Toxin - Mode of action

The A domain remains in the latent form even after uptake, gets activated only by proteolytic cleavage and reduction of disulfide bond. All form of these toxins have common mode of action - Catalyze transfer of the ADP group from the coenzyme.

Diphtheria toxin

- Its a protein of about 62,000 mol.wt
- The A and B domains are synthesized as single polypeptide chain
- The AB dimeric domain undergoes endocytosis and membrane translocation (out of vesicles with cytoplasm) to release domain A to reach cytoplasm and begin its toxic action.
- Domain A is resistant to denaturizing and is long-lived inside the cell.
- A single molecule can kill a cell
- Killing takes place by catalyzing ADP-ribosylation (addition of ADP-ribose group) to the eukaryotic elongation factor 2(EF2).
- EF2 is a protein that catalyzes the hydrolysis of GTP, which drives the movement of ribosome on eukaryotic mRNA.
- The substance for this reaction is the coenzyme NAD.

NAD+ + EF-2 =======>> ADP-ribosyl-EF2 + Nicotinamide

- The modified EF2 protein cannot participate in the elongation chain of protein synthesis and the cell dies because it can no longer synthesis protein.
- EF2 is the only known substrate for diphtheria toxin, and the specificity is that EF2 contains a rare modification in one of its histidine residues and this site is recognized by diphtheria toxin for ADP-ribosylation.
- Mutant cells cannot modify histidine and become resistant to the toxin.
- This leads to myocarditis, neuritis, necrosis of mucous membrane.

Mode of entry into the cell - Receptor mediated endocytosis

Dimeric exotoxin binds to a receptor-ligand complex that is internalized in a clathrin-coated pit that pinches off to become a coated vesicle. By the time, the molecule A and B are cleaved at protease sensitive site, but remains covalantly associated. Clathrin coat depolymerizes resulting in an uncoated vesicle (endosome). pH in the endosome decreases owing to the AT activity by reduction of disulphide bond. Low pH causes A and B component to separate, which is called as CURL ( Compartment of Uncoupling of Receptor and Ligand ). B domain is then recycled to cell surface. A domain moves through the cytosol, and inhibits protein synthesis.

Toxins - A Brief Introduction

Toxins are biological weapons and a specific soluble metabolic product of microorganisms directed at us, which causes deleterious effect on host. It is produced by Bacteria, Fungi, Protozoa and Worms. Their mode of action paves way to understand the pathophysiology of infection. Toxemia refers to the condition caused by toxins that have entered the blood of host.

Types of toxins – Exotoxin, Endotoxin.

Exotoxins

- Synthesized by specific pathogens that often has plasmids or prophages bearing the Exotoxin gene (mainly Gram +, sometimes Gram -). Ex: E.Coli, Vibrio, Shigella.
- Soluble protein, usually released in the surrounding as the pathogens grow.
- In many instances, they travel far from the site of infection to other target cells.
- Sometimes bound to Bacterial surfaces, released on cell lysis.
- Heat-labile proteins inactivated at 60-80c
- Mostly lethal – 1gm of tetanus, botulium or Shigella toxin is enough to kill 10 million people.
- Highly immunogenic and stimulate the production of neutralizing antibodies (antitoxins).
- Easily inactivated by formaldehyde, iodine to form immunogenic toxoids.
- Usually unable to produce fever.
- Categorized as neurotoxins, cytotoxins or enterotoxins based on their mechanism of action.

Structural Model

It occurs in many forms, the general structural model to which they frequently conform is AB model.
‘A’ Subunit – an enzymatic subunit, responsible for toxic effect in host cell
‘B’ Subunit – binding subunit, binds to target cells but non toxic and biologically inactive. Binds with specific receptors on target cells (or) tissue such as sialogangliosides.
Ex: Gm1 for Cholera toxin, GT1/GD1 for Tetanus toxin, GD1 for Botulinum toxin.

Entry of Subunits

Several mechanisms are proposed by the toxins. In one mechanism B subunit inserts into the membrane and creates a pore for the A subunit to enter. In another, entry is by receptor mediated endocytosis. The mode of entry is explained in the diagram given above.

Isolation & Characterization Of Leuconostoc Mesenteroides From Cheese – Part 3

RESULTS AND DISCUSSION:
Isolation of Leuconostoc:

From the cheddar cheese, two types of colonies were isolated. One colony was yellow colored shiny, smooth and entire marginated. And the other was pale white, slimy, smooth colonies. Gram staining results showed: 1) gram positive bacilli, 2) gram positive cocci, and 3) gram positive coccobacilli. So to isolate Leuconostoc from other LAB organisms, vancomycin was added to the MRS medium.

Characterization of Leuconostoc:
Cultural & Biochemical characteristics - Results
Gram’s staining Gram positive, coccobacilli
Motility Motile
Capsule staining Non-capsulated
Spore staining Non-sporing
Growth at 37oC +
Growth at 30oC +
Growth at 4oC +

On MRS medium:
At 37oC Pale yellow colored, smooth, slimy,
entire marginated, convex colonies.

At 4oC In refrigeration, colonies turned white,
and slow growth was observed.

On MacConkey agar medium Lactose fermenting, pink colored
colonies.

On blood agar medium Non-hemolytic, colorless colonies.

Biochemical tests:
Indole -
Methyl red -
Voges Proskauer +
Citrate -
Urease -
Arginine -

Carbohydrate fermentation tests:
Glucose + (acid and gas)
Arabinose +
Lactose +
Mannitol -
Sucrose +
Dextrose +
Maltose +

Discussion:

From the three different colonies isolated from cheese (fig-1), Leuconostoc was isolated separately by its specific vancomycin resistant character (fig-2). Species was identified by its biochemical and carbohydrate tests. First, Leuconostoc mesenteroides was differentiated on the basis of Arginine hydrolysis. Only L.cremoris and L.mesenteroides is Arginine hydrolyser and both are possible in cheese. Secondly, Arabinose fermenters. Between both the species, only L.mesenteroides is Arabinose fermenters (Bettache Guessas and Mebrouk Kihal, 2004). Thus the species was confirmed as Leuconostoc mesenteroides.

Isolation and Characterization of Leuconostoc Mesenteroides from Cheese – Part 2

Materials & Methods

1. Sample collection - Cheddar cheese & Mozzarella cheese
2. Serial dilution and spread plate method
3. Isolation of Lactic acid bacteria in MRS medium
4. Selective Isolation of Leuconostoc from other LAB
5. Addition of vancomycin to MRS medium
6. Leuconostoc was isolated and maintained at 4oC
7. Species identification

Biochemical characterization

Sample collection: Cheddar cheese and Mozzarella cheese used in their study were obtained from retail super market (Nilgiris), Chennai. A well vacuum packed cheese was obtained.

Bacterial strains and culture:

Isolation of Leuconostoc:

Culture Media: Leuconostoc are fastidious chemo-organotrophic bacteria. Therefore, culture media must be rich in nutrients such as carbohydrates as energy providers, amino acids as nitrogen compounds, salts and vitamins. The most widely used medium was MRS medium (Man Rogosa Sharpe). The supplements studied in MRS medium are glucose, peptone, yeast extract, beef extract, dipottasium hydrogen phosphate, ammonium citrate, magnesium sulphate, manganese sulphate sodium acetate, and tween 80. Tween 80 usually increases the growth of Leuconostoc by providing oleic acid incorporated into the cell membrane.

Composition of MRS medium

Media gm/lit

Glucose 1.0gm
Peptone 1.0gm
Beef extract 0.8gm
Yeast extracts 0.5gm
Di-potassium hydrogen phosphate
(K2HPo4) 0.2gm
Magnesium sulphate (MgSo4) 0.2gm
Manganese sulphate (MnSo4) 0.005gm
Ammonium citrate 0.2gm
Sodium acetate 0.5gm
Tween 80 0.1gm
Agar 2.0gm
pH 6.8gm

The medium was sterilized at 121oC for 15mins. Leuconostoc was enumerated by spread plate method. Serial dilutions were made from the master dilutions containing 1gm of cheddar cheese in 99ml of distilled water which gives a dilution of 10-1 (serial dilutions were made upto 10-1). 0.1ml of each dilution was spreaded on MRS medium, and incubated at 37oC for 24hrs.

From food sample, all LAB are counted since the complex nutrient requirements and optimal conditions for the growth are roughly comparable for lactobacillus, Pediococcus and Leuconostoc. So, for selective isolation of Leuconostoc from other lactic acid bacteria (vancomycin sensitive), 30mg/ml of vancomycin solution was added to the MRS medium. The Leuconostoc strain isolated was maintained by sub-culturing in MRS medium.

Indicator bacterial strains:

Salmonella typhi, ATCC-21563
Escherichia coli, ATCC-1023
Shigella flexinerrae, ATCC-11461

were received from NCIM and maintained by sub-culturing in nutrient agar slants for further biochemical tests. The incubation period for the test organisms was 37oC for 24hrs. All the cultures were stored at 4oC.

Media used for cultural characterization of Leuconostoc:

MacConkey agar medium
Blood agar medium

Biochemical characterization of Leuconostoc for species identification:

Gram’s staining
Motility test – hanging drop method
Capsule staining – negative staining
Spore staining – Scaffer-fulton method

Biochemical tests:

Indole
Methyl red
Voges Proskauer
Citrate
Nitrate
Urease
Arginine hydrolysis

Carbohydrate fermentation test:

Glucose
Arabinose
Fructose
Sucrose
Maltose
Galactose
Lactose
Mannital

All the above biochemical tests were performed and the results were noted to differentiate Leuconostoc species. The differentiating test among Leuconostoc species are catalase test, arginine test and carbohydrate fermentation tests.

Separate biochemical test were performed for the indicator organisms to confirm the strains and results were noted.

Isolation and Characterization of Leuconostoc Meseteroides from Cheese – Part 1

Leuconostoc:

This genus was called as Betacoccus by Oria – Jensen. It contains the heterofermentative lactic bacteria, which ferments sugars to produce 50% lactic acid, plus, 25% acetic acid and ethyl alcohol and 25% carbon dioxide. These other compounds are important as they impart particular tastes and aromas to the final product. The major subsps of Leuconostoc are,

Leuconostoc mesenteroides
Leuconostoc dextranicum
Leuconostoc cremoris
Leuconostoc lactis

Sources Of Leuconostic:

Leuconostoc are used as mesophilic starters in combination with other starters for making cottage and cream cheese, cultured milk, buttermilk. It produces acetic acid from citrated and may be added to cottage cheese to prevent spoilage by slime producing Pseudomonades. Leuconostoc are also found in milk and milk products, vegetables, plant surfaces. Dextrans produced by Leuconostoc mesenteroides are used as stabilizers in ice cream mixes.

Special Focus On Leuconostoc MesenteroidesS:

Leuconostoc mesenteroides differs from other lactic acid species as it can tolerate fairly high concentration of salt and sugars (unto 50% sugar). Leuconostoc mesenteroides initiate growth in vegetables more rapidly over a range of temperatures and salt concentration than any other lactic acid bacteria. It produces carbon dioxide and acids which rapidly lower the pH and inhibit the development of undesirable microorganisms. The carbon dioxide produced replaces the oxygen, making the environment anaerobic and suitable for the growth of subsequent species of lactobacillus and inhibits the growth of aerobic micro flora. Removal of oxygen also helps to preserve the color of vegetables and stabilizes any ascorbic acid that is present.

These characters of Leuconostoc mesenteroides made me to focus on its isolation and characterization, further more antimicrobial activity, bacteriocin.

Growth Requirements

Leuconostoc have a complex growth requirement with an optimum temperature of 20-30°C. They are fastidious chemo organotrophic bacteria requiring rich nutrient sources such as carbohydrates, nitrogen sources, salts, vitamins and sugars. The medium used for the isolation and enumeration of Leuconostoc was MRS medium (Man Rogosa Sharpe Medium). MRS medium contains all the essential substrates for the growth of Leuconostoc.

In isolating Leuconostoc from nature or food samples, the combination of the presence of agents inhibitory to most lactic acid bacteria other than Leuconostoc spp and the agents stimulatory for Leuconostoc spp in the medium selects for Leuconostoc spp.

The vancomycin resistant character of Leuconostoc spp. favored for the isolation from cheese sample. Other lactic acid bacteria are vancomycin sensitive. MRS medium containing 30mg/ml concentration of vancomycin was used as the special medium for Leuconostoc isolation. Comprehensive reviews of Leuconostoc differential and selective medium have been published by Garvie, Tenber & Gets and Cogan.

My first blog in microbiology

I’ve completed M.Sc., in Microbiology and I would like to share all my knowledge and experiences in my field of biology. Hope this blog will help the upcoming students and teachers too…