MECHANISMS OF BACTERIAL PATHOGENICITY: PROTEIN TOXINS

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MECHANISMS OF BACTERIAL PATHOGENICITY: PROTEIN TOXINS

© 2002 Todar University of Wisconsin-Madison Department of

Bacteriology

Bacterial Toxigenesis

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 types of bacterial toxins,

lipopolysaccharides, which are associated with the cell walls 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 lipoplysaccharide (LPS) 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 the outer membrane of Gram-negative bacteria. However,

endotoxins may be released from growing bacterial cells or from

cells which are lysed as a result of effective host defense (e.g.

lysozyme) or the activities of certain antibiotics (e.g. penicillins

and cephalosporins). Exotoxins are usually secreted by bacteria but

in some cases they are released by lysis of the bacterial cell.

Hence, either type of bacterial toxin may ultimately act in close

association with the cells that produce the toxin, or at tissue

sites remote from the original point of bacterial invasion or

growth. Some bacterial toxins may also act at the site of

colonization and play a role in invasion.

BACTERIAL PROTEIN TOXINS

Exotoxins are typically soluble proteins 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 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 Lethal toxicity

compared with:

Toxin Toxic Dose (mg) Host Strychnine Endotoxin Snake Venom

Botulism Type D 0.8x10-8 Mouse 3x106 3x107 3x105

Tetanus 4x10-8 Mouse 1x106 1x107 1x105

Shigella Neurotoxin 2.3x10-6 Rabbit 1x106 1x107 1x105

Diphtheria 6x10-5 Guinea Pig 2x103 2x104 2x102

The protein toxins resemble enzymes in a number of ways. Like

enzymes, bacterial exotoxins are denatured by heat, acid and

proteolytic enzymes; they have a high biological activity (most act

catalytically); and they exhibit specificity of action.

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 LF).

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 of Protein Toxins

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 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+ 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+ 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 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 (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 toxin

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.

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 considerations

In 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. Horizontal transfer of

genetic elements that encode virulence also occurs between species

of bacteria, which could explain how E. coli and Vibrio cholerae

produce a nearly-identical diarrhea-inducing toxin

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 disease is discussed in chapters

with the specific bacterial pathogens.

For more information on bacterial toxins go to this website:

Bacterial Toxins: Friends or Foes?

TABLE 2. ACTIVITIES OF EXTRACELLULAR BACTERIAL PROTEIN TOXINS NAME

OF TOXIN BACTERIA INVOLVED ACTIVITY

Anthrax toxin (EF) Bacillus anthracis Edema Factor (EF) is an

adenylate cyclase that causes increased levels in intracellular

cyclic AMP in phagocytes and formation of ion-permeable pores in

membranes (hemolysis)

Adenylate cyclase toxin Bordetella pertussis Acts locally to

increase levels of cyclic AMP in phagocytes and formation of ion-

permeable pores in membranes (hemolysis)

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

E. coli LT toxin Escherichia coli Similar to cholera toxin

E. coli ST toxin Escherichia coli Stimulates guanylate cyclase and

promotes secretion of water and electrolytes from intestinal

epithelium

Shiga toxin Shigella dysenteriae Enzymatically cleaves rRNA

resulting in inhibition of protein synthesis in susceptible cells

Perfringens enterotoxin Clostridium perfringens Stimulates adenylate

cyclase leading to increased cAMP in epithelial cells

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

Staphylococcus enterotoxins* Staphylococcus aureus Massive

activation of the immune system, including lymphocytes and

macrophages, leads to emesis

Toxic shock syndrome toxin (TSST-1)* Staphylococcus aureus Acts on

the vascular system causing inflammation, fever and shock

Exfoliatin toxin* Staphylococcus aureus Cleavage of epidermal cells

(intradermal separation)

Erythrogenic toxin (streptococcal pyrogenic exotoxin SPE)*

Streptococcus pyogenes Same as TSST - inflammation, fever and shock;

can cause localized erythematous reactions

* The pyrogenic exotoxins produced by Staphylococcus aureus and

Streptococcus pyogenes have been designated as superantigens. They

represent a family of molecules with the ability to elicit massive

activation of the immune system. These proteins share the ability to

stimulate T cell proliferation by interaction with Class II MHC

molecules on APCs and specific V beta chains of the T cell receptor.

The important feature of this interaction is the resultant

production of IL-1, TNF, and other lymphokines which appear to be

the principal mediators of disease processes associated with these

toxins.

TABLE 3. ENZYMATIC ACTIVITY AND BIOLOGICAL EFFECTS OF SOME BACTERIAL

EXOTOXINS TOXIN (subunit arr)* ENZYMATIC ACTIVITY BIOLOGICAL EFFECTS

Cholera toxin(A-5B) ADP ribosylates adenylate cyclase Gs regulatory

protein Activates adenylate cyclase; increased levels 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 effect 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

E. coli heat stable toxin ST ST toxins, of which there several

types, are small polypeptides ranging in size from 18 to 72 AA, and

probably lack enzymatic activity Stimulates guanylate cyclase in

epithelial cells of the GI tract resulting in intra- cellular

accumulation of cyclic GMP which has a net secretory effect on cells

and leads to diarrhea

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 cell

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+ A1 (Lethal Factor=LF) is a Zn++ dependent

protease with an unknown substrate; A2 (Edema Factor=EF) is an

adenylate cyclase B subunit, called the Protective Antigen (PA),

plus LF induces cytokine release and death of target cells or

experimental animals

Bordetella pertussis AC toxin (A/B) and Bacillus anthracis EF (A1+

Calmodulin-regulated adenylate cyclases that catalyze the formation

of cyclic AMP from ATP in susceptible cells, and the formation of

ion-permeable pores in cell membranes Increases cAMP in phagocytes

leading to inhibition of phagocytosis by neutrophils and

macrophages; hemolysis or leukolysis

* 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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Written and edited by Todar University of Wisconsin-Madison

Department of Bacteriology. All rights reserved.