David Blanco, PhD
CHS A2-087G
MIMG C106
Bordetella
Whooping cough
Whooping cough is an upper respiratory disease that is most severe in infants
and young children. Disease progresses in three stages:
1. Catarrhal stage: non-distinctive, upper respiratory, cold-like symptoms.
2. Paroxysmal stage: classic symptoms of whooping cough appear; more
frequent and more intense cough episodes; paroxysms often terminate
with a long drawn inspiratory effort that is usually accompanied by a
whoop (hence the name). This stage lasts 1 – 4 weeks.
3. Convalescent stage: less severe and less frequent paroxysms; this
stage can last for 6 months or more. In China, whooping cough is known
as the “cough of 100 days”.
Epidemiology, Vaccination, Immunity.
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Causative agent: Bordetella pertussis.
One of the most contagious infectious diseases known; household attack
rates are >90%.
Disease is most severe in infants: In a 1984-1985 study, 74% of infants
<6 months old with whooping cough were hospitalized, 20% developed
pneumonia, 2.6% had seizures, and 1% died.
Because of the severity of the disease, parents were eager to have their
children vaccinated when the first vaccines became available in the
1950’s. From 1947 until 1971 the number of cases and deaths due to B.
pertussis decreased steadily – due primarily to good compliance with
vaccination protocols. However, as a new generation of people that had
never experienced or seen whooping cough began to have children, they
focused on the risks of side effects associated with the pertussis vaccine
that subsequently resulted in a drop in vaccination compliance. Side
effects associated with the whole cell killed pertussis vaccine include
general discomfort and confusion for 1 – 2 days as well as pain and
redness around the site of injection. About 1% of infants receiving the
vaccine experience convulsions and a very small number of these cases
result in severe and irreversible brain damage and even death. Because
of the lack of compliance with vaccine protocols, the incidence of pertussis
has been increasing steadily since 1971.
Current efforts are focused on developing a more safe yet effective
vaccine – most of this effort has been on the development of “acellular”
vaccines comprised of purified bacterial proteins that are “protective
immunogens”.
Respiratory mucosal immunity.
Bordetella are non-invasive, extracellular pathogens. They bind specifically to
cilia of respiratory epithelial cells. To colonize the upper respiratory tract,
pathogens must be able to resist or evade constitutive and adaptive immune
responses effective at these locations. Constitutive defense mechanisms
operative in the trachea include the mucocilliary escaltor and the antimicrobial
action of macrophages and PMNs that survey the mucosal surface. Adaptive
immunity at the respiratory surface is mediated by IgG + complement, mucosal
IgA, and activated macrophages.
Pertussis has traditionally been viewed as a two-stage disease; colonization
followed by toxin mediated tissue damage. Two classes of virulence factors
have been identified that presumably contribute to each of these stages.
Bordetella virulence factors.
Adhesins
 Filamentous hemeagglutinin (FHA) – a large (220 kDa) hairpin shaped
molecule that is both cell-surface associated and secreted. FHA has been
shown to be critical for tracheal colonization.
 Fimbriae (pili) – Important for tracheal colonization. Fimbriae exhibit
phase variation.
 Pertactin – a 69 kDa outer membrane protein. Pertactin has RGD
sequences suggesting an integrin attachment interaction.
Toxins
 Tracheal cytotoxin (TCT) – This muramyl tetrapeptide is actually a
fragment of peptidoglycan that is release from growing cells. In most
other Gram-negative bacteria, this cell wall fragment is recycled and
reincorporated back into peptidoglycan. Bordetella do not recycle this
molecule because they lack the gene called ampG that is involved in the
recycling process. TCT causes cytopathology and extrusion of ciliated
epithelial cells from the mucosal surface of tracheal explants in vitro.
 Dermonecrotic toxin – causes necrotizing lesions when injected
intradermally into mice. Its role in pertussis pathogenesis is unknown.
 Pertussis toxin – an A-B type toxin whose structure resembles that of
cholera toxin (1 enzymatic subunits and 5 binding subunits). The S1
subunit catalyzes the ADP-ribosylation of an inhibitory G protein that down
regulates adenylate cyclase activity. The result of pertussis toxin is
therefore a dramatic increase in the intracellular concentration of cAMP.
 AC/HLY – this adenylate cyclase also has hemolytic activity. Once inside
mammalian cells, AC/HLY is activated by binding to calmodulin and
catalyzes the conversion of ATP to cAMP. Like pertussis toxin, therefore,
AC/HLY results in a dramatic increase in the intracellular concentration of
cAMP.
The development of a genetic system for Bordetella.
In 1984, Weiss and Falkow brought Bordetella research to the molecular genetic
age by developing a genetic system for mutagenizing B. pertussis with Tn5. This
system uses a suicide plasmid containing a colE1 ori (for replication in E. coli but
not in Bordetella), Tn7, which encodes trimethoprim (Tmp)-resistance,
spectinomycin (Sp)-resistance, and streptomycin (Sm)-resistance. The Tn5
encodes kanamycin (Km)-resistance. This plasmid is introduced into B. pertussis
by conjugation and B. pertussis bacteria in which Tn5 has transposed into the
bacterial chromosome are selected by plating on BG (Bordet-Gengou) plates
containing Km and naladixic acid (Nal). The strain of B. pertussis used was
resistant to Nal so Nal was used to select against the E. coli donors (E. coli is
sensitive to Nal). Km-resistant colonies were then screened for sensitivity to
Tmp, Sp, and Sm to differentiate true transposon mutants from bacterial in which
the entire plasmid had integrated into the chromosome.
Weiss and Falkow mutagenized B. pertussis and screened mutants for various
phenotypes associated with virulence:
1. Hemolytic activity was assessed by looking visually for a zone of clearing
around bacterial colonies growing on BG-blood agar.
2. Adenylate cyclase activity was determined by measuring [32P]ATP
conversion to [32P]cAMP in the presence and absence of calmodulin in
vitro.
3. pertussis toxin activity was assessed by measuring CHO cell clustering
activity and by the ability of culture supernatants to cause histamine
sensitization in mice.
4. dermonecrotic toxin activity was assessed by looking for dermonecrotizing
lesions at the site of subcutaneous injection of B. pertussis cell extracts in
mice.
5. filamentous hemagglutinin was assessed by measuring the ability of
culture supernatants to agglutinate sheep red blood cells (srbc’s) and
confirmed by Western blot analysis using anti-FHA antibody.
6. tracheal cytotoxin activity was assessed by determining the ability of
culture supernatants to induce cytopathology in hamster tracheal organ
cultures.
Many mutants with defects in one or more of these virulence phenotypes were
identified. Interestingly,
 All mutants defective for hemolytic activity were also defective for
adenylate cyclase activity (It is now known that these two activities are
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performed by a single gene product – albeit different domains of the
protein).
No mutants defective for TCT were found. (as mentioned above, TCT is a
fragment of peptidoglycan that is released because Bordetella lack
ampG). How could you construct a strain that is deficient in TCT
production?
One mutant, BP347, was defective for expression of all of the virulence
factors except TCT. It is now known that the transposon in this strain
inserted into what is called the bvgAS regulatory locus. This was the first
demonstration of trans-acting global regulation of virulence gene
expression.
The various transposon insertion mutants were tested for virulence by
determining their ability to induce lethality in infant mice. Although the
LD50s for the Ptx-, AC/HLY-, and vir(bvgAS-) mutants were increased
compared to wild type B. pertussis, the LD50 for the FHA- mutant was not
significantly different that of wild type. Does this mean that FHA is not a
Bordetella virulence gene?
Phase Variation versus Phenotypic Modulation.
Two forms of virulence gene regulation have been identified in Bordetella: phase
variation and phenotypic modulation.
Phase variation is a metastable alteration between two genetic states.
Phenotypic modulation is a reversible loss of virulence gene expression in
response to certain environmental conditions.
bvgAS encodes a two-component regulatory system. BvgS, the sensor, resides
in the cytoplasmic membrane. Its large periplasmic domain is thought to be
involved in signal recognition and is connected to three cytoplasmically located
signaling domains by a region called the “linker”. Certain mutations in the linker
lock BvgS in its active form. The three cytoplasmically located signaling domains
include a transmitter, which autophosphorylates at a conserved histidine residue,
a receiver domain, containing an aspartic acid residue that receives the
phosphoryl group from the transmitter, and a C-terminal histidine
phosphotransfer domain that is phosphorylated on a histidine residue. BvgA is a
cytoplasmically located response regulator. Its N-terminal receiver domain is
phosphorylated at an aspartic acid residue and its C terminus contains a DNA
binding motif. BvgA-phosphate is competent to both activate and repress gene
transcription.
Is bvgAS responsible for Phase Variation?
Stibitz et al., Nature 1989
At a frequency of about 10-3, vir- variants arise in a population of wild type B.
pertussis. Within this vir- population, vir+ (wild type) B. pertussis will arise at a
frequency of about 10-6. From these, vir- variants will again arise at a frequency
of 10-3. To determine the genetic basis for this variation, Scott Stibitz took a
lineage of strains, cloned their bvgAS loci from the chromosomes of each and
determined the nucleotide sequences. It was found that in the region encoding
the C-terminus of the BvgS receiver, there was a stretch of 6 cytosine (C)
nucleotides. In all of the vir- strains, there were 7 cytosines. This addition of one
nucleotide (C) caused a shift in reading frame such that the C-terminus of the
protein encoded in strains containing 7 cytosine nucleotides differed from those
containing 6 cytosine nucleotides – this rendered BvgS non-functional. Why was
the frequency of mutation 10-3 instead of 10-6? The proposed mechanism is
called slipped strand mispairing and it occurs in regions where there are long
runs of single nucleotides, or where there are repeats of specific nucleotide
sequences. Mispairing during DNA replication results in alterations in the DNA
that, depending on where these alteration occur, can lead to alterations in the
protein product that is ultimately produced.
Is bvgAS responsible for Phenotypic Modulation?
Phenotypic modulation in Bordetella is the phenotypic alteration between distinct
phenotypic phases that occurs in response to specific environmental conditions.
Under Bvg+ phase conditions (37oC and the relative absence of nicotinic acid
and MgSO4) Bordetella express all of their virulence factors. In response to low
temperature or the presence of nicotinic acid or MgSO4, BvgAS is inactive and
virulence factors are not expressed. This is a phenotypic change – not a
genotypic change.
Mutational analyses had shown that bvgAS is required for expression of
virulence genes. To determine if bvgAS is also responsible for the downregulation of virulence gene expression that occurs in response to “modulating”
conditions, Miller et al. designed a strategy to find mutants that were insensitive
to modulating conditions – i.e. mutants that expressed virulence genes even
under modulating (Bvg- phase) conditions.
1. Setting up a selection scheme:
Wild type B. pertussis (BP370) is hemolytic under Bvg+ phase conditions
(- modulators) and non-hemolytic under Bvg- phase conditions (+ modulators)
because AC/HLY is positively regulated by bvgAS. A ptx-cat fusion (cat encodes
chloramphenicol resistance) was introduced into wild type B. pertussis. This
strain (TOX35) is resistant to chloramphenicol (Cmr) under Bvg+ phase
conditions and not under Bvg- phase conditions – because ptx is positively
regulated by BvgAS. Deletion of bvgAS from this strain results in a strain
(VIR101) that is never hemolytic and never Cmr. Providing bvgAS to this strain
on a plasmid that integrates into the chromosome (strain VIR102) restores
hemolytic activity and Cmr under Bvg+ phase conditions (cis complementation of
the mutation).
2. Select spontaneous mutants.
Plate VIR102 on BG + Nicotinic acid + MgSO4 (i.e. modulating conditions) + Cm.
The following colonies were detected at a frequency of about 10 -6: CmrHLY+.
However, some at the same frequency were CmrHLY-?.
The goal was to find mutants in which BvgAS was insensitive to modulating
conditions (Bvg-constitutive or Bvgc mutants). Since HLY is controlled by BvgAS,
the colonies that were CmrHLY+ under modulating conditions must have been
expressing HLY as well a PTX under modulating conditions – they were analyzed
further. (Howver, what was the most likely explanation for the colonies that were
CmrHLY-?).
3. Recover bvgAS from the chromosome of the Bvgc mutants.
Triparental matings were used to recover the plasmids containing bvgAS from
the chromosomes of the Bvgc mutants. These plasmids were called pVIR201,
pVIR203, and pVIR204.
4. Reintroduce pVIR201, pVIR203, and pVIR204 into VIR101.
To determine if the muations conferring the mutant phenotype localized to
sequences contained on pVIR201, pVIR203, and pVIR204 (i.e. that they mapped
to bvgAS), these plasmids were reintroduced into VIR101 and Cmr and hemolytic
activity under Bvg+ and Bvg- phase conditions was determined: all plasmids
conferred the Bvgc phenotype.
5. Test the ability of these mutant bvgAS loci to activate fhaB-lacZ expression in
E. coli.
bvgAS supplied in trans on a plasmid is sufficient to activate expression of an
fhaB-lacZ fusion in E. coli and expression of this fusion is decreased when cells
are grown under modulating conditions. The bvgAS loci contained on pVIR201,
pVIR203, and pVIR204, also activated fhaB-lacZ expression in E. coli, but fhaBlacZ expression remained activated even when grown under modulating
conditions. These bvgAS loci were therefore constitutively active in E. coli –
providing further evidence that bvgAS mediates phenotypic modulation.
6. Subclone, test, sequence.
The mutations conferring the Bvgc phenotype resulted from single nucleotide
substitutions that caused single amino acid changes within the linker region of
BvgS. One single nucleotide change was therefore sufficient to render BvgS
insensitive to environmental conditions.
Does Bvg function as an ON/OFF switch for virulence gene expression?
Knapp and Mekalanos 1988, J. Bacteriology.
Knapp and Mekalanos used TnphoA to mutagenize B. pertussis. Mutants were
assayed for PhoA activity after growing cells under Bvg+ or Bvg- phase
conditions. Three mutants, SK8, SK16, and SK75, displayed higher PhoA
activity when grown under Bvg+ phase conditions compared to Bvg- phase
conditions. These loci were called vag (vir-activated genes). This expression
pattern is the same as all of the virulence genes that had been identified so far
and, in fact, some of the genes identified in this study were those encoding
previously identified virulence factors. Other mutants, however, displayed higher
PhoA activity when grown under Bvg- phase conditions than Bvg+ phase
conditions. These were called vrg loci (vir-repressed genes) and they
represented a newly discovered class of genes – those that are negatively
regulated by BvgAS.
What is the role of Bvg-mediated signal transduction in Bordetella
pathogenesis.
Since B. pertussis has never been isolated from animal or environmental
reservoirs, it has been assumed that the transition from the Bvg+ phase to the
Bvg- phase may occur in the human host during infection and may be important
for the development of infection. To determine experimentally the role of Bvg
mediated signal transduction in the Bordetella-host interaction, experiments were
performed using the closely related subspecies, B. bronchiseptica. This
organism contains a nearly identical BvgAS signal transduction system and
expresses a nearly identical set of virulence factors that are activated by BvgAS.
However, this organism expresses different Bvg- phase factors than B. pertussis.
Bvg- phase factors in B. bronchiseptica include motility, a siderophore, and
urease expression. This subspecies also differs from B. pertussis in that it
displays a broad host range, naturally infecting a variety of four-legged mammals
including rabbits, rats, and mice.
To determine the role of Bvg mediated signal transduction in vivo, mutant
derivatives of wild type B. bronchiseptica that were locked into either the Bvg+
phase or the Bvg- phase were constructed and compared with wild type B.
bronchiseptica in a rabbit model of respiratory infection.
The Bvg- phase-locked mutant was, as expected, completely avirulent – it was
unable to establish an infection even when administered at a dose of 10 6 cfu (4
logs higher than the ID50 for wild type B. bronchiseptica). The Bvg+ phase-
locked mutant, however, was indistinguishable from wild type B. bronchiseptica
in its ability to efficiently establish respiratory infection in rabbits (and other
experiments have shown the same is true in rats and mice).
When the antibody response to B. bronchiseptica infection was examined, it was
shown that high titers of anti-Bordetella antibodies were generated in animals
infected with either wild type B. bronchiseptica or the Bvg+ phase-locked mutant.
The majority of the antibodies generated were directed against Bvg+ phase
factors. Some were directed against factors that are common to the Bvg+ and
Bvg- phases (i.e. are not regulated by BvgAS). In no case, however, were
antibodies against Bvg- phase specific factors detected.
Taken together, these results indicate that the Bvg+ phase is both necessary and
sufficient for respiratory infection and strongly suggests that the switch to the
Bvg- phase does not occur in vivo.
So what is the role of the Bvg- phase?
The Bvg- phase was previously shown to be necessary and sufficient for the
ability of B. bronchiseptica to survive and multiply under nutrient-limiting
conditions. This result suggests that the role of the Bvg- phase is to survive in an
environmental reservoir.
Thus, these data support the hypothesis that the role of BvgAS is to determine if
the bacterium is within or outside a mammalian host and that the Bvg- phase
may contribute to environmental survival. However, the human pathogen B.
pertussis is only outside the host during transmission, therefore, the Bvg- phase
may represent an evolutionary remnant not required for this now adapted human
pathogen.