Clostridium perfringens Gram positive spore forming found in the - - PowerPoint PPT Presentation

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Clostridium perfringens Gram positive spore forming found in the - - PowerPoint PPT Presentation

May 18, 2023 377 likes •626 views

Clostridium perfringens Gram positive spore forming found in the gut of most animals and in soils, even desert sands types B & D cause enterotoxaemia in livestock Bacterial Pathogenicity Research Group Epsilontoxin

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SLIDE 1

Clostridium perfringens

Bacterial Pathogenicity Research Group

  • Gram positive
  • spore forming
  • found in the gut of most

animals

  • and in soils, even desert

sands

  • types B & D cause

enterotoxaemia in livestock

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SLIDE 2

Epsilon‐toxin (ε‐toxin) causes enterotoxaemia in livestock

  • Affects mainly sheep, lambs

and goats. Less often calves and other species

  • Toxin produced in the intestine
  • Crosses the gut wall.
  • causes damage in the kidneys

and brain

  • Rapidly fatal

Bacterial Pathogenicity Research Group

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SLIDE 3

Epsilon‐toxin (ε‐toxin) causes enterotoxaemia in livestock

  • Globally, almost all sheep (total 1172million animals), cows (total

996 million animals) and goats (total 862 million animals) are immunised with a crude vaccine.

  • The vaccine requires frequent boosting and is not very effective in

goats

  • There is an urgent need for an improved vaccine

Bacterial Pathogenicity Research Group

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SLIDE 4

Molecular structure of ε‐toxin monomer

Cole AR, Gibert M, Popoff M, Moss DS, Titball RW, Basak AK. Nat Struct Mol Biol. 2004 11:797‐8. Bacterial Pathogenicity Research Group

+ + +

  • 3 domains
  • Domain I is the proposed receptor

binding domain

  • Domains II and III are involved in
  • ligomerisation and pore formation
  • Possible second receptor binding

site in domain III

  • Until recently ε‐toxin was classified

as a category B bioterrorism agent by the US Department of Health

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SLIDE 5

‐toxin; mode of action

Bokori‐Brown, M. et al. (2016) Nature Communications 7, 11293 Binding Oligomerisation and pre‐pore formation Membrane insertion and pore formation

membrane cytoplasm

  • Forms heptameric pores ≥2 nm diameter in target cells
  • free passage of 1 kDa‐sized molecules
  • rapid decrease of intracellular K+
  • rapid increase of intracellular Na+ and Cl‐
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SLIDE 6

Mal is implicated in ‐toxin binding to cells

Bacterial Pathogenicity Research Group

  • Localised in compact myelin
  • Also found in T‐cells, some epithelial cells (e.g. in kidney, stomach

and thyroid gland)

  • Highly hydrophobic tetra span membrane proteolipid with 2

extracellular loops

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SLIDE 7

Binding of ε‐toxin to myelinic structures in mouse brain

7 Bacterial Pathogenicity Research Group

epsilon toxin is tagged with a green fluorescent protein Blue is a nuclear marker (TO‐PRO‐3) Co‐stained for myelin basic protein (red)

(Dorca‐Arévalo et al 2008) (Rumah et al 2013)

epsilon toxin is tagged with a Alexa 594 (red) A) Retinal section; pan‐vessel marker BSL1 (green) B) Mouse brain sections though corpus callosum; myelin marker proteolipid PLP (green)

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SLIDE 8

‐toxin and MS

8 Bacterial Pathogenicity Research Group

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SLIDE 9

The Global Distribution of MS (Marrie 2004)

9 Bacterial Pathogenicity Research Group

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SLIDE 10

MS; evidence that infection is involved

Bacterial Pathogenicity Research Group

  • The incidence of MS is linked to

geographical location

  • Individuals who move to high‐

incidence regions from low‐ incidence regions are at increased risk

  • Infection may trigger MS
  • Several candidates, but no

conclusive evidence

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SLIDE 11

11 Bacterial Pathogenicity Research Group

Evidence of a role for ‐toxin ?

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SLIDE 12

Evidence of a role for ‐toxin ?

12 Bacterial Pathogenicity Research Group

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SLIDE 13

Antibodies against epsilon‐toxin in US MS patient sera ?

13 Bacterial Pathogenicity Research Group

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SLIDE 14
  • C. perfringens type B producing ε‐toxin

isolated from a patient at first presentation

14 Bacterial Pathogenicity Research Group

  • C. perfringens producing ε‐toxin is not

considered to be a human pathogen

  • Isolation of C. perfringens producing ε‐

toxin only ever reported once before

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SLIDE 15

Serum Number of sera reactive with epsilon toxin by Western Blotting Number of sera reactive with epsilon toxin by Pepscan Combined MS patient 24% (n=129) 33% (n=43) 43% (n=43) controls 10% (n=129) 16% (n=37) 16% (n=37)

Antibodies to epsilon toxin in sera from UK MS patients

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SLIDE 16

‐toxin vaccines

16 Bacterial Pathogenicity Research Group

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SLIDE 17

Current commercial veterinary ‐toxin vaccines

  • Formaldehyde‐treated bacterial culture filtrates (toxoids)

– production requires the growth of Clostridium perfringens – contain proteins in addition to the Etx toxoid – yield of vaccine can be low & variable – immunogenicity low & variable

  • typical immunisation regimens involve an initial course of

two doses of vaccine, 2‐6 weeks apart

  • sheep are boosted at 6‐12 months
  • goats are boosted every 3‐4 months

– inflammatory responses

  • reduced feed consumption

Bacterial Pathogenicity Research Group

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SLIDE 18

Genetic toxoid

Bacterial Pathogenicity Research Group Y196A Y30A A168F

  • Y30 and Y196 mutations (in red) are

in the receptor‐binding domain

  • A168F mutation (in green) in the

pore‐forming domain

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SLIDE 19

Toxicity of the Y30AY196AA168F mutant

Bacterial Pathogenicity Research Group

tested toxicity MDCK cells toxicity (mice) toxicity CHO‐sheep MAL cells toxicity CHO‐human MAL cells Wild type toxin 9.7nM 20 ng – 200 ng 1.6nM 12.7nM Y30AY196AA168F > 6µM >20 µg † > 3µM † > 2.5µM †

† highest dose tested

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SLIDE 20

Toxicity (haemolysis) towards human red cells

10 20 30 40 50 60 70 80 90 100 Wild Type Etx Y30AY196A+A168F Tx100 PBS

% haemolysis

10µM Activated toxin 1µM Acitvated toxin Prototoxin (10µM)

  • Results expressed with respect to

1% Tx100 control (100% haemolysis)

  • PBS controls show baseline

hemolysis

  • Wild type toxin is active towards

human red cells

  • Y30AY196AA168F mutant is

inactive towards human red cells

Washed human red cells (6.6% v/v) incubated with toxin for 1 hour at 37oC and hemolysis measured as haemoglobin release

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SLIDE 21

Y30A‐Y196A‐A168F vaccine

  • 12 month old lambs
  • 200g dose given twice (at the start and week 3) with

Montanide ISA 61VG or alhydrogel adjuvant

  • Sheep bled 9 weeks following 2nd dose

Bacterial Pathogenicity Research Group

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SLIDE 22

Y30A‐Y196A‐A168F vaccine

Bacterial Pathogenicity Research Group

Treatment Group Neutralising antibody (IU/ml) measured using competition ELISA assay (week 13) Neutralising antibody (IU/ml) measured using CHO‐sMal cells (week 13) Control Y30A‐Y196A‐A168F + montanide ISA 61VG adjuvant 200 IU/ml (107 IU/ml after one dose) >160 IU/ml

12 months after the last immunisation the level of neutralising antibody was European Pharmacopoeia requires that the potency of the pooled sera is not less than 5 IU of epsilon antitoxin per millilitre.

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SLIDE 23

Conclusions

  • There is a need for an improved vaccines against ε‐toxin for

use in livestock

  • A genetic toxoid vaccine can induce high levels of protective

antibody against epsilon toxin

  • This vaccine may have a utility for the prevention or

treatment of MS

Bacterial Pathogenicity Research Group