2. Facts Known
• Bacterial spores- dormant form of life
• Nutrient Starvation, Temperature or pH extremes,
Cell crowding, Antibiotic exposure
• Survives harsh conditions
• Can survive several years (Cano, 1995)
• Bacillus and Clostridium- common endospore
formers
• B. anthracis spore
– threat commonly heard
3. Spore
• Self assembling and protective property
• Spores can survive in desiccated state
• Resist high temperatures and toxic chemicals
(Nicholson et al., 1995)
• Coat Consist of proteins- proteomeric
subunits arranged
• Bacillus and Clostridium- 2 major endospore
formers
• Bacillus- grow aerobically (Ricca et al., 2003)
4. Outer spore coat
Endospore structure
Chromosomes in core
Peptidoglycan- cortex
Lipids- cortex
Inner spore coat
Organic solvents
Lysozyme
•Hundreds of genes- for spore formation
•Bacillus subtilis > 25 coat protein present in 2 layers
8. Advantages
• Robustness – easy storage (storage
studies- recombinant protein spores also
stable)
• Ease of production
• Safe and easy genetic manipulation –
Bacillus subtilis, B. clausii, B. coagulans, B.
cereus, and B. natto (Ricca et al., 2003)
9. Carrier proteins
• Cot B and Cot C- primary targets
• Cot B- located in surface
• Cot C- found in abundance
• Both are essential for spore formation
and germination
(Ricca et al., 2003)
11. Applications of spore display
• Identification of new antibiotics
• Identification of antigens
• Delivery of vaccines and drugs
• Identifying new Receptors
• Selection of DNA-Binding Proteins
• Drug discovery (Pan et al., 2012)
12. Spore as Vaccine vehicle
• Non-toxic 459 amino acid C-terminal
fragment of the tetanus toxin (TTFC)-
encoded by the tetC gene of Clostridium
tetani
• 103 amino acid B subunit of the heat labile
toxin of enterotoxigenic strains of
Escherichia coli (LTB) -eltB gene
(Douce et al., 1997)
13. cotb
• Accessible to CotB-specific antibodies- Present in
outer coat
• CotB gene under control of σK and the DNA-binding
protein GerE
• CotB- in mother spore, assemble around forming
spore
Strategies:
1. Use of the cotB gene and its promoter for the
construction of translational fusions
2. Chromosomal integration of the cotB-tetC and cotB-
eltB gene fusions into the coding sequence of the
non-essential gene amyE (Duc et al., 2003)
14. CotB
• C-terminal, the N-terminal or in the middle of CotB
• C-terminal end- faulty assembly of proteins
• Deletion of three 27 amino acid repeats, CotBΔ105-
TTFC: correct assembly
• CotBΔ105-LTB- reduced sporulation and germination
& not resistant to lysozyme
Reason: Homology between chimeric protein & LytF, a
cell wall-associated endopeptidase
16. Payers patches
Immunity and mechanism
• CotBΔ105-TTFC used orally as vaccine
• Serum IgG and faecal sIgA- seroconversion
to TTFC
M
APC
Spores
SporeGen (UK)- SporeVax®
17. Ways to improve spore display
• Late sporulation-specific autoinduction system
• Improving expression system
• Folding of proteins should be evaluated
• Target protein- protease resistant
• Engineered protein- improve the efficiency of
display
(Pan et al., 2012)
18. As adjuvant
• Vaccines need adjuvant that can enhance broad
polyvalent adaptive immune responses
• Synthetic molecular adjuvants based on TLR agonists
• Single synthetic agonists- less immune response
• More PRR (pattern recognition receptor) activator-
good immune response
• CotM and CotP- similar to α-crystalline family of HSP-
stimulate DC activation (Barnes et al., 2007)
19. Studies with TT
• B. subtilis- non pathogenic –no mechanism to immune
system- may evoke immunity
• Spores+ TT s/c- BALB/c mice - 11 days post
immunization- > antibodies against TT than only TT
• There is dose dependent increase in antibody to
spores
• Chances of antispore antibodies- less
• i/n- IgA antibody-secreting cells seen in NALT
20. Special about spore
• Spores through i/n- also induce systemic immunity
• Spores induce both self and non self T cell
response
• Spore present antigen to both MHC I and MHC II
• Cross priming of exogenous antigen through
prolonged stimulation of CD86 and CD40L
(Barnes et al., 2007)
21. For H5N1
• Inactive, killed form of Bacillus
2 ways: 1- Virion adsorbed to spore surface
• Proteins can readily bind to negatively charged spores
• Hydrophobic bonding
• Lipid component of the viral envelope contributes to
binding
2- virion in unbound form with spore
(Song et al., 2012)
Heat killed spore
22. • i/n administration of spore+ H5N1 = good mucosal
immunity
• Both bound and unbound- same response in immunity
(Song et al., 2012) (contradictory Souza et al., 2014)
Reason- spore elicit innate immunity
• Spores stimulate maturation of DCs
• Recruit NK cells to lungs
• Induce expression of the NK-B pathway
• TLR interaction with more than one spore ligand
(Souza et al., 2014)
23. Are spores Useless
• No, spores act both as vaccine vehicle and
adjuvant (Iwanicki et al., 2014)
• As vehicle it is similar to liposomes,
ISCOMs and emulsions
Advantage:
• Easy production and stability
• Heat killed spore- as efficient as live
spore
24. Studies with spores
• Recombinant HIV gag p24 protein
• Ovalbumin (Barnes et al., 2007)
• Clostridium perfringens alpha toxin
(Hoang et al., 2008)
• Adjuvant effect with co administered DNA
vaccine
• Mycobacterium tuberculosis
• Rabies (Nascimento et al., 2012)
• Enterovirus 71 (Cao et al., 2013)
25. Biosensors
• Analytical device, used for the
detection of an analyte, that combines a
biological component with a
physicochemical detector
• Inner spore coat- small acid soluble
proteins SASPs- protect nucleic acids
26. Germination
• Requires signals: single amino acids, sugars or
purine nucleosides, combinations of nutrients
• Asparagine, glucose, fructose and K+ - spore
germination in B. subtilis
• Signals- species and strain specific
• Receptors present in inner membrane eg: GRs
of B. subtilis GerA (Rotman, 2001)
27. During germination
• Loss of refractivity
• Release of Ca2+ DPA
• Partial dehydration
• Cortex degradation- reactivation of enzymes-
synthesis of ATP from 3-PGA (3-posphoglyceric
acid)
• SASP degraded- release DNA
(Setlow, 2007)
28. Advantages of spore biosensor
• Long shelf life at room temperature
• Germination within minutes of sensing
germinants- real time response for detection
of analyte
• Production is a low priced process and its
immobilization –effortless
(Kumar et al., 2013)
30. Germinants
S.
No
Bacterium Germinants
1 Bacillus cereus Adenosine, or Inosine and L-alanine
2 Bacillus
licheniformis
Glucose or Inosine
3 Bacillus megaterium Glucose or L-proline
4 Bacillus
stearothermophilus
L-leucine or L-valine
5 Bacillus subtilis L-alanine
(Manafi et al., 1991)
31. Enzyme and substrate
S.
No
Germinogenic Enzyme Germinogenic Substrate
1 Alanine aminopeptidase L-alanoyl L-alanine
2 Pyroglutamyl
aminopeptidase
L-pyroglutamyl-L-alanine
3 Proteases Benzoyl-L-arginyl-L-alanine
4 Coagulase N-tosyl-glycyl-1-prolyl-L-arginyl-L-
alanine
5 Esterases L-alanyl-ethanol
6 Phosphatases Adenosine 3’-monophosphate
7 β-D-Galactosidase Adenosine-β-D-galactopyranoside
8 β-D-Glucuronidase Adenosine-β-D-glucuronide
9 β-Lactamase II L-alanine-cephalosporin or
Adenosine-cephalosporin
32. Aflatoxin M1 in milk
• Spore inhibition based-enzyme substrate assay (SIB-
ESA) - Bacillus megaterium
• Indoxyl acetate- Esterase release indoxyl- Indigo
colour
• *Kumar, N., Singh, N. A., Singh, V. K., Bhand, S., Malik, R. K., 2010. (Patent # 3064/DEL/2010).
Assigned to ICAR N Delhi. "Spore inhibition based enzyme substrate assay for monitoring
Aflatoxin M1 in milk". *(Published in IPO Journal - 46/ 2012 dated 16/ 11/ 2012)
For identification of Campylobacter, Helicobacter, Wolinella-
hydrolyze indoxyl acetate
33. Principle
Milk
Indoxyl acetate
Milk act as
germinant
AFM1 halts
spore
germination
Esterase act on
substrate
Indigo
colour-
negative for
AFM1
No reduction of
substrate- AFM1
positive
Spore
34. Beta lactam in milk
• B. cereus and B. licheniformis – produce β-
lactamase enzyme in presence of β-lactam
• Amount of enzyme produced = concentration of
inducer (β-lactam)
• Starch iodine- colour indicator
• Penicilloic acid - contain a non-acylated amino group
which is capable of reducing iodine to iodide
(Patent Reg No. 115/DEL/2009)
35. Similarly….
• Antibiotic residues in milk- Bacillus
Stearothermophilus
• Microbial contaminants in milk-
detecting E. coli, S. aureus and Listeria
monocytogenes
• Substrate: diacetate fluorescein (DAF)
36.
37. Biogenerators
• More RH- expand
• Less RH- Shrink
• Water absorption & release- cortex-
expansion due to cortex
• Spore responds within ~0.4 s of RH and ~
0.5 s RH
• B. subtilis- lack exosporium (Sahin et al., 2012)
• Spores over microcantilver/ latex sheets
39. Amount of current produced
• 0.5-mm-thick rubber sheet
• ~0.7 mW for 3 mg of spores
• ~233 mW/kg of spore (Chen et al., 2014)
Laser point- 5mW
40. Self healing concretes
• Bacterial spores + organic compounds packed with
concrete
• Spores remain dormant-till cracks form
• Water seeps into crack- spores activated- feed on
calcium lactate
• Calcium lactate+ oxygen
• Conversion of calcium lactate to calcium carbonate
(Calcite) (Wang et al., 2014)
41. Other uses
• Cancer treatment- Clostridium
• Probiotic
• Crude oil extraction
42. Conclusion
• Spores has multiple applications
• Studies required for its adjuvant properties
and as a vaccine vehicle
• Spore based biosensor shows promising
results
• Biogenerators & self cure concretes- needs
special attention
Spores- Life in dormant stage
