17.3: Bacterial Virulence Factors- Toxins

Learning Objectives

• Differentiate between endotoxins and exotoxins
• Describe and differentiate between various types of exotoxins
• Explain how superantigens attack the body

Toxins

In addition to exoenzymes, certain pathogens are able to produce toxins, biological poisons that assist in their ability to invade and cause damage to tissues. The ability of a pathogen to produce toxins to cause damage to host cells is called toxigenicity.

Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (Figure \(\PageIndex{4}\)). During infection and disease, gram-negative bacterial pathogens release endotoxin either when the cell dies, resulting in the disintegration of the membrane, or when the bacterium undergoes binary fission. The lipid component of endotoxin, lipid A, is responsible for the toxic properties of the LPS molecule. Lipid A is relatively conserved across different genera of gram-negative bacteria; therefore, the toxic properties of lipid A are similar regardless of the gram-negative pathogen. In a manner similar to that of tumor necrosis factor, lipid A triggers the immune system’s inflammatory response (see Inflammation and Fever). If the concentration of endotoxin in the body is low, the inflammatory response may provide the host an effective defense against infection; on the other hand, high concentrations of endotoxin in the blood can cause an excessive inflammatory response, leading to a severe drop in blood pressure, multi-organ failure, and death.

Figure \(\PageIndex{4}\): Lipopolysaccharide is composed of lipid A, a core glycolipid, and an O-specific polysaccharide side chain. Lipid A is the toxic component that promotes inflammation and fever.

A classic method of detecting endotoxin is by using the Limulus amebocyte lysate (LAL) test. In this procedure, the blood cells (amebocytes) of the horseshoe crab (Limulus polyphemus) is mixed with a patient’s serum. The amebocytes will react to the presence of any endotoxin. This reaction can be observed either chromogenically (color) or by looking for coagulation (clotting reaction) to occur within the serum. An alternative method that has been used is an enzyme-linked immunosorbent assay (ELISA) that uses antibodies to detect the presence of endotoxin.

Unlike the toxic lipid A of endotoxin, exotoxins are protein molecules that are produced by a wide variety of living pathogenic bacteria. Although some gram-negative pathogens produce exotoxins, the majority are produced by gram-positive pathogens. Exotoxins differ from endotoxin in several other key characteristics, summarized in Table \(\PageIndex{3}\). In contrast to endotoxin, which stimulates a general systemic inflammatory response when released, exotoxins are much more specific in their action and the cells they interact with. Each exotoxin targets specific receptors on specific cells and damages those cells through unique molecular mechanisms. Endotoxin remains stable at high temperatures, and requires heating at 121 °C (250 °F) for 45 minutes to inactivate. By contrast, most exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures above 41 °C (106 °F). As discussed earlier, endotoxin can stimulate a lethal inflammatory response at very high concentrations and has a measured LD₅₀ of 0.24 mg/kg. By contrast, very small concentrations of exotoxins can be lethal. For example, botulinum toxin, which causes botulism, has an LD₅₀ of 0.000001 mg/kg (240,000 times more lethal than endotoxin).

Table \(\PageIndex{3}\): Comparison of Endotoxin and Exotoxins Produced by Bacteria

Characteristic	Endotoxin	Exotoxin
Source	Gram-negative bacteria	Gram-positive (primarily) and gram-negative bacteria
Composition	Lipid A component of lipopolysaccharide	Protein
Effect on host	General systemic symptoms of inflammation and fever	Specific damage to cells dependent upon receptor-mediated targeting of cells and specific mechanisms of action
Heat stability	Heat stable	Most are heat labile, but some are heat stable
LD50	High	Low

The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and superantigens. Table \(\PageIndex{4}\) provides examples of well-characterized toxins within each of these three categories.

Table \(\PageIndex{4}\): Some Common Exotoxins and Associated Bacterial Pathogens

Category	Example	Pathogen	Mechanism and Disease
Intracellular-targeting toxins	Cholera toxin	Vibrio cholerae	Activation of adenylate cyclase in intestinal cells, causing increased levels of cyclic adenosine monophosphate (cAMP) and secretion of fluids and electrolytes out of cell, causing diarrhea
	Tetanus toxin	Clostridium teta ni	Inhibits the release of inhibitory neurotransmitters in the central nervous system, causing spastic paralysis
	Botulinum toxin	Clostridium botulinum	Inhibits release of the neurotransmitter acetylcholine from neurons, resulting in flaccid paralysis
	Diphtheria toxin	Corynebacterium diphtheriae	Inhibition of protein synthesis, causing cellular death
Membrane-disrupting toxins	Streptolysin	Streptococcus pyogenes	Proteins that assemble into pores in cell membranes, disrupting their function and killing the cell
	Pneumolysin	Streptococcus pneumoniae
	Alpha-toxin	Staphylococcus aureus
	Alpha-toxin	Clostridium perfringens	Phospholipases that degrade cell membrane phospholipids, disrupting membrane function and killing the cell
	Phospholipase C	Pseudomonas aeruginosa
	Beta-toxin	Staphylococcus aureus
Superantigens	Toxic shock syndrome toxin	Staphylococcus aureus	Stimulates excessive activation of immune system cells and release of cytokines (chemical mediators) from immune system cells. Life-threatening fever, inflammation, and shock are the result.
	Streptococcal mitogenic exotoxin	Streptococcus pyogenes
	Streptococcal pyrogenic toxins	Streptococcus pyogenes

Query \(\PageIndex{1}\)

 

The intracellular targeting toxins comprise two components: A for activity and B for binding. Thus, these types of toxins are known as A-B exotoxins (Figure \(\PageIndex{5}\)). The B component is responsible for the cellular specificity of the toxin and mediates the initial attachment of the toxin to specific cell surface receptors. Once the A-B toxin binds to the host cell, it is brought into the cell by endocytosis and entrapped in a vacuole. The A and B subunits separate as the vacuole acidifies. The A subunit then enters the cell cytoplasm and interferes with the specific internal cellular function that it targets.

Figure \(\PageIndex{5}\): (a) In A-B toxins, the B component binds to the host cell through its interaction with specific cell surface receptors. (b) The toxin is brought in through endocytosis. (c) Once inside the vacuole, the A component (active component) separates from the B component and the A component gains access to the cytoplasm. (credit: modification of work by “Biology Discussion Forum”/YouTube)

Four unique examples of A-B toxins are the diphtheria, cholera, botulinum, and tetanus toxins. The diphtheria toxin is produced by the gram-positive bacterium Corynebacterium diphtheriae, the causative agent of nasopharyngeal and cutaneous diphtheria. After the A subunit of the diphtheria toxin separates and gains access to the cytoplasm, it facilitates the transfer of adenosine diphosphate (ADP)-ribose onto an elongation-factor protein (EF-2) that is needed for protein synthesis. Hence, diphtheria toxin inhibits protein synthesis in the host cell, ultimately killing the cell (Figure \(\PageIndex{6}\)).

Figure \(\PageIndex{6}\): The mechanism of the diphtheria toxin inhibiting protein synthesis. The A subunit inactivates elongation factor 2 by transferring an ADP-ribose. This stops protein elongation, inhibiting protein synthesis and killing the cell.

Cholera toxin is an enterotoxin produced by the gram-negative bacterium Vibrio cholerae and is composed of one A subunit and five B subunits. The mechanism of action of the cholera toxin is complex. The B subunits bind to receptors on the intestinal epithelial cell of the small intestine. After gaining entry into the cytoplasm of the epithelial cell, the A subunit activates an intracellular G protein. The activated G protein, in turn, leads to the activation of the enzyme adenyl cyclase, which begins to produce an increase in the concentration of cyclic AMP (a secondary messenger molecule). The increased cAMP disrupts the normal physiology of the intestinal epithelial cells and causes them to secrete excessive amounts of fluid and electrolytes into the lumen of the intestinal tract, resulting in severe “rice-water stool” diarrhea characteristic of cholera.

Botulinum toxin (also known as botox) is a neurotoxin produced by the gram-positive bacterium Clostridium botulinum. It is the most acutely toxic substance known to date. The toxin is composed of a light A subunit and heavy protein chain B subunit. The B subunit binds to neurons to allow botulinum toxin to enter the neurons at the neuromuscular junction. The A subunit acts as a protease, cleaving proteins involved in the neuron’s release of acetylcholine, a neurotransmitter molecule. Normally, neurons release acetylcholine to induce muscle fiber contractions. The toxin’s ability to block acetylcholine release results in the inhibition of muscle contractions, leading to muscle relaxation. This has the potential to stop breathing and cause death. Because of its action, low concentrations of botox are used for cosmetic and medical procedures, including the removal of wrinkles and treatment of overactive bladder.

Link to Learning

Click this link to see an animation of how the cholera toxin functions.

Click this link to see an animation of how the botulinum toxin functions.

Query \(\PageIndex{1}\)

 

Another neurotoxin is tetanus toxin, which is produced by the gram-positive bacterium Clostridium tetani. This toxin also has a light A subunit and heavy protein chain B subunit. Unlike botulinum toxin, tetanus toxin binds to inhibitory interneurons, which are responsible for release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA). Normally, these neurotransmitters bind to neurons at the neuromuscular junction, resulting in the inhibition of acetylcholine release. Tetanus toxin inhibits the release of glycine and GABA from the interneuron, resulting in permanent muscle contraction. The first symptom is typically stiffness of the jaw (lockjaw). Violent muscle spasms in other parts of the body follow, typically culminating with respiratory failure and death. Figure \(\PageIndex{7}\) shows the actions of both botulinum and tetanus toxins.

Figure \(\PageIndex{7}\): Mechanisms of botulinum and tetanus toxins. (credit micrographs: modification of work by Centers for Disease Control and Prevention)

Membrane-disrupting toxins affect cell membrane function either by forming pores or by disrupting the phospholipid bilayer in host cell membranes. Two types of membrane-disrupting exotoxins are hemolysins and leukocidins, which form pores in cell membranes, causing leakage of the cytoplasmic contents and cell lysis. These toxins were originally thought to target red blood cells (erythrocytes) and white blood cells (leukocytes), respectively, but we now know they can affect other cells as well. The gram-positive bacterium Streptococcus pyogenes produces streptolysins, water-soluble hemolysins that bind to the cholesterol moieties in the host cell membrane to form a pore. The two types of streptolysins, O and S, are categorized by their ability to cause hemolysis in erythrocytes in the absence or presence of oxygen. Streptolysin O is not active in the presence of oxygen, whereas streptolysin S is active in the presence of oxygen. Other important pore-forming membrane-disrupting toxins include alpha toxin of Staphylococcus aureus and pneumolysin of Streptococcus pneumoniae.

Bacterial phospholipases are membrane-disrupting toxins that degrade the phospholipid bilayer of cell membranes rather than forming pores. We have already discussed the phospholipases associated with B. anthracis, L. pneumophila, and Rickettsia species that enable these bacteria to effect the lysis of phagosomes. These same phospholipases are also hemolysins. Other phospholipases that function as hemolysins include the alpha toxin of Clostridium perfringens, phospholipase C of P. aeruginosa, and beta toxin of Staphylococcus aureus.

Some strains of S. aureus also produce a leukocidin called Panton-Valentine leukocidin (PVL). PVL consists of two subunits, S and F. The S component acts like the B subunit of an A-B exotoxin in that it binds to glycolipids on the outer plasma membrane of animal cells. The F-component acts like the A subunit of an A-B exotoxin and carries the enzymatic activity. The toxin inserts and assembles into a pore in the membrane. Genes that encode PVL are more frequently present in S. aureus strains that cause skin infections and pneumonia.¹ PVL promotes skin infections by causing edema, erythema (reddening of the skin due to blood vessel dilation), and skin necrosis. PVL has also been shown to cause necrotizing pneumonia. PVL promotes pro-inflammatory and cytotoxic effects on alveolar leukocytes. This results in the release of enzymes from the leukocytes, which, in turn, cause damage to lung tissue.

The third class of exotoxins is the superantigens. These are exotoxins that trigger an excessive, nonspecific stimulation of immune cells to secrete cytokines (chemical messengers). The excessive production of cytokines, often called a cytokine storm, elicits a strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure, shock, and death. The prototype superantigen is the toxic shock syndrome toxin of S. aureus. Most toxic shock syndrome cases are associated with vaginal colonization by toxin-producing S. aureus in menstruating women; however, colonization of other body sites can also occur. Some strains of Streptococcus pyogenes also produce superantigens; they are referred to as the streptococcal mitogenic exotoxins and the streptococcal pyrogenic toxins.

Query \(\PageIndex{1}\)

Key Concepts and Summary

Virulence factors contribute to a pathogen’s ability to cause disease. Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to the macromolecule they target and exotoxins are classified based on their mechanism of action. Bacterial toxins include endotoxin and exotoxins. Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by gram-positive bacteria, but also are secreted by gram-negative bacteria. 

Footnotes

1. 1 V. Meka. “Panton-Valentine Leukocidin.” http://www.antimicrobe.org/h04c.file...L-S-aureus.asp