Modied from Uhl and Miller
[46].
FEMSRE 716 1-5-01
it contains. The membrane spanning nature of this protein

predicts it possesses both periplasmic and transmembrane
domains, and sequence homology studies conrm the absolutely conserved histidine kinase domain [25,32]. However, BvgS also contains a linker section and regions usually reserved for response regulators such as BvgA, a
receiver domain and a C-terminal output domain (Fig.
3) [25,32]. The deletion of these èxtra' domains has been
shown to make BvgS non-functional [25,43,44].
Experiments with BvgS^PhoA fusion proteins have
shown that the N-terminal region is located in the periplasm [32]. Mutations in the linker region render the bacterium incapable of sensing the environment providing
evidence that this region acts as a molecular relay between
313
periplasmic and C-terminal domains located in the cytoplasm [39,45].

The transmitter region of BvgS is necessary and sucient for autophosphorylation [43]; however, transfer of
this phosphoryl group to the C-terminal (output) domain
can only be eected via the receiver domain [46]. Not only
does the receiver domain positively regulate the phosphotransfer to the C-terminal output domain, it also has the
ability to reverse this transfer, thereby rephosphorylating
itself and postponing BvgA activation (Fig. 4). It can also
totally dephosphorylate BvgS from the receiver itself,
thereby completely halting the activation of BvgA (Fig.
4) [46]. It is the output domain (particularly His1172 ) of
the C-terminus which is responsible for the phosphorylation of BvgA [47].
3.1.3. BvgR
While BvgA exercises direct control over the expression
of bvg-activated genes by binding to promoter sequences,
control over virulence repressed genes (vrgs) is indirect,
implicating a Bvg-activated repressor protein. The gene
which controls repression of the bvg-repressed genes has
been identied and termed bvgR [48].
The DNA sequence of the 5P end of four of the ve bvgrepressed genes (vrg6, vrg18, vrg24, vrg53 and vrg73) has
brought to light a conserved consensus sequence element
present in the coding region of four of these genes [49,50].
Using a Southwestern analysis with vrg6 DNA, this sequence was recognised by a 34-kDa protein [50]. When
analysed, none of the native promoter regions of any of
the bvg-repressed genes seem necessary for modulative repression to occur [50].
The bvgR gene lies downstream of and adjacent to
bvgAS [48]. Further characterisation led to the identication of the exact location of the bvgR gene (Fig. 1) [27].
Transcribed from its own BvgA-regulated promoter, bvgR
lies 43 bp downstream of bvgS and is transcribed in the
opposite orientation. Transcriptional analysis has shown
that this ORF produces a predicted protein of 32 kDa,
close to the molecular mass (34 kDa) obtained by Beattie
and associates [27].
It has been shown that B. pertussis mutants lacking
bvgR are less ecient colonisers than wild-type strains in
a mouse aerosol challenge model, demonstrating that
BvgR regulation of the bvg-repressed genes makes a signicant contribution to infection in mice [51].
Fig. 4. The BvgS^BvgA phosphorylation cascade model. The sensor domain receives an environmental signal that causes the autophosphorylation of the histidine (H729 ) residue of the transmitter domain (open
box). The phosphoryl group is then transferred to an aspartic acid residue (D1023 ) of the receiver domain (lightly shaded box). The phosphoryl
group can then be relayed to the histidine (H1172 ) residue of the C-terminal output (heavily shaded box), or can react with water to dephosphorylate the BvgS molecule. The output domain then donates the
phosphoryl group to BvgA, leading to the transcriptional initiation of
virulence genes. The short transmembrane domains (TM; black boxes)
are also depicted. Modied from Cotter and Miller [18].
3.2. Transcriptional regulation by BvgA

As the nal part of an intricate phosphorelay process,
BvgS phosphorylates BvgA. When compared to its unphosphorylated counterpart, this activated `BvgAVP'
has a far greater anity for virulence factor promoter
BvgA binding sites [30,33,41,42]. Indeed, BvgA must be
phosphorylated for bvg-activated promoters to become
fully operational in in vitro transcription assays [30,31].
FEMSRE 716 1-5-01
314
Although BvgA-independent transcription of the fhaB

gene has been observed in vitro, expression from this alternative promoter, Pfha , only occurs at low levels [30,31].
It was theorised that the positioning of BvgAVP at a
promoter region may induce BvgA oligomerisation [29]
and raise the anity of the response regulator for the
target DNA [33]. It has been suggested that BvgAVP
may bind co-operatively to bvg-activated promoters, forming multimers along the promoter template [31,40^42]. Initially, high-anity binding occurs far upstream of the
transcriptional start site, then weaker sites are progressively bound until the site adjacent to the RNA polymerase recognition sequence is occupied.
3.3. Involvement of RNA polymerase
When bound to the promoter DNA at the correct start
site, RNA polymerase catalyses the initiation of transcription. RNA polymerase is composed of four subunits, K, L,
LP and c. The K subunit is generally the portion of RNA
polymerase that interacts with transcription factors at positively regulated prokaryotic promoters [52,53]. The c subunit is involved in promoter recognition, interaction with
transcriptional activators, DNA unwinding and setting up
the initiation complex [54]. Much research into the important roles played by these two RNA polymerase subunits
is currently under way.
The K and major c factor of B. pertussis have both been
cloned and characterised [55,56]. The major c factor of B.
pertussis is larger than the Escherichia coli c70 and has
been designated c80 [56]. The B. pertussis RNA polymerase c80 subunit confers enhanced expression of fhaB in E.
coli [56]. The authors suggest that the role played by c80
may be in positioning the RNA polymerase/BvgAVP
complex at the correct site along the promoter template.
By interacting directly with BvgAVP, the RNA polymerase c subunit is provided with an interface point through
which it can initiate transcription [56].
Studies on response regulator molecules related to BvgA
such as LuxR from V. scheri and UhpA from E. coli
have demonstrated that while still able to bind target
DNA, transcription from promoters regulated by these
proteins is severely hindered by C-terminal deletions
[23,57]. Similar experiments carried out on BvgA have
shown that a deletion of as few as two C-terminal amino
acids resulted in an avirulent phenotype [58]. When this
truncated bvgA gene was presented on a plasmid (in trans)
in otherwise wild-type B. pertussis strains, a reduced
growth rate was observed. Mutations found to remedy
the slowed growth rate were traced to the K subunit of
RNA polymerase, further suggesting that an interaction
between RNA polymerase and the C-terminus of BvgA
is required for the transcriptional activation of virulenceassociated genes in B. pertussis [58]. These authors postulate that the slowed growth rate was caused by an inappropriate interaction between the truncated BvgA protein
and the K subunit of RNA polymerase. This may have led

to a signicant loss of free RNA polymerase resulting in
slowed transcription of normal housekeeping genes, hence
a slowed growth rate.
In the absence of BvgAVP, RNA polymerase binds
upstream of the in vivo transcription start sites of the
Pfha promoter leading to low-level expression from the
BvgA-independent promoter, Pfha [30,31]. However, the
addition of BvgAVP allows transcription to be initiated
at the normal in vivo site. This is further evidence that
BvgA acts upon bvg-activated promoters by correctly positioning RNA polymerase for transcription [30,31].
Recent DNase I protection assays at bvg-activated promoters suggest that the promoter DNA exes to loop
around the BvgAVP/RNA polymerase complex
[31,40,41]. This may increase the binding anity of the
BvgAVP/RNA polymerase complex and stabilise the entire transcriptional machinery [31].
3.4. Dierential activation of virulence factors
It has been suggested that the expression of bvg-activated genes proceeds in two stages [59,60]. This dierential
activation of èarly' and `late' promoters allows the bacterium to produce adhesin molecules soon after entering the
host, whereas the `second-wave' bvg-activated genes are
not expressed until attachment is achieved.
The transcription of PT requires the presence of a
threshold concentration of BvgAVP [60]. There is actually a 10-fold higher concentration of BvgAVP required
to initiate transcription at the Pptx promoter than either
Pfha or bvg P1 [40]. Demonstration of the functional dierence between early and late expressed genes came when
mutations in the C-terminus of BvgA allowed normal levels of FHA expression while abolishing PT expression [61].
This region has been proposed as the region of the BvgA
molecule that interacts with RNA polymerase [58,61].
Binding studies at the Pptx promoter have found an
initial high-anity binding site far upstream of the 335
promoter motif [41]. This region includes a 20-bp direct
repeat which in turn encompasses heptameric inverted repeats [39,41]. Genetic analysis of this region has demonstrated that it is the heptanucleotide repeats that are crucial for promoter activation [39]. Due to the distance of
these repeats from the actual start site, Pptx may require
BvgAVP to multimerise along the promoter template
with weaker sites progressively bound until the site adjacent to the RNA polymerase recognition sequence is
reached.
A similar analysis has been undertaken at the Pfha promoter [31] at which the binding of BvgAVP to an extent
mirrors that at the Pptx promoter. Again a high-anity site
is bound initially, including an inverted repeat region homologous to that at Pptx , however it lies further downstream than the corresponding element at the Pptx promoter [31,40]. The architecture of the bvg P1 promoter,
FEMSRE 716 1-5-01
another early promoter, is very similar to that of Pfha and

interacts with BvgAVP and RNA polymerase in a similar
manner [40]. Since the expression of PT is temporally
linked to that of adenylate cyclase toxin/haemolysin it is
not surprising that the transcriptional machinery of these
two toxins is alike; the Pcya promoter has been shown to
behave in a manner which parallels that of Pptx [42].
Dierences in BvgA binding sites at the promoters of
the two classes of virulence genes suggest that the specicity of BvgA^RNA polymerase interactions may control
dierences in transcription at these promoters [31,40^
42,62].
3.5. `Bvgi '
Some of the recent research involving the co-ordinate
regulation by the bvg locus has been performed in the
closely related species B. bronchiseptica. While the bvg-repressed genes of B. pertussis were seldom studied and little
is known about their function, B. bronchiseptica has an
array of important functional bvg-repressed genes including motility, iron scavenging, urease and phosphatase activity which are at least in part repressed by the bvg locus
[63^67].
Due to this expression of functional genes under modulating growth conditions, B. bronchiseptica is sometimes
used as a model for B. pertussis. An intermediate-phase B.
bronchiseptica mutant has recently been identied which
expresses phenotypes characteristic of both bvg-activated
and bvg-repressed states [18]. Further investigation using
polyclonal antisera from animals infected by the intermediate mutant revealed that under growth conditions
described as semi-modulating, B. bronchiseptica expressed
elevated levels of a novel class of gene products. The
phase-locked `Bvgi ' mutant has a C to T transition in
the bvgS gene resulting in the threonine at position 733
of wild-type BvgS being changed to a methionine. The
close proximity of this mutation to the putative phosphorylation site (His729 ) in the transmitter domain is thought to
aect kinase activity [18].
3.6. The Ris system
The recent discovery in B. bronchiseptica of a second
two-component regulatory system distinct from bvg has
furthered our knowledge of genetic regulation in Bordetella spp. Like bvg, this system, designated the ris locus (regulator of intracellular response), also consists of genes
encoding a response regulator (RisA) and a sensor kinase
(RisS) [68]. The ris locus is essential for bacterial resistance
to oxidative stress and the production of acid phosphatase, as well as in vivo persistence. DNA sequence analysis
of the B. pertussis and B. bronchiseptica ris locus has
shown that there is only a single base dierence in each
of the risA and risS genes. However, the function of ris in
B. pertussis has not yet been determined.
315
3.7. Bvg accessory factor

The temporally dierential regulation employed by
BvgAS to control virulence factor expression in B. pertussis may require the presence of accessory factors. In E.
coli, the activation of either fhaB or bvgAS merely requires
the presence of bvgAS in trans [37,69,70]. It was thought
that this was not sucient however for expression of PT
or adenylate cyclase toxin [37,69,71]. When investigating
the factors aecting the expression of PT, it was found
that the activation of the bvg locus may depend on the
topology of the DNA [72]. These authors also alluded to
the possibility that the action of an as yet undiscovered
auxiliary protein may be required to activate expression
from the ptx promoter [72]. Stibitz [61] proposed a model
suggesting the presence of an accessory protein, which
interacts with the C-terminus of BvgA and may be required for cya and ptx activation. This protein has been
characterised and termed the Bvg accessory factor (Baf).
The expression of a ptx-lacZ fusion in E. coli requires
BvgAS and Baf in trans [73]. The expression from Pptx
in E. coli has also been demonstrated without the presence
of Baf, thereby contradicting the previous work and questioning the need for Baf [74].
4. Virulence factors of B. pertussis
The set of genes expressed by B. pertussis which allow it
to invade and persist inside the host are termed virulence
factors or virulence determinants. These include adhesins
which facilitate attachment to target host cells and toxins
which enable the bacterium to evade the host immune
system. Mutagenesis studies have shown many of these
adhesins and toxins are required for in vivo persistence
in animal models [75^78]. At present the consensus with
regard to the strategy for developing whooping cough
vaccines seems to include a few puried immunogens in
an acellular, multicomponent vaccine. Many of the virulence factors of B. pertussis have been considered for this
purpose. Candidate antigens include FHA, serotype-specic mbriae, pertactin, PT and adenylate cyclase toxin.
Other virulence determinants of B. pertussis which have
yet to be considered as vaccine antigens include dermonecrotic toxin, tracheal cytotoxin, lipopolysaccharide (LPS),
tracheal colonisation factor (Tcf) and the serum resistance
locus.
4.1. Filamentous haemagglutinin
The critical step in B. pertussis infection is attachment of
the pathogen to host cells. FHA is consistently referred to
as the major adhesin of this bacterium. It is a 220-kDa
surface-associated protein secreted to the extracellular environment to facilitate adherence to ciliated respiratory
epithelial cells, thereby initiating the pathogenic cycle.
FEMSRE 716 1-5-01
316
FHA can also work as a bridge adhesin, thereby facilitating the attachment of other micro-organisms [79]. This
might in turn explain the superinfections which usually
complicate the clinical course of whooping cough.
As the name suggests, FHA has a lamentous structure,
supported by electron microscopy studies giving the dimensions of the molecule as 2 nm wide and 45^50 nm
long [80,81]. An extensive study by Makhov and coworkers [81] culminated in the proposal of a model of FHA: a
polypeptide chain folded into a monomeric hairpin. The
hairpin comprises head, shaft and tail regions. The model
predicts that whilst the head contains the N- and C-termini, and the tail the important RGD (arginine-glycine-aspartic acid) sequence, the shaft is composed of tandem 19amino acid residue repeat regions R1 (38 cycles) and R2
(13 cycles) which maintain the structural integrity of the
molecule.
This protein is unusual in a number of respects. Firstly,
the gene for FHA, fhaB, has a coding potential of 367
kDa [82,83]. Over one third of this precursor protein is
later cleaved in a complex post-translational maturation
process to unveil the biologically active polypeptide (Fig.
5) [84]. FHA also possesses a number of possible binding
sites and functions. A further interesting point regarding
FHA is the level of regulation involved in producing and
secreting the polypeptide.
4.1.1. Binding activities
The dierent domains of FHA encompass various binding capabilities, including integrin-mediated attachment to
phagocytes, lectin-like binding to sulfated sugars found in
the extracellular matrix (ECM) and on epithelial cells, and
a carbohydrate recognition domain which allows attachment to ciliated cells present in the respiratory epithelium.
The main target binding site for B. pertussis is the ciliated epithelium of the respiratory tract [85]. Attachment to
cells in this region is mediated by the carbohydrate binding domain of FHA [67,86], which has a unique anity
for glycolipids (specically lactosylceramide residues) and
ciliated cells [87]. This carbohydrate recognition domain
was narrowed down to amino acid residues 1141^1279
using monoclonal antibodies [87]. Recent studies of this
region have enabled high-level, soluble expression in E.
coli and further characterisation of this 18-kDa polypeptide, designated fragment A. Anti-fragment A antibodies
were shown to inhibit binding of B. pertussis to ciliated
rabbit cells in the same fashion as antibodies elicited by
whole FHA, raising speculation that with further investigation, the more easily obtained fragment A may replace
FHA in component vaccines against whooping cough
[88].
The heparin binding activity of FHA may allow B. pertussis to bind to non-ciliated cells as demonstrated using
WiDr cells, HeLa cells, and Vero cells [89,90], or perhaps
facilitate interactions with the ECM. The lectin-like binding of FHA to heparin and dextran sulfate was character-
ised when it was shown that binding to epithelial cells

could be inhibited by the addition of heparin [91]. Hannah
and associates [92] extended this research by revealing that
FHA also binds specically to sulfated, but not uncharged
glycolipids and mapped the heparin binding domain to
residues 442^863 of the amino-terminal end of FHA.
The tail region of FHA includes a binding moiety specic for complement receptor type 3 (CR3, KM L2 , CD11b/
CD18) integrins present on the surface of macrophages
[93]. The FHA tail region contains an RGD sequence
which is present in a number of other bacterial adhesins
and is a region of attachment in many eukaryotic integrin
binding proteins [94,95]. It has been determined that it is
this RGD tripeptide which facilitates binding to CR3 [93].
B. pertussis docks with integrin CR3, which may initiate
entry into phagocytes without an oxidative burst. It has
since then been shown that the binding process involves
the formation of a leukocyte signal transduction complex
which upregulates CR3 binding activity, suggesting that
FHA could regulate the binding activity of its own receptor. This leukocyte signal transduction complex is composed of a receptor protein called leukocyte response integrin (LRI) and integrin-associated protein (CD47) [96].
These authors suggest that the carbohydrate/glycoconjugate binding capacity of FHA may also be involved in
the primary stages of the CR3^LRI^CD47 complex formation. This hypothesis was formulated using the example
of leukocyteêndothelial cell recognition [97] whereby a
transitory state of attachment is mediated by lectins and
this weak connection is strengthened by CR3-dependent
binding. Hazenbos and associates [98] found that a macrophage surface bronectin receptor, very late antigen-5
(VLA-5), may also be involved and proposed that binding
and cross-linking of VLA-5 by B. pertussis activates CR3,
facilitating attachment to FHA. Further study led to the
nding that VLA-5 was acting as a ligand for the minor
mbrial subunit FimD [99]. These authors suggest that
mbriae and FHA may act co-operatively to bind to macrophages, leading to uptake and intracellular survival of
B. pertussis.
The advantages gained by B. pertussis in binding to
`professional phagocytes' are concerned with evasion of
the immune system and may be threefold. Severing cellular
communication links to phagocytic cells by obstructing a
vital receptor site (CR3, VLA-5 or C4BP) may reduce the
impact of the immune system. Secondly, by attaching to
CR3, B. pertussis has taken the preliminary step in entry
and persistence in phagocytes, and `hand-to-hand combat'
using CR3 as a site from which to deliver bacterial toxins
at close range may kill macrophages and neutrophils more
economically [100,101]. Finally, B. pertussis FHA binds
the serum protein C4BP [102]. This protein is known as
a regulator of complement activation and acts to inhibit
the classical complement pathway, ceasing the formation
of the membrane attack complex. How B. pertussis is able
to utilise this binding capacity remains unclear, but once
FEMSRE 716 1-5-01
317
Fig. 5. Simplied representation of the current model of FHA secretion in B. pertussis. The large signal peptide (open box), haemolysin homologous region (lightly shaded box) and C-terminal domain (lled box) are shown. The remainder of the protein, which contains all of the binding domains, includes the repeat regions (striped boxes), the RGD sequence (open box) and the immunodominant region of the molecule (heavily shaded box). The
atypical signal peptide is cleaved from FhaB probably via a leader peptidase-dependent process as it translocates to the inner plasma membrane. The
new N-terminal glutamine residue is then converted to a pyroglutamine, indicated by the asterisk. The C-terminus is still present in the periplasmic
space, presumably to prevent incorrect folding. An interaction is then established between FhaC and the haemolysin-homologous region of FhaB. As
the precursor molecule is translocated through the FhaC L-barrel, the C-terminal domain is cleaved. Once through the outer membrane (outer) the mature FHA molecule (FHA) is then able to fold correctly into the predicted hairpin structure. Modied from Locht et al. [84] and Renauld-Mongenie et
al. [112].
again demonstrates the extent and complexity with which

B. pertussis interacts with the human immune system.
4.1.2. Genetic regulation and biogenesis
Being a virulence determinant of B. pertussis, FHA
undergoes a high level of genetic regulation. The bvg locus
controls expression of fhaB depending on the prevailing
environmental conditions. When B. pertussis is in suitable
conditions for colonisation, FHA is one of the rst proteins produced, detected in a matter of minutes [60]. Production of FHA is also dependent on the expression of at
least one accessory protein, FhaC, the gene for which lies
downstream of fhaB in a cluster of accessory genes involved in the export of both FHA and mbriae (Fig. 6)
[103,104]. FhaC is a pore-forming outer membrane protein
that interacts with FhaB allowing for secretion of the adhesin [105^107].
4.1.3. Post-translational maturation

The DNA sequence of the structural gene fhaB has been
determined [82,83,86]. The gene consists of approximately
10.1 kb and encodes a protein of 367 kDa termed FhaB.
This precursor must undergo a series of modications
which are still only partially understood before mature
FHA is secreted to the extracellular environment.
Toward the N-terminus of FhaB lies a domain of 115
residues, bearing homology to regions of the haemolysins
of Serratia marcescens and Proteus mirabilis, and which
are required for secretion of these proteins [82]. Secretion
of each of these proteins is also dependent on the presence
of a homologous accessory protein (FhaC in B. pertussis)
[107]. Recent studies have shown that the structure of
FhaC contains 19 membrane spanning L-strands that
probably allow the molecule to form a L-barrel in the
outer membrane [106,108] (Fig. 5). An FhaC-like outer
FEMSRE 716 1-5-01
318
membrane protein, HMWB, has since been found in Haemophilus inuenzae, where its function is also to aid in the
export of an adhesin, HMWA [109]. The precursors of
FHA and HMWB share an extra region of homology
comprising 22 amino acid residues, followed by a positively charged region and a hydrophobic segment. This
acts as an atypical signal peptide and is cleaved from the
molecule prior to export [83,105]. Although this is an unusually large signal peptide (71 amino acid residues), it has
recently been shown to undergo proteolysis at a leader
peptidase (Lep) consensus cleavage site, suggesting that
FhaB crosses the inner membrane via the Sec-dependent
pathway [110]. The B. pertussis lep gene has recently been
cloned and characterised [111], and future studies may
determine whether Lep is an absolute requirement for
FHA secretion in B. pertussis.
The C-terminal third of the precursor protein is also
removed leaving mature FHA comprising the N-terminal
220 kDa of FhaB. Although not part of the mature protein, the C-terminus plays an important role in secretion
[112] possibly as a type of intramolecular chaperone to
stabilise the protein and prevent incorrect folding, allowing the required secretory interactions to proceed. Thus
the current model of FHA maturation and secretion involves the precursor FhaB translocating the inner membrane to the periplasmic space, probably utilising the B.
pertussis leader peptidase (Lep) and Sec machinery. The
115-residue, haemolysin-homologous domain then interacts with FhaC (Fig. 5). As the N-terminal portion passes
through the FhaC-derived L-barrel outer membrane pore,
the 150-kDa C-terminal domain is cleaved liberating mature FHA, which then folds into the rigid hairpin structure
proposed by Makhov et al. [81].
4.2. Serotype-specic mbriae
Fimbriae, also known as pili and agglutinogens, are
long lamentous protrusions which extend from the bacterial cell surface and facilitate a variety of binding capabilities. The mbriae of B. pertussis incorporate both major and minor subunits. The major subunits form the
mbrial strand, being grouped into pentameric repeat
units, each 13 nm in length and comprising two full helical
turns [113]. This nding has been supported and extended
by more recent research [114] which also obtained an accurate value of 5.7 nm for the diameter of B. pertussis
mbriae.
B. pertussis produces two distinct types of mbriae, serotype 2 and serotype 3 (also called serotype 6). These are
constructed using major subunits, expressed from the m2
and m3 genes respectively, and minor mbrial subunit,
encoded by mD [103,104]. Major subunits are stacked to
form the long lamentous structure characteristic of mbriae and the minor subunit is located at the tip [115].
4.2.1. Major subunits
The major mbrial subunits which form the structural
helices, Fim2 and Fim3, are proteins of 22 kDa and 22.5
kDa respectively. The m2 and m3 genes which encode
these proteins have both been cloned and sequenced
[116,117] and exhibit considerable homology at both the
nucleotide and amino acid level. Two other pseudogenes
related to the major mbrial subunits have also been
found in B. pertussis, mX and mA [104,118]. The mX
gene encodes a protein of approximately 20 kDa which is
expressed at very low levels if at all [119]. The other silent
mbrial gene, mA, is located in the cluster of mbrial and
FHA accessory genes (Fig. 6) [104]. This is a region of
DNA with sequence homology to m2, m3 and mX,
however the 5P end of the gene corresponding to the Nterminal region of FimA is absent in B. pertussis. Recently, it was found that B. bronchiseptica expresses a fully
intact version of FimA [120].
The low-level expression of FimA seen in B. bronchiseptica is bvg-regulated. It has been postulated that the two
functional major mbrial subunit genes and the pseudogene mX may have arisen via gene duplication. The discovery of mA, its chromosomal location within the mbrial operon and its expression in B. bronchiseptica suggest
that mA may actually be the original, ancestral major
mbrial subunit from which m2 and m3 arose [104,120].
Since the discovery of the minor subunit and adhesin
FimD, the role of the major subunits in adherence has
been somewhat downgraded to almost being regarded as
merely the scaolding on which sits the important adhesin
molecule. Recently it has been shown, however, that the
major mbrial subunit has an anity for sulfated sugars
[121], an ability shared by the other principal adhesin
molecule, FHA. The association between B. pertussis mbriae and derivatives of heparan sulfate is highly dependent on both the number of sulfate groups present and
their placement around the disaccharide molecule [121].
The two Fim2 heparin binding regions (H1 and H2)
have since been fully characterised [122]. These regions
Fig. 6. Diagrammatic representation of the m/fha gene cluster. Genes involved in FHA production are shown as shaded boxes and those involved in
mbrial production are shown as open boxes. Modied from Willems et al. [107].
FEMSRE 716 1-5-01
share sequence and structural homology with each other

and with similar heparin binding sites of bronectin, a
eukaryotic ECM molecule.
The major antigenic domains present on Fim2 and
Fim3 mbriae have been identied using monoclonal antibodies to screen various synthetic peptides [123]. These
epitopes were again conrmed as highly immunogenic
when they were exposed to, and recognised serum antibody from, whooping cough-infected patients [124]. A
number of these antigenic epitopes in fact correspond to
the recently identied heparin binding domains [122].
4.2.2. Minor subunit and accessory proteins
The other genes found in the mbrial cluster (Fig. 6) are
designated mB, mC and mD [103,104]. The proteins
predicted within this operon are similar to proteins essential for mbrial biogenesis in both E. coli (Pap) and Klebsiella pneumoniae (Mrk). The genetic organisation of the
entire gene cluster is very similar to the mrk operon [125],
perhaps suggesting a common origin. Another similar
mbrial gene cluster has also been found in H. inuenzae
type b [126]. An even greater level of homology to mB,
mC and mD is demonstrated by these hif genes. The
most interesting aspect of this report was the inference
that the entire cluster represents an ancestral mobile genetic element and was originally inserted via a transposition
event.
The predicted amino acid sequence of FimB is homologous to the PapD superfamily of periplasmic chaperones
[103,104]. A putative signal peptide sequence directs the
newly translated FimB to the periplasm, where the signal
sequence is cleaved, leaving a mature polypeptide of approximately 24 kDa [103,104]. Southern hybridisation of
B. pertussis, B. parapertussis and B. bronchiseptica DNA
disclosed regions homologous to mB [104] in these closely
related species, suggesting that all three may utilise a similar process to escort mbrial proteins to the cell surface.
FimC is a protein with predicted homology to many
outer membrane mbrial accessory proteins, including
PapC, MrkC and HifC, all of which are essential for the
biogenesis of mbriae in their respective organisms. The
molecular mass of this predicted protein is approximately
91 kDa after signal peptide cleavage [103,104]. Based on
homology data, it was proposed that FimC is probably
located in the outer membrane, involved in mbrial transportation and anchorage [103,104].
Much of the more recent research into the mbriae of B.
pertussis has concentrated on the minor mbrial subunit
and primary adhesin, FimD. Lying directly upstream of
mC, the mD gene encodes a mbrial adhesin protein
with a molecular mass of approximately 40 kDa [127].
Like its genomic neighbours, mD exhibits homology to
genes in the mbrial clusters of H. inuenzae (hifE), K.
pneumoniae (mrkD) and E. coli (mH) [103,104,126,
128,129]. A mD homologue is also present in B. parapertussis and B. bronchiseptica [127]. In fact, the corre-
319
sponding gene in B. bronchiseptica only diers by a single

base pair change. There are 20 dierent amino acids in the
FimD polypeptide of B. parapertussis resulting from 34
base pair dierences. The dierences present in the
FimD adhesins of B. pertussis and B. parapertussis may
create an evolutionary advantage for both by eliminating
binding site competition while sharing the same human
host [127].
Binding studies involving mbrial mutant strains lacking FimD have demonstrated that B. pertussis binds to
human monocytes via FimD [99]. Known to interact
with B. pertussis mbriae, VLA-5 has since been conrmed
as the FimD receptor on the surface of monocytes [130].
The construction of `true' mbrial mutants, devoid of both
major and minor mbrial subunits, allowed further investigation [115]. A mouse colonisation model was utilised to
show that FimD plays a vital role in colonisation. The
Fim23 /33 ;FimD3 mutant colonised the lungs and trachea
of mice signicantly less eciently than wild-type, FHA3
mutant, and Fim23 /33 ;FimD mutant strains [115].
These researchers also demonstrated the binding of
FimD to the sulfated sugar heparin, also a possible target
molecule for FHA and major mbrial subunits [91,121,
131].
4.2.3. Genetic regulation
Like that of FHA, mbrial expression in B. pertussis is
regulated at a number of levels. As with other virulence
factors employed by B. pertussis to attach to host cells, the
expression of mbriae is controlled by the bvg locus. However, the correct processing and secretion of mbrial subunits requires the expression of a number of accessory
genes, which are located in the mbrial operon (Fig. 6).
B. pertussis produces two distinct mbriae: serotype 2 and
serotype 3. Some strains (serotype 2,3) express both simultaneously [132]. The serotype expressed depends on the
level of transcription of mbrial major subunit genes. Alteration of serotype involves switching promoter activation between the two mbrial subunit genes in a process
termed mbrial phase variation. The genes encoding the
major mbrial subunits, m2 and m3, with which the
mbriae are constructed have been fully characterised
[116,117] as have the homologous but transcriptionally
inactive mX and mA genes [103,104,118]. Sequence
data have revealed a high level of homology between all
of these genes which extends into the promoter regions,
unveiling some interesting features of mbrial promoter
architecture (Fig. 7).
The most noticeable aspect of the mbrial promoters is
the `C stretch', a long run of cytosine residues which has
been implicated in mbrial phase variation [133]. Insertion
or deletion of extra cytosine residues in this C stretch is
the source of mbrial phase variation [133]. It is thought
that the distance between the putative 310 box and the
activator (BvgA) binding site is vital for transcriptional
activation, and the alteration in the number of Cs in the
FEMSRE 716 1-5-01
320
Fig. 7. Promoter regions of the three mbrial genes found in B. pertussis. The 310 boxes and putative activator (BvgA) binding sites are indicated in
bold. Dashes have been introduced to increase homology. Dierences between strains in the equivalent promoter regions are underlined. The strain
from which each sequence was derived is named in parentheses. Sequence of m2 (Wellcome 28 strain) is from Livey et al. [117]. Modied from Riboli
et al. [119] and Willems et al. [133].
stretch changes this distance [133]. The reason for the lack
of transcription from the mX pseudogene may be the
short C stretch in the promoter region.
The expression of the mbrial accessory genes is also
required for the correct delivery and presentation of B.
pertussis mbriae at the cell surface [103,104]. A mutational analysis of the m/fha gene cluster has provided evidence that mC, mD and fhaC are translationally linked.
These three genes overlap slightly and there is a putative
ribosome binding (Shine^Dalgarno) site directly upstream
of the rst gene, mC [127].
4.3. Pertactin
Also known by the aliases p.69 and OMP 69 because of
its electrophoretic mobility in SDS^PAGE, pertactin is
actually a 60-kDa outer membrane protein involved in
bacterial adherence [134,135]. Similar molecules are produced by other members of the genus, p.70 in B. parapertussis [136] and p.68 in B. bronchiseptica [137].
The crystal structure of pertactin has been elucidated,
revealing a 16-stranded parallel L-helix with a V-shaped
cross-section [138]. The secondary structure of pertactin
contains two immunodominant direct repeat regions.
The rst, (Gly.Gly.Xaa.Xaa.Pro)5 , is located directly after
the important RGD tripeptide and the other,
(Pro.Gln.Pro)5 , thought to be the major immunoprotective
epitope, lies toward the C-terminal end [139,140].
Pertactin is transcribed from the prn gene, which encodes a 93.5-kDa polypeptide comprising 910 amino acids
[139]. This precursor, termed p.93, later undergoes the removal of a 34-amino acid N-terminal signal peptide [135]
and cleavage of a 30-kDa polypeptide (p.30) from the Cterminus. Although the precise role of p.30 is unclear, it is
detected in outer membrane fractions and is likely to be
involved in the export of pertactin to the outer membrane
[141]. In a comparison of the prn gene sequences of
B. pertussis, B. parapertussis and B. bronchiseptica, the

precursors of the pertactins were found to be extremely
homologous and interestingly, the most highly conserved
region is the C-terminus, suggesting the functional relevance of p.30 to these organisms [142].
The mechanism by which pertactin promotes adherence
to eukaryotic cells is unknown and no receptor has been
found. The amino acid sequence revealed an RGD motif
(amino acids 225^227), a known integrin binding moiety
present in many bacterial adhesins such as FHA and eukaryotic ECM proteins including bronectin and vitronectin [93,143]. It was assumed that this region facilitates
binding to mammalian cells. Mutants decient in pertactin
expression adhered 30^40% less well to CHO (Chinese
hamster ovary) cells and HeLa cells [134]. Synthetic peptides containing RGD derived from pertactin were found
to inhibit pertactin binding to epithelial cells and reduce B.
pertussis entry into HeLa cells [134,144]. However, a recent study has found no role for the pertactin RGD sequence as a mediator of eukaryote cell invasion [145].
These researchers used site-directed mutagenesis (aspartic
acid to glutamic acid) to obtain a pertactin molecule with
an RGE site rather than RGD. Strains expressing the
mutation displayed no dierence in their ability to promote adhesion to HEp-2 or CHO cells. It would be useful
to determine the role of pertactin in wild-type B. pertussis
infection, which would lead to a better understanding of
whooping cough and the vaccines which prevent it.
It has been demonstrated by a number of groups that
pertactin is an immunoprotective antigen, being used in
subunit and live oral vaccines to protect mice from respiratory challenge with virulent B. pertussis [146^150]. Many
human vaccine trials suggest that the inclusion of pertactin
in the formulations is imperative. Although the role of
pertactin in vaccine-based immunoprotection is unknown,
preparations including pertactin have attained promising
vaccine ecacy results [151,152].
FEMSRE 716 1-5-01
4.4. Pertussis toxin

A member of the A-B bacterial toxin superfamily, PT is
a 106-kDa, hexameric protein comprising ve distinct subunits [153]. PT is an ADP ribosyltransferase [154] with the
ability to cause a plethora of eects on host cells. It is the
A protomer or S1 subunit which possesses enzymatic capabilities, catalysing the transfer of the ADP-ribose moiety
of NAD to a family of G proteins, which are involved in
signal transduction pathways within the host cell. This
short-circuits the cell signalling machinery of epithelial
and immune system cells by breaking down G protein
interactions [154]. The B oligomer comprises the remaining subunits; S2, S3, S4 and S5 make up the B oligomer in
the ratio 1:1:2:1 [153]. This region of the protein facilitates attachment of the toxin to host cells, delivering the
toxic action of the S1 subunit [153].
The genes encoding the PT subunits are clustered together in an operon typical of many other bacterial toxins
(Fig. 8). Genetic analysis has revealed that each subunit is
translated separately with an amino-terminal signal sequence which is cleaved during transport to the periplasm
where the holotoxin is then assembled and secreted
[155,156].
A major hurdle yet to be overcome in the preparation of
acellular vaccines is the inability to obtain puried antigens in large amounts. The incompatibility of PT gene
expression signals with respect to E. coli makes the production of PT in this organism dicult to achieve
[157,158]. There are also problems associated with obtaining high yields of puried PT from virulent B. pertussis.
These include the slow growth rate, fastidious nutritional
requirements and the inherent low PT production levels of
the bacterium. There is also the risk that the co-purication of low levels of other virulence-regulated toxins may
occur in vaccine preparations. Of the Bordetella species,
only B. pertussis produces PT. A cryptic PT operon is
present in B. parapertussis and B. bronchiseptica, however
a concentration of mutations in the promoter region leaves
these operons transcriptionally silent [159].
An expression system for pertussis holotoxin has been
the goal of a number of groups, particularly stimulated by
the cloning and sequencing of the PT operon. The S1 subunit gene has upstream regions analogous to E. coli ribosomal binding sites. The remaining subunits, however,
contain novel ribosome binding site sequences upstream
of the structural genes, which bear no resemblance to
321
the E. coli consensus ribosome binding site. This, and

the fact that the PT operon is positively regulated by the
bvg locus, are major reasons why pertussis holotoxin is yet
to be expressed in E. coli. Expression of PT subunits has
been achieved in E. coli and Bacillus subtilis [158,160,161].
Unfortunately, expression of PT subunits in E. coli caused
the majority of the protein to congregate in inclusion
bodies, and not be secreted into the periplasm [158]. The
other major problem with expression of subunits for vaccine purposes is their inability to assemble into holotoxin
in vitro. The individual PT subunits have been shown to
induce antibody responses in immunised mice. However,
these mice were not protected when challenged intracerebrally with B. pertussis [158]. Simultaneous expression of
all ve PT subunits has been achieved in E. coli and Salmonella typhimurium aroA [162,163]. However, varying
levels of expression and post-translational processing, together with a lack of protection in orally immunised mice,
left these strains unsuitable for vaccine production. In a
signicant recent study the PT S1 subunit expressed in
Mycobacterium bovis BCG has been shown to induce protection against live B. pertussis in the mouse intracerebral
challenge model [164]. This reinforces that the system of
delivery and the induction of an appropriate immune response are very important elements of vaccine design.
Ecient expression of pertussis holotoxin has to date
only been achieved in Bordetella species. The major concern here is that PT production takes place only in virulent
strains of B. pertussis. Co-purication of other toxins and
thus contamination of vaccine preparations may therefore
occur. Lee and associates [165] have used recombinant
plasmids to express PT in B. parapertussis and B. bronchiseptica. Yields were comparable to those of B. pertussis
and although the majority of the PT produced was localised in the periplasm, PT was detected in the culture supernatant. B. parapertussis and B. bronchiseptica have also
been used to produce genetically altered detoxied PT
[166,167]. Bacterial strains engineered to produce a safe
pertussis `toxoid' are very good candidates for the production of acellular vaccines. Overexpression and secretion of
genetically detoxied PT has been achieved in B. pertussis
[168] by using allelic exchange techniques to introduce
multiple copies of a genetically altered PT operon into
the chromosome. Fully inducible production of PT in B.
bronchiseptica has resulted in improved growth rates and
PT yields when compared to B. pertussis [169]. Expression
of PT was achieved in a permanently avirulent strain of
Fig. 8. Schematic representation of the ptx/ptl operon. PT subunit genes (open boxes; S1^S5) and PT liberation genes (shaded boxes; AÎ) are indicated. The ptx/ptl promoter (lled arrow; Pwt ) is also depicted. Modied from Smith and Walker [171]; Farizo et al. [173].
FEMSRE 716 1-5-01
322
B. bronchiseptica using mini-transposons to clone a promoterless PT operon in front of a strong constitutive

B. bronchiseptica promoter [170]. Unfortunately, the toxin
was not secreted to the growth medium, making this strain
less than suitable as a vaccine expression system. Further
study demonstrated that neither virulent nor avirulent
strains of B. bronchiseptica were capable of PT secretion
[171].
The entire B. pertussis ptl operon, the products of which
direct the secretion of PT to the culture supernatant, has
been cloned and sequenced [172]. There were originally
thought to be eight ORFs present in the operon, designated ptlA^ptlH [172]. Evidence for the presence of a ninth
gene, ptlI, has since been found (Fig. 8) [173].
Partial ptl sequence data of B. parapertussis and B. bronchiseptica can be compared to the equivalent genomic region of B. pertussis [155,156,172]. Fusion proteins expressed in E. coli have been used to raise polyclonal
antibodies against the predicted PtlA, B, C, E, F, G and
H proteins [174]. Immunoreactive bands in Western blots
of B. pertussis whole-cell extracts were only detected using
polyclonal antibodies against PtlE, F and G. Replacement
of the B. bronchiseptica Pptx promoter with the B. pertussis
Pptx promoter results in detectable expression of PtlF in
Western blots in B. bronchiseptica [175]. These results suggested that Pptx actually drives the transcription of the
complete ptx/ptl region. This was conrmed when the
11-kb mRNA for the entire ptx/ptl operon was detected
in B. pertussis [176]. The introduction of the B. pertussis
Pptx promoter together with a portion of the PT S1 subunit gene into B. bronchiseptica and B. parapertussis via
homologous recombination results in the expression and
secretion of this heterologous toxin [177].
4.5. Adenylate cyclase toxin/haemolysin
The adenylate cyclase-haemolysin of B. pertussis is a
bifunctional protein belonging to the RTX (repeat in toxin) family. This assemblage also includes the much studied
K-haemolysin of E. coli, together with haemolysins and
leukotoxins of various pathogenic Gram-negative bacteria
including Pasteurella haemolytica, and Actinobacillus species [178]. Both the adenylate cyclase and the haemolytic
activities have been shown to be essential for the initiation
of B. pertussis infection [179]. Adenylate cyclase-haemolysin is a secreted polypeptide chain consisting of 1706 amino acid residues [180], with a molecular mass of between
175 and 220 kDa [181^183].
The adenylate cyclase activity of this protein is located
within the rst 400 amino acids at the amino-terminal end
of the protein and is activated by calmodulin [180,184].
Although the high anity of adenylate cyclase toxin/haemolysin for calmodulin was initially thought to be involved in the translocation of the toxin into host cells,
recent evidence has proven this to be incorrect [185]. The
calmodulin is, however, crucial for the enzyme once inside
Fig. 9. Operon structure of the genes encoding adenylate cyclase toxin/

haemolysin and accessory proteins. The adenylate cyclase gene (lightly
shaded box; cyaA) and accessory genes cyaB, cyaD and cyaE (heavily
shaded boxes; B, D, E) are indicated. The adenylate cyclase gene promoter (arrow; Pcya ) is depicted. The cyaC gene and the cyaB, cyaD and
cyaE genes are transcribed from two other promoters (indicated by arrows) respectively. Modied from Goyard et al. [192].
the cell, where the formation of ultraphysiological levels of

cyclic AMP is catalysed [186,187].
The remaining 1300 residues contain an independently
functional haemolysin [180,188] among other structurally
and functionally important domains. A repeat domain of
adenylate cyclase toxin/haemolysin contains 41 repeats of
the glycine- and aspartic acid-rich regions indicative of the
RTX family. Homology with other RTX members suggests that this region is involved in host cell recognition
and calcium binding [189,190]. In fact, each repeat motif
binds a single calcium ion, leading to a major conformational change and possibly the translocation of the catalytic portion of the molecule into host cells [191].
The gene encoding the adenylate cyclase toxin/haemolysin (cyaA) is contained within an operon, together with a
number of other factors involved in toxin activation and
secretion [180] (Fig. 9) [192]. Toxin secretion from B. pertussis will only proceed with the expression of cyaB, cyaD
and cyaE [193]. The nal member of the cya operon, cyaC,
is required to activate adenylate cyclase toxin/haemolysin,
and is involved in post-translational modication of the
toxin [194] (Fig. 10). This protein causes the acylation of
adenylate cyclase toxin/haemolysin resulting in the addition of a palmitoyl fatty acid residue to Lys983 of the polypeptide chain [195] (Fig. 10).
Host cell invasion by adenylate cyclase toxin/haemolysin
is not eected via a receptor-mediated endocytotic pathway [196], and instead uses a specialised calcium- and
temperature-dependent entry system which is not yet fully
understood [197].
The use of a detoxied form of adenylate cyclase toxin/
haemolysin in acellular vaccine preparations has been advocated by a number of groups [198^200]. Immunisation
with adenylate cyclase toxin/haemolysin has been shown
to be protective against bacterial colonisation in animal
models [199,201,202]. Humans infected with B. pertussis
are known to produce antibodies against adenylate cyclase
toxin/haemolysin to ght the infection [198]. The interaction between adenylate cyclase toxin/haemolysin and aspects of the human immune system has been the focus of
many research groups. Invasion and survival of B. pertussis into many dierent immune cells is now well documented [100,203,204]. The inhibition of antigen-dependent
T-cell proliferation by B. pertussis has recently been dem-
FEMSRE 716 1-5-01
323
Fig. 10. Model for the activation of adenylate cyclase toxin/haemolysin. The domain structure of the 1706-amino acid residue toxin is shown. The rst
400 amino acid residues contain the adenylate cyclase moiety (open box; adenylate cyclase domain). The haemolytic activity is contained in the remaining 1300 residues, which are also important for delivery into host cells. This region comprises a 200-residue stretch of hydrophobic amino acids (lightly
shaded box; hydrophobic region), glycine-rich, calcium binding repeat regions (heavily shaded boxes; repeat regions), and a C-terminal signal sequence
involved in secretion of the molecule (lled box; secretion signal). The palmitoylation site is shown (Lys983 ). The palmitoyl residue is represented by the
zigzag symbol. Modied from Goyard et al. [192].
onstrated and necessitates the presence of adenylate cyclase toxin/haemolysin [120]. The potential of recombinant
adenylate cyclase toxoids as vaccine carrier proteins has
recently been highlighted due to their ability to deliver
heterologous epitopes fused to their N-terminal domain
into the cytosol of antigen presenting cells [205]. This system is still being rened, however it has been used to
deliver CD8+ and CD4+ T-cell epitopes to antigen presenting cells to induce antiviral responses [205,206]. This
may be particularly useful for the presentation of vaccine
antigens when a polarised Th1 immune response is required for protection.
The role which adenylate cyclase toxin/haemolysin plays
in apoptosis has been demonstrated in vitro and in vivo in
a number of dierent cell types [207^209]. It has been
proposed that this may be a method by which B. pertussis
might escape the antibacterial eects of macrophages
[207]. Alternatively, it could be an action facilitated by
the immune system to remove the bacterium from their
possibly safer intracellular niche and expose it to the rigours of the extracellular environment, patrolled by killer
cells and antibodies.
4.6. Dermonecrotic toxin
Although known for many years [210], this toxin has
not been as extensively studied as other Bordetella viru-
lence determinants. This may be due, in part, to diculty

in the purication process. Termed heat-labile toxin because it becomes inactive at 56C, it was given its current
name due to its ability to cause necrotic skin lesions when
injected subcutaneously into mice [211,212]. This injection
is lethal when given intravenously. Dermonecrotic toxins
are produced by Bordetella species and are regulated by
the bvg locus [210,213^215]. The toxin is not secreted to
the extracellular environment and has been localised to the
cytoplasm [216]. This and its very low expression levels are
probably the major purication problems. Later studies
agree on a molecular mass of 140 kDa and, contrary to
previous reports, a single polypeptide chain [217,218].
The gene encoding the B. pertussis dermonecrotic toxin
was sectionally cloned and sequenced [219]. The toxin can
be placed by homology and functionality into a family of
toxins including the cytotoxic necrotising factors of E. coli,
botulinum C3 toxin and Pasteurella multocida toxin [220^
223]. All members of this family induce dormant cells to
begin DNA synthesis, leading to either increased cell division or multinuclear cells, as is the case with dermonecrotic toxin. The mechanism of action has recently been
determined. The C-terminal part of dermonecrotic toxin
deamidates a glutamine residue (Glu-63) of Rho protein
[224]. Although dermonecrotic toxin is considered a virulence factor, mutants decient in dermonecrotic toxin do
not exhibit any dierences in virulence in mouse studies
FEMSRE 716 1-5-01
324
[78]. The exact role of dermonecrotic toxin in the pathogenesis of B. pertussis infection is still unknown, however
recent technological advances which have allowed the purication of the toxin and the cloning of its gene may shed
further light onto the role played by dermonecrotic toxin.
4.7. Tracheal cytotoxin
The major biological activity endowed by this toxin is
the ability to damage ciliated respiratory epithelial cells
[225]. Tracheal cytotoxin is a disaccharide-tetrapeptide derivative of the cell wall peptidoglycan layer. Both structurally and functionally, tracheal cytotoxin falls into the muramyl peptide family. Gram-negative bacteria produce a
layer of peptidoglycan, however only Bordetella and Neisseria release fragments extracellularly, in the form of muramyl peptides, normally during exponential growth
[226,227].
The destruction of cilia and ciliated epithelial cells by
tracheal cytotoxin causes ciliostasis and forces the infected
individual to cough relentlessly in order to remove mucus,
a task ordinarily performed by ciliated cells. It has been
demonstrated in hamster trachea epithelial cells that the
lactyl tetrapeptide portion of the molecule is responsible
for its full toxic activity [228]. The toxicity conferred by
tracheal cytotoxin is indirect, being caused by the induction of host cells to produce interleukin-1 [229]. This activates host cell nitric oxide synthase leading to high levels
of nitric oxide radicals [230]. It is still not certain whether
it is the tracheal cytotoxin or the interleukin-1 which stimulates nitric oxide synthase. The nitric oxide acts by destroying iron-dependent enzymes, eventually inhibiting mitochondrial function and DNA synthesis in nearby host
cells [229]. Since B. pertussis is attached to the ciliated
epithelial cells, it is these which undergo the most damage.
Tracheal cytotoxin also has a toxic eect on other cells,
impairing neutrophil function at low concentrations and
conferring toxic activity in larger quantities [231]. Evolutionarily, the production of tracheal cytotoxin by B. pertussis may merely represent a breakdown by-product of
peptidoglycan manufacture. However, it is considered an
integral component in the pathogenesis of B. pertussis infection.
4.8. Lipopolysaccharide
The LPS molecule of B. pertussis is smaller than many
other bacterial LPS structures and is therefore often referred to as a lipooligosaccharide. The role which LPS
plays in the pathogenesis of B. pertussis infection is unclear; however, biological activities exhibited by B. pertussis LPS include antigenic and immunomodulating properties [232,233]. The most common action associated with
LPS is the potent endotoxin activity [234].
Present in most Gram-negative bacteria, LPS generally
comprises lipid A, core oligosaccharide and a long poly-
saccharide O antigen. The structure of B. pertussis LPS

diers in that it lacks the O antigen. B. pertussis actually
produces two types of LPS, designated A and B [235].
These molecules possess diering electrophoretic mobilities due to the presence of an extra trisaccharide moiety
on LPS-A [236]. All of the species of Bordetella express
dierent LPS molecules, which may be a factor in the high
level of species specicity demonstrated within this genus
[237]. B. parapertussis strains isolated from humans and
sheep display distinct LPS proles [237] adding to the
host-specic feature of this molecule [238,239]. The LPS
produced by B. bronchiseptica is similar to that of B. parapertussis, as they both express temperature-dependent O
antigen [237,240]. The charged O antigen of B. bronchiseptica is thought to be the element which confers protection
against antimicrobial peptides since B. pertussis, which
lacks O antigen, is not protected [241]. B. bronchiseptica
also produces host-specic LPS molecules, with isolates
from dogs having more heterogeneous LPS structures
than isolates from pigs [237]. A human isolate of B. bronchiseptica displays a dierent LPS prole than that of a
rabbit isolate [207,242]. Subsequent isolation of B. bronchiseptica from the same human patient over 2 years has
shown a variation in LPS proles during the course of
infection [207].
Transposon mutagenesis has allowed the identication
of a gene cluster encoding LPS biosynthesis in B. pertussis
[243]. Many of these genes are similar to polysaccharide
and LPS synthesis genes present in other bacteria, however
the B. pertussis LPS operon is arranged in a unique manner [243].
Unfortunately, our knowledge of the B. pertussis LPS
remains limited and further research is necessary before we
are able to dene the involvement of this molecule in both
the pathogenesis of disease caused by this bacterium, and
the immunogenicity and adjuvanticity of vaccines targeted
against whooping cough.
4.9. Tracheal colonisation factor
Originally discovered with the aid of a transposon-mutated strain which was decient in four outer membrane
proteins [244], Tcf is a bvg-regulated protein, exclusively
produced among the Bordetellae by B. pertussis. The gene
encoding Tcf, tcfA, was rst mapped using a TnphoA insertional mutation and was termed virulence-activated
gene #34 (vag34) [244]. The original TnphoA mutants
were 10-fold less able to colonise and persist in the trachea
of mice, but were not signicantly disadvantaged in the
lungs [245]. The tcfA gene has now been cloned and sequenced, with the derived amino acid sequence predicting
a 68.6-kDa precursor, the rst 39 amino acids of which
comprise a likely signal peptide, leaving a 64.4-kDa protein [245]. The C-terminal half of Tcf exhibits over 50%
homology to the C-terminus of the precursors of the pertactins of Bordetella species, including the Lys-Arg puta-
FEMSRE 716 1-5-01
tive proteolytic cleavage site. Another Bordetella protein,

the serum resistance factor BrkA, also possesses a C-terminus with signicant homology to that of Tcf [246]. The
N-terminal half of Tcf contains three RGD motifs and has
a high (16.5%) proline content, which may aect migration
in SDS^PAGE gels, perhaps explaining the discrepancy
between the predicted 68.6-kDa protein and the presumed
V90 kDa seen in PAGE gels [245]. The authors have
hypothesised that the maturation processes between pertactin and Tcf may also share common features. Thus,
their model of Tcf secretion involves the precursor molecule (68.6 kDa) being transported to the periplasm after
the cleavage of the typical signal peptide. The remaining
64.4-kDa precursor is then translocated across the outer
membrane, like pertactin, assisted by its own C-terminal
region. Cleavage at the Lys-Arg proteolysis site then occurs, liberating the mature 34-kDa Tcf protein. As a consequence of the limited research literature on Tcf, the elucidation of the exact role played by this protein in the
pathogenesis of pertussis will require subsequent scientic
scrutiny.
4.10. Serum resistance locus
Mutant strains of B. pertussis decient in the expression
of the serum resistance (brk) locus are signicantly less
virulent in infant mice than wild-type strains [78]. These
strains have also been shown to be more susceptible to
complement-activated killing in the presence of human
serum [246]. The brk locus, which has homologues in B.
parapertussis and B. bronchiseptica, consists of two genes
transcribed in opposite directions, brkA and brkB
[246,247]. The proteins predicted by these genes are
BrkA, 103 kDa, and BrkB, 32 kDa, both of which are
required for serum resistance. BrkA, translated as a precursor molecule, is proteolytically cleaved to form 73-kDa
and 30-kDa products. This cleavage pattern is reminiscent
of pertactin, the protein to which BrkA is most closely
related, and Tcf [245]. The presence of two RGD motifs
and possible roles in adherence and invasion also indicate
the similarity between these proteins.
A recent study has demonstrated that B. pertussis clinical isolates were serum-resistant, suggesting that circulating strains gain an advantage in vivo by evading complement-activated killing. It was also shown that strains
carrying an extra genetic copy of the brk locus exhibited
increased levels of serum resistance [248].
5. Elicitation of protective immune responses against
B. pertussis
The development of whooping cough vaccines has concentrated on the elicitation of high antibody titres and
protective immunity, and on the reduction of side-eects.
Recently, a number of issues have been brought to light
325
which also bear serious consideration in the preparation of

whooping cough vaccines.
5.1. Cell-mediated immunity
One of the major challenges in developing superior vaccines is our limited understanding of the mechanisms involved in the development of immunity against pertussis
following either natural infection or vaccination. Which
components of the human immune system become primed
to ght B. pertussis infection after vaccination? Defence
against intracellular pathogens is usually dependent on the
activation of a strong Th1 helper response. B. pertussis has
been shown to invade an increasing number of cell types
[100,249,250] and is now considered by some a facultative
intracellular pathogen. Intracellular bacteria may be protected from host clearance mechanisms resulting in the
infection persisting. It is therefore reasonable to assume
that cell-mediated immunity (CMI) may play a key role
in eliminating B. pertussis infection, at the very least by
facilitating the release of bacteria surviving within phagocytes. Studies have demonstrated the importance of CMI
in natural and vaccine-based immunity [251,252]. Immunisation of mice with whole-cell vaccine or recovery from
natural B. pertussis infection induces a Th1 response,
whereas immunisation with an acellular vaccine elicits a
Th2 response [252]. Production of interleukin-12 is induced by native infection or whole-cell vaccination, but
not by acellular vaccination [253].
Recent studies have shown that protection after respiratory challenge in the murine model is an excellent correlate for ecacy in humans of acellular and whole-cell
vaccines, and that humoral and cell-mediated immunity
play complementary roles in vaccine-elicited protection
[254]. Using dierent knockout mice, Mills and associates
[255] found that both humoral and cell-mediated responses
are required for the stimulation of protective immunity
following vaccination. The initial response may be antibody-mediated and is involved in restricting the extent
of infection and minimising the damage to epithelial and
immune cells. To achieve complete bacterial clearance
however, the induction of a CMI response is also required.
The Th2 response elicited after immunisation with an acellular vaccine results in delayed bacterial clearance in the
murine respiratory challenge model. In contrast, natural
infection or immunisation with killed whole cells, inducing
a Th1 response, was associated with rapid clearance [255].
However, analyses of the CMI responses of children in
recent vaccine ecacy trials revealed that acellular vaccines induce Th1 responses [256,257] equivalent to responses exhibited after natural infection [258].
5.2. Development of vaccine resistance
Despite the time spent on research into mechanisms of
immunity, ecacy and reactogenicity of vaccines against
FEMSRE 716 1-5-01
326
B. pertussis, the bacterium itself has been ghting back.

Recent data from The Netherlands reported a dramatic
increase in the incidence of pertussis in that country in
1996, despite high levels of immunisation [259]. An extensive study has traced the problem to the changing population structure of B. pertussis itself [260]. The antigenic
variation of the protective antigens pertactin and the S1
subunit of PT was analysed using circulating clinical isolates collected over a long period. Three dierent variants
each of pertactin and S1 were discovered and the presence
of these variants showed temporal shifts. Polymorphism in
pertactin was found to occur in the midst of a repeat
region (Gly.Gly.Xaa.Xaa.Pro)5 termed region 1. The mutations in the S1 subunit were similarly conned to a small
region of the polypeptide. The mutations in S1 and pertactin are concentrated in regions dened as T-cell and Bcell epitopes respectively [140,261]. This highlights the importance of maintaining a constant global monitoring of
circulating isolates and the necessity to adapt present formulations according to temporal antigenic shifts.
6. Concluding remarks
B. pertussis is exquisitely ne-tuned to inhabit the human respiratory tract. This has been achieved via an evolutionary process. The twentieth century had caught up
with this pathogen somewhat, with the advent of antimicrobials and immunisation, and it was on the way out.
Now it is again evolving and re-emerging as a dangerous
pathogen. Together with the attention to vaccine reactogenicity, the induction of appropriate immune response
mechanisms and raising the protective ecacy of future
anti-pertussis vaccines, we must also consider the rapid
evolution of B. pertussis in the development of future
whooping cough vaccines.
An exciting development in B. pertussis research is the
ongoing genome sequencing project of this bacterium by
the Sanger Centre, details of which are available on their
website [262]. This will undoubtedly facilitate the development of better vaccines against whooping cough.
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FEMS Microbiology Reviews 25 (2001) 309^333

The virulence factors of Bordetella pertussis: a matter of control

, Carlos A. Guzman b , Mark J. Walker

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4.10. Serum resistance locus . . . . . . . . . . .

ponent into line with the less reactogenic constituents of

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Updated and modied from Gross et al. [10].

tract, a subset of B. pertussis genes are expressed which

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and P4 , and one bvg-independent promoter, P2 (Fig. 1)

Fig. 2. Domain structure of the 209-amino acid transcriptional activator

Fig. 3. Domain structure of the 1236-amino acid protein BvgS. The

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it contains. The membrane spanning nature of this protein

periplasmic and C-terminal domains located in the cytoplasm [39,45].

3.2. Transcriptional regulation by BvgA

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Although BvgA-independent transcription of the fhaB

and the K subunit of RNA polymerase. This may have led

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another early promoter, is very similar to that of Pfha and

3.7. Bvg accessory factor

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ised when it was shown that binding to epithelial cells

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again demonstrates the extent and complexity with which

4.1.3. Post-translational maturation

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share sequence and structural homology with each other

sponding gene in B. bronchiseptica only diers by a single

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B. pertussis, B. parapertussis and B. bronchiseptica, the

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4.4. Pertussis toxin

the E. coli consensus ribosome binding site. This, and

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B. bronchiseptica using mini-transposons to clone a promoterless PT operon in front of a strong constitutive

Fig. 9. Operon structure of the genes encoding adenylate cyclase toxin/

the cell, where the formation of ultraphysiological levels of

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lence determinants. This may be due, in part, to diculty

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saccharide O antigen. The structure of B. pertussis LPS

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tive proteolytic cleavage site. Another Bordetella protein,

which also bear serious consideration in the preparation of