Endotoxins

Endotoxin is the lipopolysaccharide (LPS) of the outer membrane of gram negative bacteria. They are bound to bacterium and are released only when the organism lyses since it is a part of cell wall. In contrast to Exotoxin, they are
- heat stable
- toxin only at high dose
- weakly immunogenic
- generally similar, regardless of source
- cannot be toxoided
- Induces many and different pharmacological and immunological changes at low and high concentration.
- At low amounts, elicits a series of reaction : fever, activation of complement by alternative pathway, activation of macrophages and stimulation of B-cells
- In large amounts, it produces shock and hypotension and even death.

Chemistry of Endotoxin:

Bacterial LPS is composed of three parts:

è A glycophospholipid called lipid A
- is a complex array of lipid sources
- Water insoluble since it is hydrophobic
- Responsible for toxicity
- Even when paired with artificial carriers, its activity is restored.

è A Core Polysaccharide with ethanolamine and phosphate
- Common to all gram negative
- serve as carrier

è ‘O’ Antigen
- A long side chain of species specific
- Unusual polysaccharide
- serve as carriers for lipid A

Major effects of Endotoxin:

- At low concentration it sets of series of alarm reactions.
- At high range, it induces shock.
- Overlapping of these complex events depends on the amount of Endotoxin, route of infection and previous exposure of host to those substances.
- The primary target cells of Endotoxin are mono-nuclear phagocytes (monocytes, macrophages of spleen, bone marrow, lung alveoli, peritoneal cavity and kuffer cells), neutrophils, platelets and B- lymphocytes since they have specific Endotoxin receptor.

Pharmacological toxins

Pharmacological toxins – Function by elevating or depressing normal cell functions but which do not result in death of their target cell.

Toxins that elevate cyclic AMP – Cholera toxin

These toxins raise the concentration of cyclic AMP (cAMP) without damaging the cell. The excess cAMP inhibits chemotaxis and phagocytosis, thus reducing their power to kill microorganisms. cAMP can be increased in several ways,
- some pathogens pour out cAMP themselves
- some secrete adenyl cyclase to make more cAMP from ATP
- secrete toxin that alters the activity of adenyl cyclase of host cells.

Cholera toxins (Best example)

It is an enterotoxin, a protein of mol.wt 90,000. Target tissue is the epithelium of the small intestine. It has separate A and B subunits
B component has specific affinity for the intestinal epithelial mucosa via gangliosiodic receptor.
A subunit has affinity to ADP-ribosylate of the target protein.
The target protein is part of a complex that makes cAMP. Synthesis of cAMP becomes unregulated and is made in large amount. This provokes loss of fluid and copious diarrhea which is the characteristic of Cholera.

Synthesis of cAMP

- The cyclic AMP is synthesized by the enzyme adenylate cyclase.
- It is composed of 3 proteins - Gs, R and cyclic itself.
- Gs protein is a GTP binding protein that has 2 conformational states.
- When it binds GTP, it stimulates adenylate cyclase to make cAMP.
- This effect is normally of short duration because Gs protein is also a GTPase that cleaves GTP to GDP.
- The activity of adenyl cyclase is thus determined by the balance of binding and hydrolysis of GTP by Gs protein.
- The balance of binding of GTP by Gs proteins is determined by R-protein which stimulates Gs.
- R-protein is a receptor for one of several hormones (adrenergic).
- Hence when R-protein binds one of these hormones, it interacts with Gs protein to increase its binding of GDP.
- Gs protein becomes active to stimulate adenyl cyclase.

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.