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Toxigenesis, or the ability to produce toxins, is an underlying mechanism by which many bacterial pathogens produce disease. At a chemical level, there are two main types of bacterial toxins, lipopolysaccharides, which are associated with the cell wall of Gram-negative bacteria, and proteins, which are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth. The cell-associated toxins are referred to as endotoxins and the extracellular diffusible toxins are referred to as exotoxins.

Endotoxins are cell-associated substances that are structural components of bacteria. Most endotoxins are located in the cell envelope. In the context of this article, endotoxin refers specifically to the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) located in the outer membrane of Gram-negative bacteria. Although structural components of cells, soluble endotoxins may be released from growing bacteria or from cells that are lysed as a result of effective host defense mechanisms or by the activities of certain antibiotics. Endotoxins generally act in the vicinity of bacterial growth or presence.

Exotoxins are usually secreted by bacteria and act at a site removed from bacterial growth. However, in some cases, exotoxins are only released by lysis of the bacterial cell. Exotoxins are usually proteins, minimally polypeptides, that act enzymatically or through direct action with host cells and stimulate a variety of host responses. Most exotoxins act at tissue sites remote from the original point of bacterial invasion or growth. However, some bacterial exotoxins act at the site of pathogen colonization and may play a role in invasion.

BACTERIAL PROTEIN TOXINS

Exotoxins are usually secreted by living bacteria during exponential growth. The production of the toxin is generally specific to a particular bacterial species that produces the disease associated with the toxin (e.g. only Clostridium tetani produces tetanus toxin; only Corynebacterium diphtheriae produces the diphtheria toxin). Usually, virulent strains of the bacterium produce the toxin while nonvirulent strains do not, and the toxin is the major determinant of virulence (e.g. tetanus and diphtheria). At one time, it was thought that exotoxin production was limited mainly to Gram-positive bacteria, but clearly both Gram-positive and Gram-negative bacteria produce soluble protein toxins.

Bacterial protein toxins are the most powerful human poisons known and retain high activity at very high dilutions. The lethality of the most potent bacterial exotoxins is compared to the lethality of strychnine, snake venom, and endotoxin in Table 1 below.

TABLE 1. LETHALITY OF BACTERIAL PROTEIN TOXINS

Toxin	Toxic Dose (mg)	Host	Lethal toxicity	compared with:
			Strychnine	Endotoxin (LPS)	Snake Venom
Botulinum toxin	0.8x10-8	Mouse	3x106	3x107	3x105
Tetanus toxin	4x10-8	Mouse	1x106	1x107	1x105
Shiga toxin	2.3x10-6	Rabbit	1x106	1x107	1x105
Diphtheria toxin	6x10-5	Guinea pig	2x103	2x104	2x102

Bacterial protein toxins are strongly antigenic. In vivo, specific antibody neutralizes the toxicity of these bacterial exotoxins (antitoxin). However, in vitro, specific antitoxin may not fully inhibit their activity. This suggests that the antigenic determinant of the toxin may be distinct from the active portion of the protein molecule. The degree of neutralization of the active site may depend on the distance from the antigenic site on the molecule. However, since the toxin is fully neutralized in vivo, this suggests that other host factors must play a role in toxin neutralization in nature.

Protein exotoxins are inherently unstable. In time they lose their toxic properties but retain their antigenic ones. This was first discovered by Ehrlich who coined the term "toxoid" for this product. Toxoids are detoxified toxins which retain their antigenicity and their immunizing capacity. The formation of toxoids can be accelerated by treating toxins with a variety of reagents including formalin, iodine, pepsin, ascorbic acid, ketones, etc. The mixture is maintained at 37 degrees at pH range 6 to 9 for several weeks. The resulting toxoids can be used for artificial immunization against diseases caused by pathogens where the primary determinant of bacterial virulence is toxin production. Toxoids are effective immunizing agents against diphtheria and tetanus that are part of the DPT (DTP) vaccine.

Toxins with Enzymatic Activity

As proteins, many bacterial toxins resemble enzymes in a number of ways. Like enzymes, they are denatured by heat, acid and proteolytic enzymes, they act catalytically, and they exhibit specificity of action. The substrate (in the host) may be a component of tissue cells, organs or body fluid.

A plus B Subunit Arrangement

Many protein toxins, notably those that act intracellularly (with regard to host cells), consist of two components: one component (subunit A) is responsible for the enzymatic activity of the toxin; the other component (subunit B) is concerned with binding to a specific receptor on the host cell membrane and transferring the enzyme across the membrane. The enzymatic component is not active until it is released from the native (A+B) toxin. Isolated A subunits are enzymatically active but lack binding and cell entry capability. Isolated B subunits may bind to target cells (and even block the binding of the native toxin), but they are nontoxic.

There are a variety of ways that toxin subunits may be synthesized and arranged: A + B indicates that the toxin is synthesized and secreted as two separate protein subunits that interact at the target cell surface; A-B or A-5B indicates that the A and B subunits are synthesized separately, but associated by noncovalent bonds during secretion and binding to their target; 5B indicates that the binding domain of the protein is composed of 5 identical subunits. A/B denotes a toxin synthesized as a single polypeptide, divided into A and B domains that may be separated by proteolytic cleavage.

Attachment and Entry of Toxins

There are at least two mechanisms of toxin entry into target cells.

In one mechanism called direct entry, the B subunit of the native (A+B) toxin binds to a specific receptor on the target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into the cell cytoplasm.

In an alternative mechanism, the native toxin binds to the target cell and the A+B structure is taken into the cell by the process of receptor-mediated endocytosis (RME). The toxin is internalized in the cell in a membrane-enclosed vesicle called an endosome. H⁺ ions enter the endosome lowering the internal pH which causes the A+B subunits to separate. The B subunit affects the release of the A subunit from the endosome so that it will reach its target in the cell cytoplasm. The B subunit remains in the endosome and is recycled to the cell surface.

In both cases above, a large protein molecule must insert into and cross a membrane lipid bilayer, either the cell membrane or the endosome membrane. This activity is reflected in the ability of most A+B or A/B toxins, or their B components, to insert into artificial lipid bilayers, creating ion permeable pathways. If the B subunit contains a hydrophobic region (of amino acids) that insert into the membrane (as in the case of the diphtheria toxin), it may be referred to as the T (translocation) domain of the toxin.

A few bacterial toxins (e.g. diphtheria) are known to utilize both direct entry and RME to enter into host cells, which is not surprising since both mechanisms are variations on a theme. Bacterial toxins with similar enzymatic mechanisms may enter their target cells by different mechanisms. Thus, the diphtheria toxin and Pseudomonas exotoxin A, which have identical mechanisms of enzymatic activity, enter their host cells in slightly different ways. The adenylate cyclase toxin of Bordetella pertussis (pertussis AC) and anthrax EF produced by Bacillus anthracis, act similarly to catalyze the production of cAMP from host cell intracellular ATP reserves. However, the anthrax toxin enters cells by receptor mediated endocytosis, whereas the pertussis adenylate cyclase traverses the cell membrane directly.

The specific receptors for the B subunit of toxins on target cells or tissues are usually sialogangliosides (glycoproteins) called G-proteins on the cell membrane. For example, the cholera toxin utilizes the ganglioside GM1, and tetanus toxin utilizes ganglioside GT1 and/or GD1b as receptors on host cells.

The best known and studied bacterial toxin is the diphtheria toxin, produced by Corynebacterium diphtheriae. Diphtheria toxin is a bacterial exotoxin of the A/B prototype. It is produced as single polypeptide chain with a molecular weight of 60,000 daltons. The function of the protein is distinguishable into two parts: subunit A, with a m.w. of 21,000 daltons, contains the enzymatic activity for inhibition of elongation factor-2 involved in host protein synthesis; subunit B, with a m.w. of 39,000 daltons, is responsible for binding to the membrane of a susceptible host cell. The B subunit possesses a region T (translocation) domain which inserts into the endosome membrane thus securing the release of the enzymatic component into the cytoplasm.

Figure 1. Diphtheria Toxin (Dtx). A (red) is the catalytic domain; B (yellow) is the binding domain which displays the receptor for cell attachment; T (blue) is the hydrophobic domain responsible for insertion into the endosome membrane to secure the release of A. The protein is illustrated in its "closed" configuration.

In vitro, the native toxin is produced in an inactive form which can be activated by the proteolytic enzyme trypsin in the presence of thiol (reducing agent). The enzymatic activity of Fragment A is masked in the intact toxin. Fragment B is required to enable to enable Fragment A to reach the cytoplasm of susceptible cells. The C terminal end of Fragment B is hydrophilic and contains determinants that interact with specific membrane receptors on sensitive cell membranes and the N-terminal end of Fragment B (called the T domain) is strongly hydrophobic. The specific membrane receptor for the B fragment has been shown to be a transmembranous heparin-binding protein on the susceptible cell's surface.

The diphtheria toxin enters its target cells by either direct entry or receptor mediated endocytosis. The first step is the irreversible binding of the C-terminal hydrophilic portion of Fragment B (AA 432-535) to the receptor. During RME, the whole toxin is then taken up in an endocytic vesicle. In the vesicle, the pH drops to about 5 which allows unfolding of the A and B chains. This exposes hydrophobic regions of both the A and B chains that can insert into the vesicle membrane. The result is exposure of the A chain to the cytoplasmic side of the membrane. There, reduction and proteolytic cleavage releases the A chain in the cytoplasm. The A fragment is released as an extended chain but regains its active (enzymatic) globular conformation in the cytoplasm. The A chain catalyzes the ADP ribosylation of elongation factor-2 (EF-2) as shown in Figure 2.

Figure 2. Entry and activity of diphtheria toxin (Dtx) in susceptible cells. The B domain of the toxin binds to a cognate receptor on a susceptible cell. The toxin is taken up in an endosome by receptor mediated encocytosis. Acidification of the endocytic vesicle allows unfolding of the A and B chains exposing the hydrophobic T domain of the toxin. The T domain inserts into the endosome membrane translocating the A fragment into the cytoplasm where it regains its enzymatic configuration. The enzymatic A component utilizes NAD as a substrate. It catalyzes the attachment of the ADP-ribose portion of NAD to elongation factor (EF-2) which inactivates its function in protein synthesis.

TABLE 2.  BIOLOGICAL EFFECTS OF SOME BACTERIAL EXOTOXINS WITH ENZYMATIC ACTIVITY

TOXIN (subunit arr)*	ENZYMATIC ACTIVITY	BIOLOGICAL EFFECTS
Cholera toxin (A-5B)	ADP ribosylates eucaryotic adenylate cyclase Gs regulatory protein	Activates adenylate cyclase; increased level of intracellular cAMP promote secretion of fluid and electrolytes in intestinal epithelium leading to diarrhea
Diphtheria toxin (A/B)	ADP ribosylates elongation factor 2	Inhibits protein synthesis in animal cells resulting in death of the cells
Pertussis toxin (A-5B)	ADP ribosylates adenylate cyclase Gi regulatory protein	Blocks inhibition of adenylate cyclase; increased levels of cAMP affect hormone activity and reduce phagocytic activity
E. coli heat-labile toxin LT (A-5B)	ADP ribosylates adenylate cyclase Gs regulatory protein	Similar or identical to cholera toxin
Shiga toxin (A/5B	Glycosidase cleavage of ribosomal RNA (cleaves a single Adenine base from the 28S rRNA)	Inactivates the mammalian 60S ribosomal subunit and leads to inhibition of protein synthesis and death of the susceptible cells; pathology is diarrhea, hemorrhagic colitis (HC) and/or hemolytic uremic syndrome (HUS)
Pseudomonas Exotoxin A (A/B)	ADP ribosylates elongation factor-2 analogous to diphtheria toxin	Inhibits protein synthesis in susceptible cells, resulting in death of the cells
Botulinum toxin (A/B)	Zn++ dependent protease acts on synaptobrevin at motor neuron ganglioside	Inhibits presynaptic acetylycholine release from peripheral cholinergic neurons resulting in flaccid paralysis
Tetanus toxin (A/B)	Zn++ dependent protease acts on synaptobrevin in central nervous system	Inhibits neurotransmitter release from inhibitory neurons in the CNS resulting in spastic paralysis
Anthrax toxin LF (A2+B)	Metallo protease that cleaves MAPKK (mitogen-activated protein kinase kinase) enzymes	Combined with the B subunit (PA), LF induces cytokine release and death of target cells or experimental animals
Bordetella pertussis AC toxin (A/B) and Bacillus anthracis EF (A1+B)	Calmodulin-regulated adenylate cyclases that catalyze the formation of cyclic AMP from ATP in susceptible cells, as well as the formation of ion-permeable pores in cell membranes	Increases cAMP in phagocytes leading to inhibition of phagocytosis by neutrophils and macrophages; also causes hemolysis and leukolysis
Staphylococcus aureus Exfoliatin B	Cleaves desmoglein 1, a cadherin found in desmosomes in the epidermis (also a superantigen)	Separation of the stratum granulosum of the epidermis, between the living layers and the superficial dead layers.

Pore-forming Toxins

Pore-forming toxins, as the name suggests, insert a transmembranous pore into a host cell membrane, thereby disrupting the selective influx and efflux of ions across the membrane. This group of toxins includes the RTX toxins of Gram-negative bacteria, streptolysin O produced by S. pyogenes, and S. aureus alpha toxin. Generally, these toxins are produced as subunits that self-assemble as a pore on the eucaryotic membrane.

S. aureus alpha-toxin is considered the model of oligomerizing pore-forming cytotoxins. The alpha-toxin is synthesized as a 319 amino acid precursor molecule that contains an N-terminal signal sequence of 26 amino acids. The secreted mature toxin, or protomer, is a hydrophilic molecule with a molecular weight of 33 kDa. Seven toxin protomers assemble to form a 232 kDa mushroom-shaped heptamer comprising three distinct domains. The cap and rim domains of the heptamer are situated at the surface of the plasma membrane, while the stem domain serves as a transmembranous ion channel through the membrane.

TABLE 3. SOME PORE-FORMING BACTERIAL TOXINS

The regulation of synthesis and secretion of many bacterial toxins is tightly controlled by regulatory elements that are sensitive to environmental signals. For example, the production of diphtheria toxin is totally repressed by the availability of adequate amounts of iron in the medium for bacterial growth. Only under conditions of limiting amounts of iron in the growth medium does toxin production become derepressed. The expression of cholera toxin and related virulence factors (adhesins) is controlled by environmental osmolarity and temperature. In B. pertussis, induction of different virulence components is staggered, such that attachment factors are produced initially to establish the infection, and toxins are synthesized and released later to counter the host defenses and promote bacterial survival.

The processes by which protein toxins are assembled and secreted by bacterial cells are also variable. Many of the classic exotoxins are synthesized with an NH terminal leader (signal) sequence consisting of a few (1-3) charged amino acids and a stretch of (14-20) hydrophobic amino acids. The signal sequence may bind and insert into the cytoplasmic membrane during translation such that the polypeptide is secreted while being synthesized. The signal peptide is cleaved as the toxin (protein) is released into the periplasm. Alternatively, the toxin may be synthesized intracytoplasmically, then bound to a leader sequence for passage across the membrane. Frequently, chaperone proteins are required to guide this process. Some multicomponent toxins, such as the cholera toxin, have their subunits synthesized and secreted separately into the periplasm where they are assembled. In Gram-negative bacteria, the outer membrane poses an additional permeability barrier that a protein toxin usually has to mediate if it is to be released in a soluble form. It has been proposed that some Gram-negative exotoxins (e.g. E. coli ST enterotoxin) might be released in membrane vesicles composed of outer membrane components. Since these vesicles possibly possess outer membrane-associated attachment factors, they could act as "smart bombs" capable of specifically interacting with and possibly entering target cells to release their contents of toxin.

Other considerations

The genetic ability to produce a toxin, including regulatory genes, may be found on the bacxterial chromosome, plasmids and lysogenic bacteriophages. Sometimes they occur within pathogenicity islands. In any case, the processes of genetic exchange in bacteria, notably conjugation and transduction, can mobilize genetic elements between strains and species of bacteria. Horizontal gene transfer  (HGT) of genes that encode virulence is known to occur between species of bacteria. This explains how E. coli and Vibrio cholerae produce a nearly identical diarrhea-inducing toxin, as well as how E. coli O157:H7 acquired ability to produce shiga toxin form Shigella dysenteriae. The intestinal tract is probably an ideal habitat for bacteria to undergo HGT with one another.

There is conclusive evidence for the pathogenic role of diphtheria, tetanus and botulinum toxins, various enterotoxins, staphylococcal toxic shock syndrome toxin, and streptococcal pyrogenic exotoxins. And there is good evidence for the pathological involvement of pertussis toxin, anthrax toxin, shiga toxin and the necrotizing toxins of clostridia, in bacterial disease. But why certain bacteria produce such potent toxins is mysterious and is analogous to asking why an organism should produce an antibiotic. The production of a toxin may play a role in adapting a bacterium to a particular niche, but it is not essential to the viability of the organism. Most toxigenic bacteria are free-living in nature and in associations with humans in a form which is phenotypically identical to the toxigenic strain but lacking the ability to produce the toxin.

A summary of bacterial protein toxins and their activities is given in Tables 4. Details of the mechanisms of action of these toxins and their involvement in the pathogenesis of disease is discussed in chapters with the specific bacterial pathogens.

For more information and references on bacterial toxins go to this website:  Bacterial Toxins: Friends or Foes?

TABLE 4. SUMMARY: ACTIVITIES OF EXTRACELLULAR BACTERIAL  TOXINS

NAME OF TOXIN	BACTERIA INVOLVED	ACTIVITY
Anthrax toxin (EF)	Bacillus anthracis	An adenylate cyclase enzyme that increases levels in intracellular cyclic AMP in phagocytes and formation of ion-permeable pores in cell membrane. Leads to edema and decreased phagocytic responses
Adenylate cyclase toxin (pertussis AC)	Bordetella pertussis	Acts locally to increase levels of cyclic AMP in phagocytes and formation of ion-permeable pores in cell membranes
Alpha toxin	Staphylococcus aureus	Protein subunits assemble into an oligomeric structure that forms an ion channel (pore) in the cell plasma membrane
Cholera enterotoxin (Ctx)	Vibrio cholerae	ADP ribosylation of G proteins stimulates adenlyate cyclase and increases cAMP in cells of the GI tract, causing secretion of water and electrolytes leading to diarrhea
E. coli LT toxin	Escherichia coli	Similar to cholera toxin
E. coli ST toxins	Escherichia coli	Binding of the heat-stable enterotoxins (ST) to a guanylate cyclase receptor results in an increase in cyclic GMP (cGMP) that adversely effects electrolyte flux. Promotes secretion of water and electrolytes from intestinal epithelium leading to diarrhea.
Shiga toxin	Shigella dysenteriae E. coli O157:H7	Enzymatically cleaves eucaryotic 28S rRNA resulting in inhibition of protein synthesis in susceptible cells. Results in diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS)
Perfringens enterotoxin	Clostridium perfringens	Stimulates adenylate cyclase leading to increased cAMP in epithelial cells. Result is diarrhea
ToxinA/ToxinB	Clostridium difficile	Modifies Rho, a subfamily of small GTP-binding proteins that are regulators of the actin cytoskeleton. Deamidation of the glutamine residue at position 63 of Rho to a glutamic acid produces a dominant active Rho protein unable to hydrolyze bound GTP. Pathological result is cell necrosis and bloody diarrhea associated with colitis
Botulinum toxin	Clostridium botulinum	Zn++ dependent protease that inhibits neurotransmission at neuromuscular synapses resulting in flaccid paralysis
Tetanus toxin	Clostridium tetani	Zn++ dependent protease that Inhibits neurotransmission at inhibitory synapses resulting in spastic paralysis
Diphtheria toxin (Dtx)	Corynebacterium diphtheriae	ADP ribosylation of elongation factor 2 leads to inhibition of protein synthesis in target cells
Exotoxin A	Pseudomonas aeruginosa	Inhibits protein synthesis; similar to diphtheria toxin
Anthrax toxin (LF)	Bacillus anthracis	Lethal Factor (LF) is a Zn++ dependent protease that induces cytokine release and is cytotoxic to cells by an unknown mechanism
Pertussis toxin (Ptx)	Bordetella pertussis	ADP ribosylation of G proteins blocks inhibition of adenylate cyclase in susceptible cells
Exfoliatin toxin*	Staphylococcus aureus	Cleavage within epidermal cells (intraepidermal separation); also acts as a superantigen
Staphylococcus enterotoxins*	Staphylococcus aureus	Superantigen causes massive activation of the immune system, including lymphocytes and macrophages; exact role in in emesis not not known
Toxic shock syndrome toxin (TSST-1)*	Staphylococcus aureus	Superantigen acts on the vascular system causing inflammation, fever and shock
Erythrogenic toxin [streptococcal pyrogenic exotoxin (SPE)]*	Streptococcus pyogenes	Super antigen same as TSST - inflammation, fever and shock; can cause localized erythematous reactions (scarlet fever)