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Bacterial spore
This document discusses bacterial spores and their potential applications. It provides background on spore structure and the genes involved in spore formation. Spores can survive harsh conditions for many years due to their protective coat. This protective property makes spores useful for vaccine delivery, as they can display foreign antigens on their coat and induce immune responses when administered. The document explores using spores that display tetanus and E. coli antigens as oral vaccines. It also discusses using spores as biosensors, where germination in response to certain molecules could generate detectable signals. Finally, the document briefly outlines additional applications of spores in energy generation, self-healing concrete, and cancer treatment.
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Bacterial spore
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- 2. Facts Known • Bacterialspores- 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 assemblingand 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 Endosporestructure 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
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- 6. Spore coat • Outer-electron dense- 5 principal polypeptides, CotA (65 kDa), CotB (59 kDa), CotG (24 kDa), CotC (11 kDa) and CotF (8 kDa) (Martins et al., 2002) • CotA- multi-copper oxidase (Zilhao et al., 2004) • CotG and CotC- unusual amino acid sequences: 12–13 amino acids rich in lysines and tyrosines (Hullo et al., 2001) • Spore coat- flexible- contract and expand
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- 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 • CotB 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)
- 10. Genes and stageof spore formation
- 11. Applications of sporedisplay • 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 Vaccinevehicle • 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 toCotB-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, theN-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
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- 16. Payers patches Immunity andmechanism • CotBΔ105-TTFC used orally as vaccine • Serum IgG and faecal sIgA- seroconversion to TTFC M APC Spores SporeGen (UK)- SporeVax®
- 17. Ways to improvespore 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 • Vaccinesneed 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 administrationof 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 • Lossof 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 sporebiosensor • 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)
- 29. Spore Germinant Enzymes Substrate Principle of sporebased biosensor
- 30. Germinants S. No Bacterium Germinants 1 Bacilluscereus 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 GerminogenicEnzyme 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 inmilk • 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 actas germinant AFM1 halts spore germination Esterase act on substrate Indigo colour- negative for AFM1 No reduction of substrate- AFM1 positive Spore
- 34. Beta lactam inmilk • 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 residuesin milk- Bacillus Stearothermophilus • Microbial contaminants in milk- detecting E. coli, S. aureus and Listeria monocytogenes • Substrate: diacetate fluorescein (DAF)
- 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
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- 39. Amount of currentproduced • 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 • Cancertreatment- Clostridium • Probiotic • Crude oil extraction
- 42. Conclusion • Spores hasmultiple 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
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