Upload
karthik-kumaragurubaran
View
181
Download
6
Embed Size (px)
DESCRIPTION
Citation preview
K. Karthik,1689
Bacterial spores and their microbiological applications
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
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)
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
Spore display
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
Spore coat proteins
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)
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)
Genes and stage of spore formation
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)
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)
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 fusions2. 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)
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
The construct
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®
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)
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)
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
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)
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
• 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)
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
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)
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
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)
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)
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)
Spore
Germinant
Enzymes
Substrate
Principle of spore based biosensor
GerminantsS. No
Bacterium Germinants
1 Bacillus cereus Adenosine, or Inosine and L-alanine2 Bacillus
licheniformisGlucose 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)
Enzyme and substrateS. No
Germinogenic Enzyme
Germinogenic Substrate
1 Alanine aminopeptidase L-alanoyl L-alanine2 Pyroglutamyl
aminopeptidaseL-pyroglutamyl-L-alanine
3 Proteases Benzoyl-L-arginyl-L-alanine4 Coagulase N-tosyl-glycyl-1-prolyl-L-arginyl-L-
alanine5 Esterases L-alanyl-ethanol6 Phosphatases Adenosine 3’-monophosphate7 β-D-Galactosidase Adenosine-β-D-galactopyranoside8 β-D-Glucuronidase Adenosine-β-D-glucuronide9 β-Lactamase II L-alanine-cephalosporin or
Adenosine-cephalosporin
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
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
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)
Similarly….
• Antibiotic residues in milk- Bacillus
Stearothermophilus
• Microbial contaminants in milk- detecting E. coli, S. aureus and Listeria
monocytogenes
• Substrate: diacetate fluorescein (DAF)
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
Mechanism
RHN S
Current produced
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
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)
Other uses• Cancer treatment- Clostridium• Probiotic• Crude oil extraction
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
SPORES
Thank you