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.