|Endotoxins are part of the outer cell wall of bacteria.
Endotoxins are invariably associated with Gram-negative bacteria as
constituents of the outer membrane of the cell wall. Although the term endotoxin
is occasionally used to refer to any "cell-associated" bacterial
toxin, it should be reserved for the lipopolysaccharide complex associated
with the outer envelope of Gram-negative bacteria such as E. coli, Salmonella,
Shigella, Pseudomonas, Neisseria, Haemophilus,
and other leading pathogens.
The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory responses in an animal. Because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of Gram-negative bacterial infections.
The relationship of endotoxins to the bacterial cell surface is
illustrated in Figure 1 below.
Figure 1. Structure of the cell surface of a Gram-negative bacterium
Gram-negative bacteria probably release minute amounts of endotoxin while growing. For example, it is known, that small amounts of endotoxin may be released in a soluble form, especially by young cultures. However, for the most part, endotoxins remain associated with the cell wall until disintegration of the bacteria. In vivo , this results from autolysis of the bacteria, external lysis mediated by complement and lysozyme, and phagocytic digestion of bacterial cells.
Compared to the classic exotoxins of bacteria, endotoxins are less potent and less specific in their action, since they do not act enzymatically. Endotoxins are heat stable (boiling for 30 minutes does not destabilize endotoxin), but certain powerful oxidizing agents such as superoxide, peroxide and hypochlorite, degrade them. Endotoxins, although strongly antigenic, cannot be converted to toxoids. A comparison of the properties of bacterial endotoxins and classic exotoxins is shown in Table 1.
Lipopolysaccharides participate in a number of outer membrane functions that are essential for bacterial growth and survival, especially within the context of a host-parasite interaction. An intact outer membrane exerts several vital functions in Gram-negative bacteria:
Chemical Nature of Endotoxin. Most of the work on the chemical structure of endotoxin has been done with species of Salmonella and E. coli. LPS can be extracted from whole cells by treatment with 45% phenol at 90o. Mild hydrolysis of LPS yields Lipid A plus polysaccharide.
Lipopolysaccharides are complex amphiphilic molecules with a mw of about 10kDa, that vary widely in chemical composition both between and among bacterial species The general architecture of LPS is shown in Figure 2. The general structure of Salmonella LPS is shown in Figure 3 and the complete structure of Salmonella lipid A is illustrated in Figure 4.
Glc = glucose; GlcNac = N-acetyl- glucosamine; Gal = galactose; Hep = heptose; P = phosphate; Etn = ethanolamine; R1 and R2 = phoshoethanolamine or aminoarabinose. Ra to Re indicate incomplete forms of LPS. The Rd2 phenotype (not shown) would have only a single heptose unit. The Rc, Rd2, and Rd1 mutants lack the phosphate group attached to Hep.
LPS consists of three components or regions:
The structure of LPS in Salmonella typhimurium and E. coli is seen in Figure 3). The elucidation of the structure of LPS relied heavily on the availability of mutants each blocked at a particular step in LPS synthesis. The biosynthesis of LPS is strictly sequential. The core sugars are added sequentially to Lipid A by successive additions, and the O side chain is added last, one preassembled subunit at a time. The properties of mutants producing incomplete LPS molecules suggests the nature and biological functions performed by various parts of the LPS molecule:
LPS and virulence of Gram-negative bacteria
Both Lipid A (the toxic component of LPS) and the polysaccharide side chains (the nontoxic but immunogenic portion of LPS) act as determinants of virulence in Gram-negative bacteria. Virulence and the property of "smoothness" (associated with an intact O polysaccharide) are regularly associated in many bacterial infections. The polysaccharide chain must also be important for virulence as shown by the fact that small changes in the sugar sequences in the side chains of LPS, result in major changes in virulence. How are the polysaccharide side chains involved in the expression of virulence? There are a number of possibilities:
a. Smooth antigens could allow organisms to adhere specifically to certain tissues, especially epithelial tissues.
b. Smooth antigens probably allow resistance to phagocytes, since rough mutants are more readily engulfed and destroyed by phagocytes.
c. The hydrophilic O polysaccharides could act as water-solubilizing carriers for toxic Lipid A. It is known that the exact structure of the polysaccharide can greatly influence water binding capacity at the cell surface.
d. The O antigens could provide protection from damaging reactions with antibody and complement. Rough strains of Gram-negative bacteria derived from virulent strains are generally non virulent. Smooth strains have polysaccharide "whiskers" which bear O antigens projecting from the cell surface. The O antigens are the key targets for the action of host antibody and complement, but when the reaction takes place at the tips of the polysaccharide chains, a significant distance external to the general bacterial cell surface, complement fails to have its normal lytic effect. Such bacteria are virulent because of this resistance to immune forces of the host. If the projecting polysaccharide chains are shortened or removed, antibody reacts with antigens on the general bacterial surface, or very close to it, and complement can lyse the bacteria (Thus, "rough" colonial strains are non virulent.).
Biological Properties of Endotoxins
Endotoxins are toxic to most mammals. Even though endotoxins are strong antigens, they seldom elicit immune responses which gives full protection to the animal against secondary challenge with the endotoxin. They cannot be toxoided. Regardless of the bacterial source, all endotoxins produce the same range of biological effects in the animal host.
Most of our knowledge of the biological activities of endotoxins derives not from the study of natural disease but by challenge of experimental animals.
The injection of living or killed Gram-negative cells, or purified LPS, into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions such as:
Injection of large doses of endotoxin results in death in most mammals. The sequence of events follows a regular pattern: (1) latent period; (2) physiological distress (diarrhea, prostration, shock); (3) death. How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal. Animals vary in their susceptibility to endotoxin
Since Lipid A is embedded in the outer membrane of bacterial cells, it probably only exerts its toxic effects when released from multiplying cells in a soluble form, or when the bacteria are lysed as a result of autolysis, complement and the membrane attack complex (MAC), ingestion and killing by phagocytes, or killing with certain types of antibiotics. It is thought that LPS released into the bloodstream by lysing Gram-negative bacteria is first bound by certain plasma proteins identified as LPS-binding proteins. The LPS-binding protein complex interacts with CD14 receptors on monocytes and macrophages and other types of receptors on endothelial cells. In monocytes and macrophages three types of events are triggered during their interaction with LPS (See also Handout 11 Figure 5):
During infectious disease caused by Gram-negative bacteria, endotoxins released from, or part of, multiplying cells have similar effects on animals and significantly contribute to the symptoms and pathology encountered. The range of inflammatory effects caused by LPS during Gram-negative bacteremia or septicemia are outlined below.
These physiological activities of endotoxins are mediated mainly by the Lipid A component of LPS. The primary structure of Lipid A has been elucidated and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO4 groups, the acyl chains, and also the KDO-containing inner core. Thus Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system.