lecture 15 - bordetella pertussis

what is the disease associated with bordetella pertussis?

whooping cough - respiratory illness

what are some characteristics of bordetella pertussis?

• gram (-)

• coccobacillus

• aerobe → NEEDS oxygen (as a respiratory pathogen)

how can we detect the growth of B.P.?

• culturing → growth takes about 3-5 days

• PCR (16S rRNA)

• direct fluorescent monoclonal antibody staining

does B.P. infect upper or lower respiratory tract?

• the upper respiratory tract

what are some important cells of note in terms of a B.P. infection?

• goblet cells → produce mucin protein → makes up mucus

• ciliated cells → sweep and help clear out debris (mucus + dead bacteria + dust etc)

what cells does B.P. tend to prefer or favour during pathogenesis?

• the ciliated cells

  • it prevents the clearance of mucus and debris → a lot of coughing → whooping cough

what are the 3 stages of whooping cough?

1. catarrhal

2. paroxysmal

3. convalescent

what is the catarrhal stage?

the first stage:

• incubation period is ~5-10 days

• think its just a cold

what is the paroxysmal stage?

second stage:

• 1-2 weeks

• brutal coughing - severe + rapid

• whooping sound

convalescent stage

last and final stage

• 2 weeks to several months

• “recovery” but can get worse if get a secondary infection

  • immune system is active

when was the first vaccine made for whooping cough?

1940’s

what type of vaccine was the first type of whooping cough vaccine?

whole cell

when was the acellular vaccine introduced?

the late 1990s

how many virulence factors does B.P. have?

7

how many B.P. virulence factors are in the pertussis vaccine?

5

what are the virulence factors present in the aP (acellular pertussis) vaccine?

1) pertactin (Prn)

2) pertussis toxin (PT)

3) fimbria 2

4) fimbria 3

5) filamentous hemagglutinin (FHA)

how effective is the pertussis vaccine?

• not very effective → requires multiple booster shots over the years (it’s very short-term)

what was a new vaccine campaign in 2014 against whooping cough?

• vaccinating pregnant mothers so that the fetus could receive the dose and perhaps build stronger immunity and not require as many boosters

• still acellular vaccine

vaccines and B.P. evolution?

with the rise of vaccines, we also saw a lot of diversity arise in B.P.

what are some of the virulence factors of B.P. with HIGH mutability?

• Prn

• PT

• Fim

what are the 7 virulence factors of B.P.?

1. PT (pertussis toxin)

2. Prn (pertactin)

3. Fim 2 (fimbrae 2)

4. Fim 3 (fimbrae 3)

5. FHA (filamentous hemagglutinin)

6. TCT (tracheal cytotoxin)

7. ACT (adenylate cyclase toxin)

pertussis toxin (PT) - what is it

• an A-B₅ toxin BUT 2 of the B subunits are the same!

  • eg. instead of S1, S2, S3, S4, S5, S6

    • we have: S1, S2, S3, S4, S4, S5

• bind to sugars on the receptor

• uses retrograde transport (golgi → ER)

in the vaccine!

pertussis toxin (PT) - what class of toxin is it?

• a class III toxin (A-B₅)

pertussis toxin (PT) - what does it do?

• steals ADP-ribosyl from NAD+

• ADP-ribosylates the Gi subunit of G protein

• Gi subunit usually inhibits AC (adenylase cyclase) so no cAMP made

• so PT locks AC into active state (it got rid of inhibitor)

• too much cAMP causes more fluid secretion in lung

  • sticky mucus that cilia can’t get rid of → excessive coughing → whooping cough

pertactin (Prn) - what is it?

• outer membrane protein

• secreted via T5SS autotransporter system

• acts as an adhesin (helps B.P. bind to the ciliary cells)

• has an RGD (arginine-glycine-aspartate) loop on the protein that binds to integrin on host cells

what is special about pertactin in regards to vaccines?

strains are emerging without pertactin

→ could be because of vaccines

regardless, this could make the current vaccine less effective if strains don’t have pertactin

what are the adhesive cell surface virulence factors?

• (Prn) pertactin

• fimbrae 2

• fimbrae 3

• FHA (filamentous hemagglutinin)

FHA (filamentous hemagglutinin)

• HUGE protein

• NOT a pilus

• adhesin binds to glycolipids on ciliated cells

• secreted via T5SS 2 partner system

FIM2 and FIM 3 (fimbrae 2 and fimbrae 3)

basically like pili

surface attachment

same function for both

• small subunits built up to make non-hollow thin structure

ACT (adenylate cyclase toxin/ CyaA)

• encoded by the gene cyaA

• secreted by T1SS

• inhibits immune cells

N terminal → adenylate cyclase activity → increase cAMP → more sticky mucus → more bad coughing → whooping cough

C terminal → forms pores in membrane from aggregation

TCT (tracheal cytotoxin)

• it is an endotoxin (made from peptidoglycan)

• prevents cilia movement

• causes shedding of ciliated cells

can be the cause of paroxysmal cough (whooping cough bc cannot clear airways of debris and constant coughing)

how is B.P. pathogenicity regulated?

• virulent state → Bvg +

  • more adhesion factors expressed

  • less flagella and motility genes expressed

• avirulent state → Bvg -

• run via BvgAS system and is temporally regulated between the two states

B.P. virulence gene class 1

• motility genes

B.P. virulence gene class 2

• adhesion factors

B.P. virulence gene class 3

• the intermediate genes

B.P. virulence gene class 4

• toxin production genes

BvgSA - 2 component regulatory system

BvgS = 3 part histidine sensor kinase (it autophosphorylates and hands the phosphate down its own chain and then passes it to BvgA)

BvgA = gets phosphorylated from BvgS and gets activated then binds to the promoter regions and tells the cell to make the genes

what are some activators for the BvgSA system?

→ we don’t know for in vivo

→ in vitro:

1) temperature

2) calcium

mediated by phosphorylation!! eg. temp/calcium triggers phosphorylation → activation

BvgSA and phase variation

phase variation = the switch between virulent/avirulent states

switch up rate is much higher than spontaneous mutation, so it must be on purpose

• caused by a frameshift mutation:

→ insertion mutation to go avirulent, and deletion mutation to go virulent

→ can be visualized on a plate with colony morphology (virulence factors are not expressed in Bvg -

is B.P. infectious once infected a human?

yes, its highly infectious