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