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6.2C: Type III Toxins: A-B Toxins and other Toxins that Interfere with Host Cell Function - Biology

6.2C: Type III Toxins: A-B Toxins and other Toxins that Interfere with Host Cell Function - Biology


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Learning Objectives

  1. Define A-B toxins and state the functions of the A component and the B component.
  2. State how the following exotoxins cause harm and name a bacterium producing each:
    1. diphtheria exotoxin
    2. cholera exotoxin
    3. enterotoxins
    4. shiga toxin
    5. anthrax lethal toxin and edema toxin
    6. botulism exotoxin
    7. tetanus exotoxin

Highlighted Bacterium

  1. Read the description of Corynebacterium diphtheriae andmatch the bacterium with the description of the organism and the infection it causes.
  2. Read the description of Bacillus anthracis andmatch the bacterium with the description of the organism and the infection it causes.

The classic type III toxins are A-B toxins that consist of two parts (see Figure (PageIndex{1})):

  1. An "A" or active component that enzymatically inactivates some host cell intracellular target or signalling pathway to interfere with a host cell function; and
  2. a "B" or binding component (see Figure (PageIndex{2})) that binds the exotoxin to a receptor molecule on the surface of the host cell membrane and determines the type of host cell to which the toxin is able to affect.

Once the exotoxin binds, it is translocated across the host cell membrane. Some A-B toxins enter by endocytosis (see Figure (PageIndex{3})), after which the A-component of the toxin separates from the B-component and enters the host cell's cytoplasm. Other A-B toxins bind to the host cell and the A component subsequently passes directly through the host cell's membrane and enters the cytoplasm (see Figure (PageIndex{4})).

The A components of most A-B toxins then catalyze a reaction by which they remove the ADP-ribosyl group from the coenzyme NAD and covalently attach it to some host cell protein, a process called ADP- ribosylation (see Figure (PageIndex{5})). This interferes with the normal function of that particular host cell protein that, in turn, determines the type of damage that is caused. Some A-B toxins work differently.

The body's major defense against exotoxins is the production of antitoxin antibodies. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.

Examples of A-B toxins include:

  1. Diphtheria exotoxin, produced by Corynebacterium diphtheriae (inf). This toxin interferes with host cell protein synthesis by catalyzing the ADP-ribosylation of host cell elongation factor 2 (EF-2), necessary in order for tRNA to insert new amino acids into the growing protein chain. This results in cell death. Initially cells of the throat are killed by the toxin. The toxin is also released into the blood where it damages internal organs and can lead to organ failure. The "D" portion of the DTP vaccine contains diphtheria toxoid to stimulate the body to make neutralizing antibodies against the binding component of the diphtheria exotoxin. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.

, and be able to match the bacterium with its description on an exam.

  1. Cholera exotoxin (choleragen), produced by Vibrio cholerae (inf). This exotoxin catalyzes the ADP-ribosylation of a host cell protein called Gs that turns the synthesis of a metabolic regulator molecule called cyclic AMP (cAMP) on and off. In this case, synthesis stays turned on. High levels of cAMP block intestinal epithelial cells from taking in sodium from the lumen of the intestines and stimulates them to secrete large quantities of chloride. Water and other electrolytes osmotically follow. This causes loss of fluids, diarrhea, and severe dehydration. For a movie of showing the effect of cholera exotoxin on human cells, see the Theriot Lab Website at Stanford University Medical School. Click on "Vibrio cholerae colonizing human cells."
  2. Enterotoxins. A number of bacteria produce exotoxins that bind to the cells of the small intestines. Most of these toxins catalyze the ADP-ribosylation of host cell proteins that turn the synthesis of the metabolic regulator molecules cyclic AMP (cAMP) or cyclic GMP on and off in intestinal mucosal cells. High levels of cAMP and cGMP cause loss of electrolytes and water that results in diarrhea. Organisms producing enterotoxins include Clostridium perfringens (inf),and Bacillus cereus (inf). (As mentioned under Type I toxins, the enterotoxins of Staphylococcus aureus (inf) and enterotoxogenic E. coli (inf) work differently, functioning as superantigens.)
  3. Pertussis exotoxin, produced by Bordetella pertussis (inf). The pertussis exotoxin catalyzes the ADP-ribosylation of a host cell protein called Gi leading to high intracellular levels of cAMP. This disrupts cellular function. In the respiratory epithelium, the high levels of cAMP results in increased respiratory secretions and mucous production and contribute to coughing. In the case of phagocytes, excessive cAMP decreases phagocytic activities such as chemotaxis, engulfment, killing. In the blood, the toxin results in increased sensitivity to histamine. This can result in increased capillary permeability, hypotension and shock. It may also act on neurons resulting in encephalopathy.
  4. Pseudomonas aeruginosa produces a variety of toxins that lead to tissue damage in the host. Type II toxins include:
    1. Exotoxin A: interferes with host cell protein synthesis by catalyzing the ADP-ribosylation of host cell elongation factor 2 (EF-2), necessary in order for tRNA to insert new amino acids into the growing protein chain; is also immunosuppressive.
    2. Exotoxin S: inhibits host cell protein synthesis causing tissue damage; is immunosuppressive.
  5. Shiga toxin, produced by species of Shigella (inf) and enterohemorrhagic Escherichia coli (EHEC) such as such as E. coli O157:H7. This toxin is an A-B toxin that cleaves host cell rRNA and prevents the attachment of charged tRNAs thus stopping host cell protein synthesis. The shiga toxin also enhances the LPS-mediated release of cytokines such as Il-1 and TNF-alpha and appears to be responsible for a complication of shigellosis and E. coli O157:H7 infection called hemolytic uremic syndrome (HUS), probably by causing blood vessel damage.
  6. Anthrax toxins, produced by Bacillus anthracis. In the case of the two anthrax exotoxins, two different A-components known as lethal factor (LF) and edema factor (EF) share a common B-component known as protective antigen (PA). Protective antigen, the B-component, first binds to receptors on host cells and is cleaved by a protease creating a binding site for either lethal factor or edema factor.
    1. Lethal factor is a protease that inhibits mitogen-activated kinase-kinase. At low levels, LF inhibits the release of proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha, (TNF-alpha), and NO. This may initially reduce immune responses against the organism and its toxins. But at high levels, LF is cytolytic for macrophages, causing release of high levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and NO. Excessive release of these cytokines can lead to a massive inflammatory response and the shock cascade, similar to septic shock.
    2. Edema factor is an adenylate cyclase that generates cyclic AMP in host cells. It impairs phagocytosis, and inhibits production of TNF and interleukin-6 (IL-6) by monocytes. This most likely impairs host defenses.

, and be able to match the bacterium with its description on an exam.

There are a number of other bacterial exotoxins that cause damage by interfering with host cell function. They include the following.

  1. Botulinal exotoxin, produced by Clostridium botulinum (inf). This is a neurotoxin that acts peripherally on the autonomic nervous system. For muscle stimulation, acetylcholine must be released from the neural motor end plate of the neuron at the synapse between the neuron and the muscle to be stimulated. The acetylcholine then induces contraction of the muscle fibers. The botulism exotoxin binds to and enters the presynaptic neuron and blocks its release of acetylcholine. This causes a flaccid paralysis , a weakening of the involved muscles. Death is usually from respiratory failure. While two exotoxins of C. botulinum catalyze ADP-ribosylation of host cell proteins, the botulinal toxin that affects neurons does not. Since the botulinal toxin is able to cause a weakening of muscles, it is now being used therapeutically to treat certain neurologic disorders such as dystonia and achalasia that result in abnormal sustained muscle contractions, as well as a treatment to remove facial lines.
  1. Tetanus exotoxin (tetanospasmin), produced by Clostridium tetani (inf). This is a neurotoxin that binds to inhibitory interneurons of the spinal cord and blocks their release of inhibitor molecules. It is these inhibitor molecules from the inhibitory interneurons that eventually allow contracted muscles to relax by stopping excitatory neurons from releasing the acetylcholine that is responsible for muscle contraction. The toxin, by blocking the release of inhibitors, keeps the involved muscles in a state of contraction and leads to spastic paralysis , a condition where opposing flexor and extensor muscles simultaneously contract. The "T" portion of the DTP vaccine contains tetanus toxoid to stimulate the body to make neutralizing antibodies against the binding component of the diphtheria exotoxin. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.
  1. Neutrophil activating protein, produced by Helicobacter pylori (inf). Neutrophil activating protein promotes the adhesion of human neutrophils to endothelial cells and the production of reactive oxygen radicals. The toxin induces a moderate inflammation that promote H. pylori growth by the release of nutrients factors from the inflamed tissue.

Explain the adaptive immune mechanism by which this immunization confers protection.

Summary

The classic type III toxins are A-B toxins that consist of two parts: an “A” or active component that enzymatically inactivates some host cell protein or signalling pathway to interfere with a host cell function; and a “B” or binding component that binds the exotoxin to a receptor molecule on the surface of the host cell membrane and determines the type of host cell to which the toxin is able to affect.

Examples include the diphtheria exotoxin produced by Corynebacterium diphtheria, the cholera exotoxin produced by Vibrio cholerae, certain enterotoxins that cause loss of electrolytes and water resulting in diarrhea, the pertussis exotoxin produced by Bordetella pertussis, shiga toxin, produced by species of Shigella and enterohemorrhagic Escherichia coli (EHEC), the anthrax toxins produced by Bacillus anthracis, the tetanus exotoxin of Clostridium tetani, and the botulism exotoxin of Clostridium botulinum.

Questions

Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.

  1. State the functions of the A component and the B component in A-B toxins. (ans).
  2. Match the following descriptions with the exotoxin:

    _____ Produced by certain strains of Escherichia coli such as E. These toxins kill intestinal epithelial cells of the colon and cause bloody diarrhea. Less commonly, the toxins enter the blood and are carried to the kidneys where they damage endothelial cells of the blood vessels and cause hemolytic uremic syndrome (HUS). (ans)

    _____ Produced by a species of Clostridium. This toxin binds to and enters the presynaptic neuron and blocks its release of acetylcholine. This causes a flaccid paralysis, a weakening of the involved muscles. This is a neurotoxin that binds to inhibitory interneurons of the spinal cord and blocks their release of inhibitor molecules.The toxin, by blocking the release of inhibitors, keeps the involved muscles in a state of contraction and leads to spastic paralysis, a condition where opposing flexor and extensor muscles simultaneously contract. (ans)

    _____ At low levels, this toxin inhibits the release of proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha, (TNF-alpha), and NO. But at high levels, it is cytolytic for macrophages, causing release of high levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and NO. Excessive release of these cytokines can lead to a massive inflammatory response and the shock cascade, similar to septic shock. (ans)

    1. diphtheria exotoxin
    2. cholera exotoxin
    3. enterotoxins
    4. pertussis exotoxin
    5. shiga toxin
    6. anthrax lethal toxin
    7. botulism exotoxin
    8. tetanus exotoxin
  3. Multiple Choice (ans)

An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp

Bacteria have evolved sophisticated mechanisms to inhibit the growth of competitors 1 . One such mechanism involves type VI secretion systems, which bacteria can use to inject antibacterial toxins directly into neighbouring cells. Many of these toxins target the integrity of the cell envelope, but the full range of growth inhibitory mechanisms remains unknown 2 . Here we identify a type VI secretion effector, Tas1, in the opportunistic pathogen Pseudomonas aeruginosa. The crystal structure of Tas1 shows that it is similar to enzymes that synthesize (p)ppGpp, a broadly conserved signalling molecule in bacteria that modulates cell growth rate, particularly in response to nutritional stress 3 . However, Tas1 does not synthesize (p)ppGpp instead, it pyrophosphorylates adenosine nucleotides to produce (p)ppApp at rates of nearly 180,000 molecules per minute. Consequently, the delivery of Tas1 into competitor cells drives rapid accumulation of (p)ppApp, depletion of ATP, and widespread dysregulation of essential metabolic pathways, thereby resulting in target cell death. Our findings reveal a previously undescribed mechanism for interbacterial antagonism and demonstrate a physiological role for the metabolite (p)ppApp in bacteria.

Figures

Extended Data Figure 1 |. Homologs of…

Extended Data Figure 1 |. Homologs of PA14_01140 and Tse6 are enriched in P. aeruginosa…

Extended Data Figure 2 |. Characterization of…

Extended Data Figure 2 |. Characterization of the PA14_01140-PA14_01130- tsi6 gene cluster.

Extended Data Figure 3 |. PA14_01140 tox…

Extended Data Figure 3 |. PA14_01140 tox possesses remote homology to characterized (p)ppGpp synthetases.

Extended Data Figure 4 |. The C-terminal…

Extended Data Figure 4 |. The C-terminal domain of PA14_01140 (PA14_01140 tox ) is toxic…

Extended Data Figure 5 |. Interaction with…

Extended Data Figure 5 |. Interaction with PA14_01130 distorts the predicted nucleotide acceptor site of…

Extended Data Figure 6 |. Tas1 pyrophosphorylates…

Extended Data Figure 6 |. Tas1 pyrophosphorylates the 3’ hydroxyl group of adenosine nucleotides.

Extended Data Figure 7 |. Purified Tas1…

Extended Data Figure 7 |. Purified Tas1 tox can use pppApp as a pyrophosphate donor…

Extended Data Figure 8 |. Tas1 tox…

Extended Data Figure 8 |. Tas1 tox overexpression in E. coli leads to (p)(p)pApp accumulation…

Extended Data Figure 9 |. The pmf…

Extended Data Figure 9 |. The pmf uncoupling ionophore CCCP but not the ppGpp-hydrolase domain…

Extended Data Figure 10 |. (p)ppApp binds…

Extended Data Figure 10 |. (p)ppApp binds to and inhibits PurF in a similar manner…

Figure 1 |. A unique T6SS effector-immunity…

Figure 1 |. A unique T6SS effector-immunity pair is encoded within the H1-T6SS of P.…

Figure 2 |. Tas1 tox adopts a…

Figure 2 |. Tas1 tox adopts a RelA-SpoT Homolog (RSH) fold found in enzymes that…

Figure 3 |. Tas1 intoxication depletes cellular…

Figure 3 |. Tas1 intoxication depletes cellular ADP and ATP resulting in dysregulation of central…

Figure 4 |. (p)ppApp interacts with PurF…

Figure 4 |. (p)ppApp interacts with PurF and inhibits de novo purine biosynthesis.


Contents

The term Type III secretion system was coined in 1993. [1] This secretion system is distinguished from at least five other secretion systems found in Gram-negative bacteria. Many animal and plant associated bacteria possess similar T3SSs. These T3SSs are similar as a result of divergent evolution and phylogenetic analysis supports a model in which gram-negative bacteria can transfer the T3SS gene cassette horizontally to other species. The most researched T3SSs are from species of Shigella (causes bacillary dysentery), Salmonella (typhoid fever), Escherichia coli (Gut flora, some strains cause food poisoning), Vibrio (gastroenteritis and diarrhea), Burkholderia (glanders), Yersinia (plague), Chlamydia (sexually transmitted disease), Pseudomonas (infects humans, animals and plants) and the plant pathogens Erwinia, Ralstonia and Xanthomonas, and the plant symbiont Rhizobium.

The T3SS is composed of approximately 30 different proteins, making it one of the most complex secretion systems. Its structure shows many similarities with bacterial flagella (long, rigid, extracellular structures used for motility). Some of the proteins participating in T3SS share amino-acid sequence homology to flagellar proteins. Some of the bacteria possessing a T3SS have flagella as well and are motile (Salmonella, for instance), and some do not (Shigella, for instance). Technically speaking, type III secretion is used both for secreting infection-related proteins and flagellar components. However, the term "type III secretion" is used mainly in relation to the infection apparatus. The bacterial flagellum shares a common ancestor with the type III secretion system. [2] [3]

T3SSs are essential for the pathogenicity (the ability to infect) of many pathogenic bacteria. Defects in the T3SS may render a bacterium non-pathogenic. It has been suggested that some non-invasive strains of gram-negative bacteria have lost the T3SS because the energetically costly system is no longer of use. [4] Although traditional antibiotics were effective against these bacteria in the past, antibiotic-resistant strains constantly emerge. Understanding the way the T3SS works and developing drugs targeting it specifically have become an important goal of many research groups around the world since the late 1990s.

The hallmark of T3SS is the needle [5] [6] (more generally, the needle complex (NC) or the T3SS apparatus (T3SA) also called injectisome when the ATPase is excluded see below). Bacterial proteins that need to be secreted pass from the bacterial cytoplasm through the needle directly into the host cytoplasm. Three membranes separate the two cytoplasms: the double membrane (inner and outer membranes) of the Gram-negative bacterium and the eukaryotic membrane. The needle provides a smooth passage through those highly selective and almost impermeable membranes. A single bacterium can have several hundred needle complexes spread across its membrane. It has been proposed that the needle complex is a universal feature of all T3SSs of pathogenic bacteria. [7]

The needle complex starts at the cytoplasm of the bacterium, crosses the two membranes and protrudes from the cell. The part anchored in the membrane is the base (or basal body) of the T3SS. The extracellular part is the needle. A so-called inner rod connects the needle to the base. The needle itself, although the biggest and most prominent part of the T3SS, is made out of many units of a single protein. The majority of the different T3SS proteins are therefore those that build the base and those that are secreted into the host. As mentioned above, the needle complex shares similarities with bacterial flagella. More specifically, the base of the needle complex is structurally very similar to the flagellar base the needle itself is analogous to the flagellar hook, a structure connecting the base to the flagellar filament. [8] [9]

The base is composed of several circular rings and is the first structure that is built in a new needle complex. Once the base is completed, it serves as a secretion machine for the outer proteins (the needle). Once the whole complex is completed the system switches to secreting proteins that are intended to be delivered into host cells. The needle is presumed to be built from bottom to top units of needle monomer protein pile upon each other, so that the unit at the tip of the needle is the last one added. The needle subunit is one of the smallest T3SS proteins, measuring at around 9 kDa. 100−150 subunits comprise each needle.

The T3SS needle measures around 60−80 nm in length and 8 nm in external width. It needs to have a minimal length so that other extracellular bacterial structures (adhesins and the lipopolysaccharide layer, for instance) do not interfere with secretion. The hole of the needle has a 3 nm diameter. Most folded effector proteins are too large to pass through the needle opening, so most secreted proteins must pass through the needle unfolded, a task carried out by the ATPase at the base of the structure. [10]

The T3SS proteins can be grouped into three categories:

  • Structural proteins: build the base, the inner rod and the needle.
  • Effector proteins: get secreted into the host cell and promote infection / suppress host cell defences.
  • Chaperones: bind effectors in the bacterial cytoplasm, protect them from aggregation and degradation and direct them towards the needle complex.

Most T3SS genes are laid out in operons. These operons are located on the bacterial chromosome in some species and on a dedicated plasmid in other species. Salmonella, for instance, has a chromosomal region in which most T3SS genes are gathered, the so-called Salmonella pathogenicity island (SPI). Shigella, on the other hand, has a large virulence plasmid on which all T3SS genes reside. It is important to note that many pathogenicity islands and plasmids contain elements that allow for frequent horizontal gene transfer of the island/plasmid to a new species.

Effector proteins that are to be secreted through the needle need to be recognized by the system, since they float in the cytoplasm together with thousands of other proteins. Recognition is done through a secretion signal—a short sequence of amino acids located at the beginning (the N-terminus) of the protein (usually within the first 20 amino acids), that the needle complex is able to recognize. Unlike other secretion systems, the secretion signal of T3SS proteins is never cleaved off the protein.

Contact of the needle with a host cell triggers the T3SS to start secreting [11] not much is known about this trigger mechanism (see below). Secretion can also be induced by lowering the concentration of calcium ions in the growth medium (for Yersinia and Pseudomonas done by adding a chelator such as EDTA or EGTA) and by adding the aromatic dye Congo red to the growth medium (for Shigella), for instance. These methods and other are used in laboratories to artificially induce type III secretion.

Induction of secretion by external cues other than contact with host cells also takes place in vivo, in infected organisms. The bacteria sense such cues as temperature, pH, osmolarity and oxygen levels, and use them to "decide" whether to activate their T3SS. For instance, Salmonella can replicate and invade better in the ileum rather than in the cecum of animal intestine. The bacteria are able to know where they are thanks to the different ions present in these regions the ileum contains formate and acetate, while the cecum does not. The bacteria sense these molecules, determine that they are at the ileum and activate their secretion machinery. Molecules present in the cecum, such as propionate and butyrate, provide a negative cue to the bacteria and inhibit secretion. Cholesterol, a lipid found in most eukaryotic cell membranes, is able to induce secretion in Shigella.

The external cues listed above either regulate secretion directly or through a genetic mechanism. Several transcription factors that regulate the expression of T3SS genes are known. Some of the chaperones that bind T3SS effectors also act as transcription factors. A feedback mechanism has been suggested: when the bacterium does not secrete, its effector proteins are bound to chaperones and float in the cytoplasm. When secretion starts, the chaperones detach from the effectors and the latter are secreted and leave the cell. The lone chaperones then act as transcription factors, binding to the genes encoding their effectors and inducing their transcription and thereby the production of more effectors.

Structures similar to Type3SS injectisomes have been proposed to rivet gram negative bacterial outer and inner membranes to help release outer membrane vesicles targeted to deliver bacterial secretions to eukaryotic host or other target cells in vivo. [12]

T3SS effectors enter the needle complex at the base and make their way inside the needle towards the host cell. The exact way in which effectors enter the host is mostly unknown. It has been previously suggested that the needle itself is capable of puncturing a hole in the host cell membrane this theory has been refuted. It is now clear that some effectors, collectively named translocators, are secreted first and produce a pore or a channel (a translocon) in the host cell membrane, through which other effectors may enter. Mutated bacteria that lack translocators are able to secrete proteins but are not able to deliver them into host cells. In general each T3SS includes three translocators. Some translocators serve a double role after they participate in pore formation they enter the cell and act as bona fide effectors.

T3SS effectors manipulate host cells in several ways. The most striking effect is the promoting of uptake of the bacterium by the host cell. Many bacteria possessing T3SSs must enter host cells in order to replicate and propagate infection. The effectors they inject into the host cell induce the host to engulf the bacterium and to practically "eat" it. In order for this to happen the bacterial effectors manipulate the actin polymerization machinery of the host cell. Actin is a component of the cytoskeleton and it also participates in motility and in changes in cell shape. Through its T3SS effectors the bacterium is able to utilize the host cell's own machinery for its own benefit. Once the bacterium has entered the cell it is able to secrete other effectors more easily and it can penetrate neighboring cells and quickly infect the whole tissue.

T3SS effectors have also been shown to tamper with the host's cell cycle and some of them are able to induce apoptosis. One of the most researched T3SS effector is IpaB from Shigella flexneri. It serves a double role, both as a translocator, creating a pore in the host cell membrane, and as an effector, exerting multiple detrimental effects on the host cell. It had been demonstrated that IpaB induces apoptosis in macrophages—cells of the animal immune system—after being engulfed by them. [13] It was later shown that IpaB achieves this by interacting with caspase 1, a major regulatory protein in eukaryotic cells. [14]

Another well characterized class of T3SS effectors are Transcription Activator-like effectors (TAL effectors) from Xanthomonas. When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. [15] TAL effector-DNA recognition has recently been demonstrated to comprise a simple code [16] [17] and this has greatly improved the understanding of how these proteins can alter the transcription of genes in the host plant cells.

Hundreds of articles on T3SS have been published since the mid-nineties. However, numerous issues regarding the system remain unresolved:

  • T3SS proteins. Of the approximately 30 T3SS proteins less than 10 in each organism have been directly detected using biochemical methods. The rest, being perhaps rare, have proven difficult to detect and they remain theoretical (although genetic rather than biochemical studies have been performed on many T3SS genes/proteins). The localization of each protein is also not entirely known.
  • The length of the needle. It is not known how the bacterium "knows" when a new needle has reached its proper length. Several theories exist, among them the existence of a "ruler protein" that somehow connects the tip and the base of the needle. Addition of new monomers to the tip of the needle should stretch the ruler protein and thereby signal the needle length to the base.
  • Energetics. The force that drives the passage of proteins inside the needle is not completely known. An ATPase is associated with the base of the T3SS and participates in directing proteins into the needle but whether it supplies the energy for transport is not clear.
  • Secretion signal. As mentioned above, the existence of a secretion signal in effector proteins is known. The signal allows the system to distinguish T3SS-transported proteins from any other protein. Its nature, requirements and the mechanism of recognition are poorly understood, but methods for predicting which bacterial proteins can be transported by the Type III secretion system have recently been developed. [19]
  • Activation of secretion. The bacterium must know when the time is right to secrete effectors. Unnecessary secretion, when no host cell is in vicinity, is wasteful for the bacterium in terms of energy and resources. The bacterium is somehow able to recognize contact of the needle with the host cell. How this is done is still being researched, and the method may well be dependent on the pathogen. Some theories postulate a delicate conformational change in the structure of the needle upon contact with the host cell this change perhaps serves as a signal for the base to commence secretion. One method of recognition has been discovered in Salmonella, which relies on sensing host cell cytosolic pH through the pathogenicity island 2-encoded T3SS in order to switch on secretion of effectors. [20]
  • Binding of chaperones. It is not known when chaperones bind their effectors (whether during or after translation) and how they dissociate from their effectors before secretion.
  • Effector mechanisms. Although much was revealed since the beginning of the 21st century about the ways in which T3SS effectors manipulate the host, the majority of effects and pathways remains unknown.
  • Evolution. As mentioned, the T3SS is closely related to the bacterial flagellum. [21] There are three competing hypotheses: [22] first, that the flagellum evolved first and the T3SS is derived from that structure, second, that the T3SS evolved first and the flagellum is derived from it, and third, that the two structures are derived from a common ancestor. There was some controversy about the different scenarios, [2][22] since they all explain protein homology between the two structures, as well as their functional diversity. [23] Yet, recent phylogenomic evidence favours the hypothesis that the T3SS derived from the flagellum by a process involving initial gene loss and then gene acquisition. [24] A key step of the latter process was the recruitment of secretins to the T3SS, an event that occurred at least three times from other membrane-associated systems.

Since the beginning of the 1990s new T3SS proteins are being found in different bacterial species at a steady rate. Abbreviations have been given independently for each series of proteins in each organism, and the names usually do not reveal much about the protein's function. Some proteins discovered independently in different bacteria have later been shown to be homologous the historical names, however, have mostly been kept, a fact that might cause confusion. For example, the proteins SicA, IpgC and SycD are homologs from Salmonella, Shigella and Yersinia, respectively, but the last letter (the "serial number") in their name does not show that.

Below is a summary of the most common protein-series names in several T3SS-containing species. Note that these names include proteins that form the T3SS machinery as well as the secreted effector proteins:

  • Yersinia
    • Yop: Yersinia outer protein
    • Ysc: Yersinia secretion (component)
    • Ypk: Yersinia protein kinase
    • Spa: Surface presentation of antigen
    • Sic: Salmonella invasion chaperone
    • Sip: Salmonella invasion protein
    • Prg: PhoP-repressed gene
    • Inv: Invasion
    • Org: Oxygen-regulated gene
    • Ssp: Salmonella-secreted protein
    • Iag: Invasion-associated gene
    • Ipg: Invasion plasmid gene
    • Ipa: Invasion plasmid antigen
    • Mxi: Membrane expression of Ipa
    • Spa: Surface presentation of antigen
    • Osp: Outer Shigella protein
    • Tir: Translocated intimin receptor
    • Sep: Secretion of E. coli proteins
    • Esc: Escherichia secretion (component)
    • Esp: Escherichia secretion protein
    • Ces: Chaperone of E. coli secretion
    • Hrp: Hypersensitive response and pathogenicity
    • Hrc: Hypersensitive response conserved (or Hrp conserved)
    • Nop: Nodulation protein
    • Rhc: Rhizobium conserved
    • Vir: Virulence

    Following those abbreviations is a letter or a number. Letters usually denote a "serial number", either the chronological order of discovery or the physical order of appearance of the gene in an operon. Numbers, the rarer case, denote the molecular weight of the protein in kDa. Examples: IpaA, IpaB, IpaC MxiH, MxiG, MxiM Spa9, Spa47.

    Several key elements appear in all T3SSs: the needle monomer, the inner rod of the needle, the ring proteins, the two translocators, the needle-tip protein, the ruler protein (which is thought to determine the needle's length see above) and the ATPase, which supplies energy for secretion. The following table shows some of these key proteins in four T3SS-containing bacteria:

    ↓ Function / Genus → Shigella Salmonella Yersinia Escherichia
    Needle monomer MxiH PrgI YscF EscF
    Inner rod MxiI PrgJ YscI EscI
    Needle-tip protein IpaD SipD LcrV EspA
    Translocator IpaB SipB YopB EspD
    Translocator IpaC SipC YopD EspB
    Chaperone for the two translocators IpgC SicA SycD CesD
    ATPase Spa47 InvC YscN SepB (EscN)
    Ruler protein Spa32 InvJ YscP Orf16
    Switch Spa40 SpaS YscU EscU
    Gatekeeper MxiC InvE YopN (TyeA) SepL

    Isolation of T3SS needle complexes Edit

    The isolation of large, fragile, hydrophobic membrane structures from cells has constituted a challenge for many years. By the end of the 1990s, however, several approaches have been developed for the isolation of T3SS NCs. In 1998 the first NCs were isolated from Salmonella typhimurium. [26]

    For the isolation, bacteria are grown in a large volume of liquid growth medium until they reach log phase. They are then centrifuged the supernatant (the medium) is discarded and the pellet (the bacteria) is resuspended in a lysis buffer typically containing lysozyme and sometimes a detergent such as LDAO or Triton X-100. This buffer disintegrates the cell wall. After several rounds of lysis and washing, the opened bacteria are subjected to a series of ultracentrifugations. This treatment enriches large macromolecular structures and discards smaller cell components. Optionally, the final lysate is subjected to further purification by CsCl density gradient.

    An additional approach for further purification uses affinity chromatography. Recombinant T3SS proteins that carry a protein tag (a histidine tag, for instance) are produced by molecular cloning and then introduced (transformed) into the researched bacteria. After initial NC isolation, as described above, the lysate is passed through a column coated with particles with high affinity to the tag (in the case of histidine tags: nickel ions). The tagged protein is retained in the column, and with it the entire needle complex. High degrees of purity can be achieved using such methods. This purity is essential for many delicate assays that have been used for NC characterization.

    Type III effectors were known since the beginning of the 1990s, but the way in which they are delivered into host cells was a complete mystery. The homology between many flagellar and T3SS proteins led researchers to suspects the existence of an outer T3SS structure similar to flagella. The identification and subsequent isolation of the needle structure enabled researchers to:

    • characterize the three-dimensional structure of the NC in detail, and through this to draw conclusions regarding the mechanism of secretion (for example, that the narrow width of the needle requires unfolding of effectors prior to secretion),
    • analyze the protein components of the NC, this by subjecting isolated needles to proteomic analysis (see below),
    • assign roles to various NC components, this by knocking out T3SS genes, isolating NCs from the mutated bacteria and examining the changes that the mutations caused.

    Microscopy, crystallography and solid-state NMR Edit

    As with almost all proteins, the visualization of T3SS NCs is only possible with electron microscopy. The first images of NCs (1998) showed needle structures protruding from the cell wall of live bacteria and flat, two-dimensional isolated NCs. [26] In 2001 images of NCs from Shigella flexneri were digitally analyzed and averaged to obtain a first semi-3D structure of the NC. [5] The helical structure of NCs from Shigella flexneri was resolved at a resolution of 16 Å using X-ray fiber diffraction in 2003, [27] and a year later a 17-Å 3D structure of NCs from Salmonella typhimurium was published. [28] Recent advances and approaches have allowed high-resolution 3D images of the NC, [29] [30] further clarifying the complex structure of the NC.

    Numerous T3SS proteins have been crystallized over the years. These include structural proteins of the NC, effectors and chaperones. The first structure of a needle-complex monomer was NMR structure of BsaL from "Burkholderia pseudomallei" and later the crystal structure of MixH from Shigella flexneri, which were both resolved in 2006. [31] [32]

    In 2012, a combination of recombinant wild-type needle production, solid-state NMR, electron microscopy [33] and Rosetta modeling revealed the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. [34] It was shown that the 80-residue PrgI subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellum of Salmonella typhimurium. The model also revealed an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen. [34]

    Proteomics Edit

    Several methods have been employed in order to identify the array of proteins that comprise the T3SS. Isolated needle complexes can be separated with SDS-PAGE. The bands that appear after staining can be individually excised from the gel and analyzed using protein sequencing and mass spectrometry. The structural components of the NC can be separated from each other (the needle part from the base part, for instance), and by analyzing those fractions the proteins participating in each one can be deduced. Alternatively, isolated NCs can be directly analyzed by mass spectrometry, without prior electrophoresis, in order to obtain a complete picture of the NC proteome.

    Genetic and functional studies Edit

    The T3SS in many bacteria has been manipulated by researchers. Observing the influence of individual manipulations can be used to draw insights into the role of each component of the system. Examples of manipulations are:

    • Deletion of one or more T3SS genes (gene knockout). of one or more T3SS genes (in other words: production in vivo of a T3SS protein in quantities larger than usual).
    • Point or regional changes in T3SS genes or proteins. This is done in order to define the function of specific amino acids or regions in a protein.
    • The introduction of a gene or a protein from one species of bacteria into another (cross-complementation assay). This is done in order to check for differences and similarities between two T3SSs.

    Manipulation of T3SS components can have influence on several aspects of bacterial function and pathogenicity. Examples of possible influences:

    • The ability of the bacteria to invade host cells, in the case of intracellular pathogens. This can be measured using an invasion assay (gentamicin protection assay).
    • The ability of intracellular bacteria to migrate between host cells.
    • The ability of the bacteria to kill host cells. This can be measured by several methods, for instance by the LDH-release assay, in which the enzyme LDH, which leaks from dead cells, is identified by measuring its enzymatic activity.
    • The ability of a T3SS to secrete a specific protein or to secrete at all. In order to assay this, secretion is induced in bacteria growing in liquid medium. The bacteria and medium are then separated by centrifugation, and the medium fraction (the supernatant) is then assayed for the presence of secreted proteins. In order to prevent a normally secreted protein from being secreted, a large molecule can be artificially attached to it. If the then non-secreted protein stays "stuck" at the bottom of the needle complex, the secretion is effectively blocked.
    • The ability of the bacteria to assemble an intact needle complex. NCs can be isolated from manipulated bacteria and examined microscopically. Minor changes, however cannot always be detected by microscopy.
    • The ability of bacteria to infect live animals or plants. Even if manipulated bacteria are shown in vitro to be able to infect host cells, their ability to sustain an infection in a live organism cannot be taken for granted.
    • The expression levels of other genes. This can be assayed in several ways, notably northern blot and RT-PCR. The expression levels of the entire genome can be assayed by microarray. Many type III transcription factors and regulatory networks were discovered using these methods.
    • The growth and fitness of bacteria.

    Inhibitors of the T3SS Edit

    A few compounds have been discovered that inhibit the T3SS in gram-negative bacteria, including the guadinomines which are naturally produced by Streptomyces species. [35] Monoclonal antibodies have been developed that inhibit the T3SS too. [36] Aurodox, an antibiotic capable of inhibiting the translation of T3SS proteins has been shown to able to prevent T3SS effectors in vitro and in animal models [37] [38]


    Types of Toxins

    A toxin can come in many different shapes and sizes. It can be as simple as a charged particle, running rampant through the system affecting other reactions, to specific proteins that target the nervous system of a prey animal. Because “toxin” is such a broad category, it is impossible to define their size and shape. Toxins produced by animals are typically used to subdue prey or defend against attack. As such, they have evolved to specifically effect certain animals.

    For instance, many pesticides are designed to kill insects, but not to harm other organisms. These typically work by targeting a portion of the insect anatomy that other organisms do not have. Many pesticides are generally safe to use, and there are even some very natural pesticides which are toxin to insects but not to other organisms. However, some of these toxins have unknown effects on other organisms which can cause be very damaging. For instance, the pesticide DDT was invented for use against insects on crops. The molecule was found to be safe for other organisms and was put into widespread use. It wasn’t until decades later that environmental scientists found that the toxin had been slowly weakening the shells of birds at the top of the food chain. DDT was responsible for a massive loss of raptors across the nation, including the Bald Eagle.

    In general, there are 3 main types of toxin. A toxin can be produced by an organism, making it a biological toxin. It may be a single atom or complex molecule produced in nature or in a laboratory, making it a chemical toxin. Lastly, radiation is a special form of toxin which is emitted from radioactive molecules in the environment. Like other toxins, radioactivity disrupts the processes of cells and can lead to death. The many millions of different toxins are classified and categorized differently by different branches of science, but in general they are classified towards their toxicity to humans.


    Type-III-secreted toxins mimicking the Rho GTPases and apoptosis

    Certain Gram-negative bacterial pathogens are equipped with a secretory machinery termed the type-III secretion apparatus, which is highly conserved in both animal and plant pathogens. 37 Bacteria use this protein secretion pathway to deliver a set of effector proteins into the host cell cytosol, where they can usurp host cell signal transduction pathways or mimic host cell molecules. Such type-III-secreted proteins mediate a variety of events, such as macropinocytosis and bacterial invasion in epithelial cells and antiphagocytosis and apoptosis in macrophages. 38 Macropinocytosis is triggered by a vast number of type-III-secreted toxins that, acting either directly on Rho GTPases or mimicking the host cell GEFs, 39 favor the bacterial entry into epithelial cells (Figure 3b). However, since the promotion of this cellular activity in epithelial cells is so far not connected to apoptosis, this matter is out of the scope of this review.

    The general strategy employed by these pathogenic bacteria is the block of phagocytosis and the subsequent elimination of the harmful phagocytes by apoptosis. To gain this result, a vast number of bacteria coerce the Rho GTPases that are central to the organization of the actin cytoskeleton (a scheme on the mechanisms employed is shown in Figure 5). Exoenzyme S (ExoS) plays an, as yet, undefined role in the pathogenesis of the opportunistic pathogen Pseudomonas aeruginosa. 40 It is a bifunctional protein, with two distinct domains, both able to mediate changes in the cytoskeleton. The C-terminus moiety of ExoS ADP-ribosylates Ras at Arg-41 and Arg-128 residues, thus inactivating Ras and blocking Ras-dependent signaling pathways. The N-terminus domain exhibits an ‘arginine finger’ sequence that acts as a GAP for Rac and Cdc42. 40 The ExoS N-terminus has a limited amino acid homology to YopE from Yersinia pseudotubercolosis that also acts as a GAP stimulating the GTPase activity of Cdc42 and Rac but not of other small GTPases. 41 Yersinia YopT is a cystein metalloprotease that cleaves the carboxy terminus end of Rho GTPases, causing their release from the plasma membrane and the consequent disruption of the actin cytoskeleton. 42 In all cases, the inactivation of Rho interrupts signaling events that are necessary for the actin cytoskeleton reshape, thus potentially contributing to antiphagocytosis.

    Type-III-secreted toxins: molecular strategies to foil macrophages. Several bacterial pathogens utilize type-III-secreted effector molecules that usurp the control of Rho GTPases to exert an antiphagocytic activity. YopE and ExoS, which mimic the host cell GAP, interact with Rho-GTP, to induce GTP hydrolysis and the release of Rho-GDP from the plasma membrane. YopT cleaves directly Rho-GTP causing its detachment from the plasma membrane. In both cases, the consequent Rho inactivation interrupts signaling events necessary for the actin cytoskeleton reorganization and thus potentially contributes to antiphagocytosis. Besides impairing their own capture by macrophages, certain bacteria also activate the apoptotic pathway by type-III-secreted toxins. YopT, besides inducing the actin cytoskeleton breakdown, commits cells to apoptosis by directly modifying the Rho GTPases. IpaB and SipB can bind to and presumably activate caspase-1. How caspase-1 can trigger apoptosis is still under debate. In addition to caspase-1, SipB directly binds to caspase-2 switching on an apoptotic pathway that also involves the activation of caspase-3, -6, -8 and the release of cytochrome c from mitochondria. YopP/J also induces macrophage apoptosis probably by preventing the nuclear translocation of the antiapoptotic factor NF-κB. YopP/J also generates a truncated form of the proapoptotic protein BID (tBID) that, upon translocation to mitochondria, induces the release of cytochrome c, activation of executioner caspases and apoptosis

    The first bacterium shown to induce apoptosis was Shigella flexneri, which specifically kills cultured macrophages but not epithelial cells. 43 Macrophages undergo apoptosis and release large quantities of the proinflammatory cytokine interleukin (IL)-1. The IpaB protein is required for such effects, independent of its role in cell entry. Inside cells, IpaB binds to caspase-1, a protease that cleaves pro-IL-1 to release the mature protein and, presumably, such a binding also activates apoptosis. 44 Salmonella spp. also induce phagocyte apoptosis by using a type-III-secreted protein, SipB, which directly activates the host cell's apoptotic and inflammatory pathways by targeting caspase-1, 45 in the same vein of IpaB. More recent evidence, however, shows that SipB activates the apoptotic machinery by also regulating caspase-2 as well as caspase-3, -6, -8 and the release of cytochrome c from mitochondria. 46 Mitochondria, however, are untouched in Salmonella-infected macrophages. It is possible that cytochrome c feeds back on the caspase cascade and accelerates the apoptotic process.

    Within the host cell, type-III-secreted toxins from Yersinia act to inhibit phagocytosis and induce apoptosis. 47 The plant virulence factor AvrPphB (which does not target the Rho GTPases) acts as YopT, modifying a target molecule that blocks the infection by triggering apoptosis of the plant defense system (hypersensitive response). 48 In general, modifications of target molecules by plant virulence factors are recognized by a detection system of the host (R factor), which responds by inducing cell death. As stated by Schneider, 48 the pathogen may double-cross the host and turn that to its own advantage. This could be also true for bacterial virulence factors targeting mammalian cells. For instance, YopP/J from Yersinia plays an anti-inflammatory role beneficial to the bacteria by preventing the activation of NF-κB, but also induces apoptosis because NF-κB is required for cell survival. 49 YopP/J also generates a truncated form of the proapoptotic protein BID (tBID) that, upon translocation to mitochondria, induces the release of cytochrome c, the activation of executioner caspases and apoptosis. 50 Therefore, modification of Rho GTPases themselves, or their downstream effects (e.g. actin cycloskeleton changes), might be detected by the host cell, which will hence induce apoptosis.


    Conclusion

    The use of botulinum toxins has revolutionised the treatment of various ophthalmic spastic disorders, facial dystonias and periocular wrinkles. A precise knowledge and understanding of the functional anatomy of the mimetic muscles is absolutely necessary to correctly use botulinum toxins in clinical practice. Adverse effects are usually mild and transient. The most common substantive complication is excessive or unwanted weakness, and this resolves as the action of the toxin is lost. Brow ptosis, eyelid ptosis, neck weakness, dysphagia, and diplopia may occur. Knowledge of the functional anatomy and experience with the procedure help injectors avoid complications. In future, the development of new potent toxins with increasing effectiveness and duration of effect will further aid this expanding and interesting field of chemodenervation.


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    OVERVIEW

    S. dysenteriae and the Stx were identified in the 19 th century by Drs. Neisser and Shiga (1) and Conradi (2). Approximately 80 years later the same toxin (now called Stx1 to distinguish it from the toxin produced by S. dysenteriae) was found in a group of E. coli isolates. These bacteria caused bloody diarrhea and a serious sequelae, the hemolytic uremic syndrome (HUS), a condition characterized by thrombocytopenia, hemolytic anemia, and kidney failure (3, 4). Some E. coli strains were later shown to produce a highly related toxin, Stx2, that has the same mode of action as Stx/Stx1 but that is immunologically distinct. The Stxs (also known as Vero toxins, and previously as Shiga-like toxins) are a group of bacterial AB5 protein toxins of about 70 kDa that inhibit protein synthesis in sensitive eukaryotic cells. Protein synthesis is blocked by the Stxs through the removal of an adenine residue from the 28S rRNA of the 60S ribosome. This N-glycosidase activity of the toxin resides in the A subunit. The pentamer of identical B subunits mediates toxin binding to the cellular receptor globotriaosylceramide (Gb3). Additional commonalities between the Stx groups are that the subunit genes are encoded in an operon with the A subunit gene 5’ to that of the B subunit, that the stx operon is usually found within the sequence for an inducible, lysogenic, lambda-like bacteriophage, and that the toxins utilize a retrograde pathway to reach the cytoplasm. Differences between the two toxin groups include the fact that the genes for stx/stx1a are repressed by the Fur when high levels of iron are present (5𠄷), and that E. coli strains that encode stx2 are epidemiologically linked to more severe disease than those that carry stx1 (8, 9).


    Chapter One - Antivirulence Properties of Probiotics in Combating Microbial Pathogenesis

    Probiotics are nonpathogenic microorganisms that confer a health benefit on the host when administered in adequate amounts. Ample evidence is documented to support the potential application of probiotics for the prevention and treatment of infections. Health benefits of probiotics include prevention of diarrhea, including antibiotic-associated diarrhea and traveler's diarrhea, atopic eczema, dental carries, colorectal cancers, and treatment of inflammatory bowel disease. The cumulative body of scientific evidence that demonstrates the beneficial effects of probiotics on health and disease prevention has made probiotics increasingly important as a part of human nutrition and led to a surge in the demand for probiotics in clinical applications and as functional foods.

    The ability of probiotics to promote health is attributed to the various beneficial effects exerted by these microorganisms on the host. These include lactose metabolism and food digestion, production of antimicrobial peptides and control of enteric infections, anticarcinogenic properties, immunologic enhancement, enhancement of short-chain fatty acid production, antiatherogenic and cholesterol-lowering attributes, regulatory role in allergy, protection against vaginal or urinary tract infections, increased nutritional value, maintenance of epithelial integrity and barrier, stimulation of repair mechanism in cells, and maintenance and reestablishment of well-balanced indigenous intestinal and respiratory microbial communities. Most of these attributes primarily focus on the effect of probiotic supplementation on the host. Hence, in most cases, it can be concluded that the ability of a probiotic to protect the host from infection is an indirect result of promoting overall health and well-being. However, probiotics also exert a direct effect on invading microorganisms.

    The direct modes of action resulting in the elimination of pathogens include inhibition of pathogen replication by producing antimicrobial substances like bacteriocins, competition for limiting resources in the host, antitoxin effect, inhibition of virulence, antiadhesive and antiinvasive effects, and competitive exclusion by competition for binding sites or stimulation of epithelial barrier function. Although much has been documented about the ability of probiotics to promote host health, there is limited discussion on the above mentioned effects of probiotics on pathogens. Being in an era of antibiotic resistance, a better understanding of this complex probiotic–pathogen interaction is critical for development of effective strategies to control infections. Therefore, this chapter will focus on the ability of probiotics to directly modulate the infectious nature of pathogens and the underlying mechanisms that mediate these effects.


    Engineering of bacterial toxins for research and medicine

    INTRODUCTION

    Bacterial toxins are proteins capable of achieving multiple remarkable tasks ( Menetrey et al., 2005 Parker and Feil, 2005 ). They function as autonomous molecular devices, targeting specific cells in an organism, punching holes in their membranes, or modifying intracellular components. Intoxication processes involve highly specialized steps of great complexity. It is thus tempting for the biochemist, the protein engineer, the biotechnologist, or the medical scientist to exploit the sophisticated properties of bacterial toxins to design new toxin-derived molecules for research, biotechnology, or medical treatments. Some toxins or toxin subunits are used in their natural form as biochemical and cell biology tools or for the treatment of specific diseases. For instance, streptolysin O is used to punch transient holes into cells for the delivery of oligonucleotides ( Broughton et al., 1997 ) or proteins ( Fawcet et al., 1998 Walev et al., 2001 ) into their cytoplasm. Cholera toxin B pentamer is used as a marker of lipid rafts due to its binding specificity for gangliosides sequestered in these membrane microdomains ( Harder et al., 1998 ). It is also used as an adjuvant for mucosal vaccines ( Freytag and Clements, 2005 ). Clostridium toxins are used to study actin and G proteins ( Richard et al., 1999 ) botulinum toxin is used in the treatment of dystonies ( Jankovic, 2004 ). Also, natural bacterial toxins inactivated by chemical treatment are used as vaccines. These include diphtheria, tetanus, and pertussis toxins.

    Besides the use of native toxins as tools, therapeutics, or vaccines, toxins or toxin fragments can be engineered or combined with other protein domains to build rationally new proteins with new defined activities. This review focuses on the vast possibilities offered by toxin engineering for the design of new tools and therapeutics. The applications of native toxins are described in other chapters of this book. Also, the mechanisms of action of the toxins used for engineering are not detailed here. The reader is invited to return to the corresponding chapters of this book to find all precisions on those mechanisms.

    A brief overview of the properties of bacterial toxins helps to understand the fascinating possibilities they offer as building blocks to tailor new proteins with desired activities ( Table 60.1 ). Toxins are usually secreted by bacteria in a soluble form. The toxins are then able to diffuse in the aqueous environment of body fluids such as digestive or respiratory secretions, interstitial fluid surrounding infected tissues, lymph, blood, etc. Some toxins are capable of crossing epithelial barriers or penetrating deeply inside organs. For instance, botulinum toxin crosses the digestive wall from the intestine lumen by transcytosis to the blood circulation, and tetanus toxin reaches inhibitory interneurons in the central nervous system by retrograde transport through peripheral neurons. Cell intoxication proceeds with a series of successive steps involving different domains or subunits of the toxin ( Figure 60.1 ). Activation of the toxin by proteolytic cleavage of a terminal peptide, an interdomain loop, or even an intradomain site is necessary for many toxins. This process may involve bacterial or host proteases and may occur before or after binding of the toxin to the surface of target cells. It is likely that the need for an activation mechanism prevents the toxins from interacting inappropriately with bacterial or host membranes. Bacterial toxins select their target cells by recognizing specific cell surface receptors. Most toxins recognize receptors found on any cell types, thus having a broad spectrum of targets, while others recognize receptors found on very specific cells, such as neurons (Clostridium neurotoxins) or dendritic cells of the immune system (Bordetella pertussis adenylate cyclase). Pore-forming toxins act directly at the cell surface, in most cases after oligomerization ( Parker and Feil, 2005 ). Toxins with intracellular targets of the ABRT and AB5 types act usually as individual molecules (monomers or preassembled oligomers), while toxins of the AnB7 type assemble as oligomers following binding to the cell surface ( Menetrey et al., 2005 ). These toxins penetrate the cells by the internalization pathways followed by their receptors. They are directed to given intracellular compartments depending on their receptors or on internal targeting sequences. Most toxins of both types (pore-forming or with intracellular targets) undergo major conformational changes involving one or several domains in order to interact with and penetrate into the membrane of the cell surface or of the intracellular compartments to which they are targeted. They create channels or pores, some with small sizes and narrow selectivity, some with very large sizes permeable to macromolecules. Toxins with intracellular targets translocate their catalytic domain (C) or subunit through the membrane of given cell compartments into the cell cytoplasm. This process involves major structural changes of the catalytic components (Falnes et al., 1994). Finally, these components refold in the cytoplasm and exert their enzymatic activities towards specific substrate molecules participating in key cellular processes ( Menetrey et al., 2005 ).

    TABLE 60.1 . Properties of bacterial toxins that can be exploited for engineering

    PropertiesToxins concerned a
    Diffusion in body fluidsPore-forming b and intracellular c
    Crossing of epithelial barriersBotulinum toxins, some AB5 toxins
    Penetration into central nervous systemTetanus
    Proteolytic activationPore-forming b and intracellular c
    Targeting of cell surface receptorsPore-forming b and intracellular c
    Action on the cell surface (permeabilization)Pore-forming b
    Translocation from the cell surfaceB. pertussis adenylate cyclase
    Internalization and intracellular traffickingIntracellular c except B. pertussis adenylate cyclase
    Conformational change upon oligomerizationPore-forming b , AnB7 toxins
    Conformational change upon acidificationABRT toxins, AnB7 toxins
    Membrane penetrationPore-forming b , ABRT, AnB7 toxins
    Pore formation (large size, poor or no selectivity)Pore-forming b (except RTX toxins)
    PermeabilizationRTX toxins, some ABrt
    Channel formation (small size, high selectivity, voltage gated)ABRT, AnB7 toxins and colicins
    Translocation from cell compartmentsIntracellular c except B. pertussis adenylate cyclase
    Enzymatic activities (see Menetrey et al., 2005 for list)Intracellular c

    FIGURE 60.1 . Structure/function organization of toxins with intracellular targets ( Ménétrey et al., 2005 ). In principle, each module (chain or domain) may be isolated and used for engineering in combination with other proteins. In all cases, chain A carries the catalytic domain (C) of the toxin. For ABrt toxins, chain B is formed of a receptor-binding domain (R) responsible for binding to the cell surface, receptor-mediated internalization, and intracellular trafficking and a translocation domain (T) responsible for passage of the C domain inside the cytoplasm. For AB5 toxins, chain B is responsible for binding to the cell surface, receptor-mediated internalization, and intracellular trafficking. For AnB7 toxins, up to three A chains may bind to one B heptamer. Chain A contains a domain responsible for binding to the B heptamer and a C domain. Chain B is responsible for binding to the cell surface, receptor-mediated internalization, intracellular trafficking, and translocation of chain A into the cytoplasm. Among ABrt toxins are: diphtheria toxin (C. diphtheria), exotoxin A (P. aeruginosa), botulinum toxins (C. botulinum), tetanus toxin (C. tetani), large clostridial toxins (C. difficile, C. sordellii, C. novyi), and dermonecrotic toxins (E. coli, B. pertussis). The adenylate cyclase of B. pertusis resembles ABrt toxins, although its chain B is derived from an RTX toxin. Among AB5 toxins are cholera toxin (V. cholerae), heat-labile toxins (E. coli), pertussis toxin (B. pertussis), Shiga toxin (S. dysenteriae), and Shiga-like toxins (E. coli). Among AnB7 toxins are anthrax toxin (B. anthracis), VIP toxin (B. cereus), C2 toxin (C. botulinum), iota toxin (C. perfringens), and actin-ADP-ribosylating toxins (C. spiroforme and C. difficile).

    All these steps, all these complex activities are frequently associated with individual domains, chains, or subunits of toxins ( Figure 60.1 ). This is particularly true for toxins with intracellular activities. Many of these domains can be isolated from the rest of the toxin by recombinant DNA technology and used for their properties, isolated or combined with other proteins. Also, they can be modified by protein engineering. They may retain entirely or partially their original function, or even express new unexpected capabilities. Even genes encoding fragments of toxins or toxin receptors can be used to engineer cells or animals. Possibilities are infinite. This review intends to illustrate the concept of toxin engineering with a series of examples but it cannot be exhaustive. These examples are sorted according to the two principal types of bacterial toxins (toxins with intracellular activities and pore-forming toxins) and to the molecular functions or mechanisms that are exploited ( Table 60.1 ). However, most examples of toxin engineering involve toxins with intracellular targets, especially diphtheria toxin, which is certainly one of the most extensively manipulated toxin ( Chenal et al., 2002a ) ( Figure 60.2 ).

    FIGURE 60.2 . Examples of diphtheria toxin engineering. See text for references.