Home » Urology, nephrology » Characteristic properties of endotoxin. Chemical structure of endotoxins

Characteristic properties of endotoxin. Chemical structure of endotoxins

Toxigenesis involves the production of toxins by pathogenic bacteria. This is one of the main methods of delivering diseases and diseases caused by bacteria. 2 categories of toxins that lead to various infections and diseases; endotoxins and exotoxins, and they differ depending on their chemical nature. Endotoxins are bacterial toxins composed of lipids (lipopolysaccharides), while exotoxins are composed of proteins.

What are endotoxins?

Endotoxins are lipopolysaccharides produced by gram-negative bacteria. Endotoxins are bound by cells and are produced only when cells are lysed. Entotoxins are present in the outer membrane of the cell wall in Gram bacteria. Endotoxins are also called lipopolysaccharides and are present in E coli, Shigella, Salmonella, Pseudomonas, Haemophilus influenza, Neisseria and Vibrio cholerae cells. Endotoxins are usually secreted by bacterial development due to the actions of certain antibiotics or by the action of phagocytic digestion.

Endotoxins exhibit less activity and are not very active on their substrate. They are heat resistant. The outer wall of bacteria is impermeable to large molecules and molecules that cannot dissolve in water and protect themselves from the external environment.

These toxins are part of this protective activity. It acts on the host during colonization. In addition, endotoxins exhibit weak antigenicity.

What are exotoxins?

Exotoxins are toxins that are released extracellularly as the organism develops. Exotoxins are contagious toxins that spread from the site of infection to other parts of the body and cause damage. They are soluble proteins that act as enzymes. The exotoxin is capable of causing damage to the host by destroying cells or disrupting normal cellular metabolism. Exotoxins are very potent and can cause harm to the host. Exotoxins are released due to their rapid growth or during cell lysis. Both gram+ and gram-bacteria produce exotoxins.

Exotoxins are more toxic than endotoxins and differ from certain strains of bacteria. Exotoxins cause diseases specific only to this infection. Eg. Clostridium tetani produces tetanus toxin. There are 3 main categories of exotoxins: enterotoxins, neurotoxins and cytotoxins. These types indicate the location of the activity. Enterotoxic activity can be observed on gastrointestinal tract. Neurotoxins exert their functions on neurons, and cytotoxins disrupt the functioning of the host cell. Some of the health disorders caused by exotoxins include cholera, tetanus and diphtheria. The antigenicity of exotoxins is quite high. Exotoxins trigger the immune system and secrete antitoxins to nullify the toxin.

exotoxin

Metabolic product of a developing cell.

illnesses

endotoxins

Infections urinary tract, typhoid fever, meningococcal meningitis, coronary artery disease, neonatal necrotizing enterocolitis, Crohn's disease and ulcerative colitis, cystic fibrosis, meningococcemia, sepsis with gram-negative rods, hemorrhagic shock

Exotoxins

Gas gangrene, Scarlet fever, Diphtheria, Botulism, tetanus, antibiotic associated diarrhea, skin syndrome.

Summary of Endotoxins vs. Exotoxins

The differences between endotoxins and exotoxins are given below:

Comparison table for endotoxins and exotoxins

Endotoxin (ET) is a lipopolysaccharide (LPS) that is an obligate component of the outer membrane of all Gram-negative bacteria. Endotoxin is released into the intestinal lumen as a result of self-renewal of the cellular pool of saprophytic microflora and/or violent destruction as a result antibacterial therapy, food poisoning, dysbiosis, intestinal toxic infections, etc. One of the models of the structure of ET, namely Salmonella typhimurium LPS, proposed by O. Westphal, is presented in the diagram (Fig. 1).

The LPS subunit consists of three large parts: O-chain, R-core and lipid A. The outer part of LPS - O-chain - is built of repeating oligosaccharide units, which consist of 3-4 sugars. This part of LPS determines the specificity of the O-antigen of bacteria and varies significantly among different types gram-negative bacteria.

The middle region, the R-core, is an oligosaccharide whose structure is less variable than the structure of the O-chain. The most constant components of R-core are sugars adjacent to the lipid part of LPS.

Lipid A is a conservative chemical structure and determines the common biological properties of LPS of all gram-negative bacteria. Under natural conditions of endotoxin synthesis, lipid A exists in complex with three molecules of ketodeoxyoctulonic acid. This complex is part of the biochemical structure of all LPS. It is synthesized in isolation in genetically defective strains of gram-negative microorganisms, the so-called Re-mutants, and is called Re-glycolipid. It is this enzyme LPS that is associated with almost the entire spectrum of biological activity of endotoxin.

Fig.1. Scheme of the structure of LPS from gram-negative bacteria

Endotoxin has a number of biological properties. List of types of biological activity of endotoxin:

- activation of leukocytes and macrophages ;

- stimulation of the production of endogenous pyrogen, antagonist

glucocorticoids, interferon, interleukins,

tumor necrotizing factor (cachexin) and other mediators;

- activation of the synthesis of acute phase proteins, including amyloid

squirrel;

- mitogenic effect;

- activation of myelopoiesis;

- polyclonal activation of B cells;

- induction of provirus development;

- suppression of tissue respiration;

- development of hyperlipidemia;

- activation of the complement system;

- activation of platelets and blood clotting factors;

- cell death;

- local and generalized Shvartsman phenomenon;

- disseminated intravascular coagulation (DIC);

- endotoxin shock and the development of acute multiorgan

insufficiency

The great interest of researchers in LPS is due not only to its unique structure and wide variety of biological activity caused by the effects, but also to the fact that a person is in constant contact with ET, since a fairly large number of Gr bacteria live in the intestines. Until recently, it was believed that the intact mucosa of the colon of a healthy person is a fairly reliable barrier that prevents LPS from entering the bloodstream in large quantities. In the experiment, pure ET did not penetrate the intestinal epithelium. In this regard, the generally accepted opinion was that LPS from the intestine under normal conditions does not penetrate into the bloodstream or penetrates in small quantities only into the portal vein system, but not into the systemic circulation. However, in recent years this point of view is changing significantly. Research conducted under the leadership of M. Yu. Yakovlev in the laboratory of pathological anatomy of extreme conditions of the Institute of Human Morphology of the USSR Academy of Medical Sciences, for the first time established the fact of the presence of intestinal LPS in the general bloodstream almost healthy people. Subsequent studies showed that ET penetrates into the general bloodstream of the newborn already in the first hours of life, and this process is synchronous with the colonization of the infant’s intestines with gram-negative microflora. Moreover, data have been obtained indicating that LPS can penetrate into the blood of the fetus already in utero.

The process of penetration of ET into the bloodstream is enhanced by damage to the intestinal mucosa, dysbacteriosis and various influences that are accompanied by the translocation of bacteria and their metabolic products from the intestine to other organs and tissues.

LPS can interact with almost all cells of the macroorganism. On the surface of mammalian cells there are ET-specific protein receptors CD 14, CD 18, Toll receptors and others. The functions of these receptors are different. When bound to the CD18 receptor protein, endotoxin does not cause activation of polymorphonuclear leukocytes (PMNs). At the same time, when bound to the LBP protein (lipopolysaccharid binding protein) of blood plasma, LPS, in combination with this protein, reacts with the CD14 receptor on the cell surface, which leads to activation of leukocytes. The binding of endotoxin to the Toll receptor leads to activation of the innate immune system.

To a large extent, the biological activity of LPS is due to its interaction with leukocytes, macrophages, endothelial cells, etc. The main ET-accepting cellular element in human blood is polymorphonuclear leukocytes (PMN). Several types of interaction between LPS and leukocytes are known. The interaction of hydrophobic structures of LPS with membrane components of cells may depend on the appearance under the influence of ET and the content of endothelial-leukocyte adhesion molecules (ELAM) on the surface of neutrophils. In particular, selectins are classified as ELAMs. E-selectin (ELAM-1) is present on the plasma membrane of neutrophils and other phagocytes. L-selectin (VCAM-1 vascular adhesion molecule) is found on monocytes and lymphocytes and is not found on granular leukocytes. The ligand for the adhesion molecule VCAM-1 are slow-reacting antigens - VLA (a4, b4), which are also found on lymphocytes and monocytes. PMN respond to the action of LPS by releasing cytokines, interleukin-1b (IL-Ib) and tumor necrosis factor (TNF-a), and increasing the synthesis of VCAM-1. VCAM-1 is involved in adhesion various types lymphocytes, including B cell binding. The adhesion of non-granular leukocytes is ensured by membrane immunoglobulins (ICAM-1, ICAM-2), which bind to the lymphocyte-associated antigen LFA-1. Like E-selectin and VCAM-1, ICAM-1 is produced on agranulocytes only after they are stimulated by IL-1 and TNF-a in response to exposure to ET. In experiments on Lewis rats, endothelial damage was induced by endotoxin through the expression of ICAM-1 upon treatment with IL-2, TNF-a and IFN-g. The enhanced effect of ICAM-1 is the adhesion of leukocytes, among which monocytes (about 80%) and T-lymphocytes (8% to 20%) predominate. Maximum adhesion of leukocytes is observed at 6 hours from the moment of exposure to ET and continues up to 72 hours. Then monocytes and lymphocytes actively penetrate into the vascular wall through the intercellular channels of even intact endothelial cells.

The next feature of the interaction of ET with leukocytes is the Fc-dependent binding of LPS by antibodies localized on the Fc receptors of leukocytes. This type of interaction leads to phagocytosis and inactivation of ET.

After administration of ET to rabbits at a dose of 0.25 mg, LPS is detected after 1-1.5 hours on 40% of circulating PMNs. At the same time, they are not destroyed, as was previously believed, but are redistributed into the marginal pool of the microvasculature.

ET can be found on the surface of granulocytes in the blood of apparently healthy adults, newborns and their mothers. The use of enzyme-linked immunosorbent assay (ELISA) has made it possible to show that in thin blood smears of healthy people, about 3-4% of PMNs that have bound LPS in the bloodstream are detected. In addition, about 5% of PMNs are able to bind ETs in vitro when smears are treated with LPS, i.e. healthy people have reserves for endotoxin binding by granulocytes.

Bibliography

Westphal O. Bacterial Endotoxins // Int.Arch.Allergy Appl.Immunol. 1975. V.49.
Likhoded V.G., Yushchuk N.D., Yakovlev M.Yu. The role of endotoxin of gram-negative bacteria in infectious and non-infectious pathology // Archives of Pathology. 1996. No. 2.
AU-Benoit R., Rowe S., Boyle P., Garret M. Alber S., Wiener J., Rowe M.I. Pure endfotoxin does not pass across the intestinal epithelium in vitro // Shock. 1998.V.10.
Yakovlev M.Yu. The role of intestinal microflora and insufficiency of the barrier function of the liver in the development of endotoxemia and inflammation // Kazan. honey. zhur. 1988. No. 5.
Yakovlev M.Yu. Systemic endotoxemia in human physiology and pathology. // Author's abstract. diss. ...Dr. med. Sci. M., 1993.
Likhoded V.G., Chkhaidze I.G., Galdavadze M.A. and others. Development of intestinal dysbiosis in newborns with a deficiency of antibodies to Re-glycolipid // Microbiology. 1998. No. 4.
Tabolin V.A., Belchik Yu.F., Chabaidze Zh.L. and others. Indicators of antiendotoxin immunity in newborns in normal and pathological conditions // International. magazine immunorehab. 2000. No. 1.
Anikhovskaya I.A., Oparina O.N., Yakovleva M.M., Yakovlev M.Yu. Intestinal endotoxin as a universal factor of adaptation and pathogenesis of general adaptation syndrome // Human Physiology. 2006. T.32. No. 2.
Heumann D. CD14 and LPB in endotoxinemia and infections caused by Gram-negative bacteria // J. Endotox. Res. 2001. V. (6).
Pugin J., Ulevitch R.J., Tobias P.S. A critical role for monocytes and CD14 in endotoxin-induced endothelial cell activation // J. Exp. Med. 1998.V.178.
Amberger A., Maczek C., Jurgens G., Michaelis D. et al. Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins // Cell. Stress. Chaperones. 1997. V. 2(2).
Seitz C.S., Kleindienst R., Xu Q., Wick G. Coexpression of heat-shock protein 60 and intercellular-adhesion molecule-1 is related to increased adhesion of monocytes and T cells to aortic endothelium of rats in response to endotoxin // Lab . Invest. 1996. V. 74(1).
Likhoded V.G., Anikhovskaya I.V., Apollonin A.V. and others. Fc-dependent binding of endotoxins of gram-negative bacteria by polymorphonuclear leukocytes in human blood // Microbiology. 1996. No. 2.

Number of views of the publication: Please wait

Endotoxins are found only in gram-negative bacteria. They are represented by lipopolysaccharides and associated proteins. The peculiarity of endotoxins is that they are heat stable and are released from bacterial cells after their destruction. Endotoxins, unlike exotoxins, do not have specific action. Their toxicity and pyrogenicity are caused by lipid A, which is part of LPS and has a similar structure in different gram-negative bacteria. The pyrogenic effect of endotoxins is not associated with their direct effect on the thermoregulatory centers of the brain. They induce the release of some pyrogenic substance from polymorphonuclear leukocytes. Endotoxins are inflammatory agents; they increase capillary permeability and have a destructive effect on cells. Their inflammatory and pyrogenic effects are nonspecific. The variety of manifestations of endotoxin poisoning is due not only to LPS itself, but also to the release of numerous biologically active compounds, the synthesis of which it induces in humans and animals (histamine, serotonin, prostaglandins, leukotrienes, etc., more than 20 in total). These substances cause disorders in various organs and tissues.

All three components of LPS - lipid A, the polysaccharide core and its side chain of repeating sugars - have pronounced antigenic properties. LPS stimulates the synthesis of interferons, activates the complement system along the classical pathway, has a mitogenic effect on lymphocytes, as well as an allergenic effect. Its toxic properties, unlike exotoxins, are not removed by treatment with formaldehyde, and LPS is not converted into toxoid.

Exotoxins. They are produced by both gram-positive and gram-negative bacteria. In Gram-positive bacteria, exotoxins are actively secreted through the CM and cell wall into the environment using special secretion systems. In gram-negative bacteria (Vibrio cholerae, toxigenic E. coli, Salmonella), some exotoxins (enterotoxins) are synthesized only under certain conditions directly in the infected organism and are often stored in the cytoplasm, released from the cell only after its destruction.

All known bacterial exotoxins are proteins, among them there are heat-labile and heat-stable ones. Their main properties are associated with the protein nature of exotoxins: they have a high potency (the strongest toxins in nature are of microbial origin), high selectivity and associated specificity of action (the picture of tetanus in laboratory animals is the same, both when they are infected with the pathogen and its exotoxin), which they manifest after a certain latent period. Exotoxins are strong antigens, and some are even superantigens. They induce the formation of antibodies in the body, i.e. antitoxins, which neutralize their effect. When treated with formalin, exotoxins are neutralized and converted into toxoids. Toxoids are deprived toxic properties, but retain their ability to induce the synthesis of antitoxins, therefore they are widely used to create artificial immunity against diphtheria, tetanus, botulism and other diseases.

Rice. Schematic structure of the cell wall of gram-negative bacteria

Gram-negative bacteria They have a double-layered cell wall that surrounds a cytoplasmic membrane. The first layer is a very thin (1 nm thick) non-lipid membrane consisting of peptidoglycan. It is also called glycopeptide or mucopeptide. It is a complex matrix containing polysaccharide chains linked to each other by cross-linking short peptide chains. The second layer of the cell wall is lipid membrane 7.5 nm thick. It is on this outer membrane that endotoxins (lipopolysaccharides) are located. Endotoxin molecules provide structural integrity, are responsible for many physiological functions, including determining the pathogenic and antigenic properties of bacteria.

Structurally, the endotoxin molecule is divided into three parts - Lipid A, Core and O-specific chain (Fig. below).

O-specific chain lipopolysaccharides are built from repeating oligosaccharides. The most common sugars that make up the O-specific chain are glucose, galactose, and rhamnose. This part of the molecule gives it hydrophilic properties, due to which LPS is highly soluble in water. The polysaccharide part is the most variable part of the LPS molecule. This fragment of the molecule is often called the O-antigen, since it is responsible for the antigenic activity of gram-negative bacteria

Core- the central part of the molecule that binds the O-antigen to Lipid A. Formally, the structure of the core is divided into external and internal parts. The inner part of the core usually contains residues of L-glycero-O-mannoheptose and 2-keto-3-deoxyoctonic acid (KDO). KDO contains 8 carbon atoms and is found almost nowhere else in nature.

Lipid A consists of a disaccharide, phosphate and fatty acids. The Lipid A region is the most constant region of the LPS molecule, and its structure is similar in many bacteria.

In addition to lipopolysaccharides The outer wall of gram-negative bacteria also includes proteins (the outer membrane is ¾ LPS, and only ¼ is protein components). These proteins, together with LPS, form protein-lipopolysaccharide complexes of different sizes and molecular weights. These complexes are called bacterial endotoxins . The purified preparations that are used as standards are devoid of peptide fragments and represent a pure preparation of lipopolysaccharide. However, the term “bacterial endotoxins” is applied equally well to natural endotoxins that appear in solution as a result of bacterial destruction and to pure LPS preparations.

The outer wall of one gram-negative bacterium can contain up to 3.5 million LPS molecules. After her death, they all end up in solution. Endotoxins of gram-negative bacteria remain biologically active molecules even after the death of the bacteria. The endotoxin molecule is heat stable and easily withstands the autoclave sterilization cycle. The small size of endotoxin molecules allows them to easily pass through membranes used to sterilize solutions (0.22 microns). Therefore, endotoxins may be present in finished products. dosage forms, even those produced under aseptic conditions and undergoing final sterilization.

Bacterial endotoxins are extremely active (strong) pyrogens. For the development of a febrile attack, the presence of bacterial endotoxins in infusion solution at a concentration of 1 ng/ml (see http://forums. rusmedserv.com/archive/index.php/t-98927.html). Other pyrogens are less active, and for the development of a pyrogenic response their concentration must be 100-1000 times higher. Typically, the terms “pyrogens” and “endotoxins” are used interchangeably and, although not all pyrogens are endotoxins, the most significant are the endotoxins of gram-negative bacteria.

Pore-forming toxins . These include bacterial toxins that function by inserting into the host plasma membrane and forming transmembrane pores in it, leading to cell lysis. Such toxins are also called the RTX family due to the presence of a large number of repeats in their molecules. The mechanism of their action can be clearly seen in the example of S.aureus alpha-toxin, considered as a prototype of an oligomerizing pore-forming cytotoxin

Organization and mechanism of action of a toxic molecule. Most toxins have an A-B structure. This structure suggests the presence of two components - the B subunit, which is involved in the binding of the toxin to the receptor on the surface of the host cell and facilitates the transport of the toxin into the host cell; and the A-subunit - exhibiting enzymatic (toxic) activity in the host cell. The structure of B domains depends on the structure of the target receptors with which the toxin interacts. A subunits are more conserved than B subunits, especially in regions critical for their enzymatic activity

Rice. Mechanism of action of bacterial toxins

A. Damage to cell membranes by S. aureus alpha toxin. After the cell is released into the cell, a stalk of mushroom-like alpha-toxin is inserted into the target cell and causes an influx or outflow of ions from the cell (indicated as dark and light circles, respectively). B. Inhibition of cell protein synthesis by Shiga toxin (Stx). Holotoxin, which consists of an enzymatically active subunit (A), enters the cell through a receptor (Gb3). Then the A subunit, which has N-glycosidic activity, cuts off the adenosine residue from the 28S ribosomal RNA, which stops protein synthesis. C. Examples of bacterial toxins that activate second messenger pathways*. The binding of thermostable enterotoxin (ST) to the guanylate cyclase receptor leads to an increase in the amount of GMP, which converts into reverse side current of electrolytes. Through ADP-ribosylation or glycosylation (respectively), C. botulinum exoenzyme C3 and C. difficile toxins A (CdA) and B (CdB) inactivate small GTP-binding proteins. Cytotoxic necrotizing factor (CNF) from E. coli and dermonecrotic toxin (DNT) from the genus Bordetella activate blockade of effectors through deamination.

* ̶ Secondary messengers (second messengers) are small signaling molecules, components of the signal transmission system in the cell.