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Protein Toxins |
At a chemical level there are two types of bacterial
toxins:
The lipopolysaccharide (LPS) component of the Gram-negative bacterial outer membrane bears the name endotoxin because of its association with the cell wall of bacteria. Most of the protein toxins are thought of as exotoxins, since they are "released" from the bacteria and act on host cells at a distance. A subclass of exotoxins is Enterotoxins, which are usually (there are important exceptions) extracellular bacterial proteins that exert their activity in the GI tract. BACTERIAL PROTEIN TOXINSExotoxins are typically soluble proteins secreted by living bacteria during exponential growth. The production of protein toxins is generally specific to a particular bacterial species (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 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
The protein toxins resemble enzymes in a number of ways. Like enzymes, bacterial exotoxins:
As enzymes attack specific substrates, so bacterial protein toxins are highly specific in the substrate utilized and in their mode of action. The substrate (in the host) may be a component of tissue cells, organs, or body fluid. Usually the site of damage caused by the toxin indicates the location of the substrate for that toxin. Terms such as enterotoxin, neurotoxin, leukocidin or hemolysin are sometimes used to indicate the target site of some well-defined protein toxins. Certain protein toxins have very specific cytotoxic activity (i.e., they attack specific types of cells). For example, tetanus or botulinum toxins attack only neurons. But some toxins (as produced by staphylococci, streptococci, clostridia, etc.) have fairly broad cytotoxic activity and cause nonspecific death of all sorts of cells and tissues, eventually resulting in necrosis. Toxins that are phospholipases act in this way. They cleave phospholipids which are regular components of host cell membranes, resulting in the death of the cell by leakage of cellular contents. This is also true of pore-forming hemolysins and leukocidins. A few bacterial toxins that obviously bring about the death of an animal are known simply as lethal toxins, and even though the tissues affected and the target sites may be known, the precise mechanism by which death occurs is not understood (e.g. anthrax toxin). Bacterial protein toxins are strongly antigenic. In vivo, specific antibody (antitoxin) neutralizes the toxicity of these bacterial proteins. However, in vitro, specific antitoxin may not fully inhibit their enzymatic activity. This suggests that the antigenic determinant of the toxin may be distinct from the active (enzymatic) portion of the protein molecule. The degree of neutralization of the enzymatic 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 nature. Protein toxins are inherently unstable: in time they lose their toxic properties but retain their antigenic ones. This was first discovered by Ehrlich and he 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 use for artificial immunization against diseases caused by pathogens where the primary determinant of bacterial virulence is toxin production. Toxoids are the immunizing agents against diphtheria and tetanus that are part of the DPT vaccine. 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 2 separate protein subunits that interact at the target cell surface; A-B or A-5B indicates that the 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 ToxinsThere are at least two mechanisms of toxin entry into target cells (See Figure 2). 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. Somehow, 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.
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 and the anthrax toxin (Edema Factor) of 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 the toxin 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.
Control of Synthesis and the Release of Protein ToxinsThe 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 (14-20) of 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 presumably would possess the 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.
Diphtheria toxinThe 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.
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 is strongly hydrophobic. The specific membrane receptor for the B fragment has recently 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 endocytic 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. A 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). Diphtheria toxin is very potent in its action; a single molecule of subunit A within a cell is lethal and a single diphtheria bacillus is thought to be able to produce about 5,000 molecules per hour. The toxin (subunit A) utilizes NAD as a substrate: it catalyzes the attachment of the ADP- ribose portion of NAD to the elongation factor which inactivates its function in protein synthesis.
Other considerationsIn keeping with the observation that genetic information for functions not involved in viability of bacteria is frequently located extrachromosomally, the genes encoding toxin production are generally located on plasmids or in lysogenic bacteriophages. Thus the processes of genetic exchange in bacteria, notably conjugation and transduction, can mobilize these genetic elements between strains of bacteria, and therefore may play a role in determining the pathogenic potential of a bacterium.
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. There is conclusive evidence for the pathogenic role of diphtheria, tetanus and botulinum toxins, various enterotoxins, staphylococcal toxic shock syndrome toxin, and streptococcal erythrogenic toxin. 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. A summary of bacterial protein toxins and their activities is given in Tables 2 and 3. Details of the mechanisms of action of these toxins and their involvement in the pathogenesis of bacterial disease will be discussed in subsequent lectures. TABLE 2. ACTIVITIES OF EXTRACELLULAR BACTERIAL PROTEIN TOXINS
* toxin subunit arrangements: A-B or A-5B indicates subunits synthesized separately and associated by noncovalent bonds; A/B denotes subunit domains of a single protein that may be separated by proteolytic cleavage; A+B indicates subunits synthesized and secreted as separate protein subunits that interact at the target cell surface; 5B indicates that the binding domain is composed of 5 identical subunits. |
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