Chapter 17 Bacterial Toxins

• Bacterial toxins are substances produced by bacteria that are toxic to human cells, causing disease symptoms.
• Toxins were the only virulence factors that could be clearly isolated and defined as having a role in pathogenicity for a long time.
• Modern technologies have made it easier to determine the cellular targets of toxins and how they exert their toxic effects.
• Toxins have proven useful reagents for scientists studying cell biology, probing signaling pathways, and metabolic processes.
• Toxins are being used for beneficial purposes in medicine like treating neurological conditions with botulinum neurotoxin and agriculture like controlling pests with insecticidal toxins.
• The mechanisms behind why bacteria acquire toxin genes and benefit from producing toxins remain mysterious.
• Toxins that kill or impair the action of neutrophils and macrophages can protect bacteria from phagocytic cells.
• Toxins that kill human cells can release iron stores or carbon sources that bacteria need to survive and multiply.
• Toxins can dampen the immune response, enabling bacteria to survive in the host by evading the immune system.
• There are cases where it's difficult to discern the benefits for bacteria to produce toxins.
• Botulinum neurotoxin (BoNT) attacks neurons, causing the disease botulism, but bacteria are often eliminated before the toxin exerts its effects.
• C. botulinum survives as spores in soil or as vegetative cells in anaerobic environments, where it may kill fish and small mammals for nutrients.
• In many toxin-producing bacteria, toxin genes are associated with pathogenicity islands on mobile genetic elements.
• Horizontal gene transfer (HGT) is a major driving force in the evolution of toxins and toxin-producing pathogens.
• Toxin genes carried on lysogenic bacteriophages lead to toxin production only in bacteria that have acquired the phage.
• Widespread genetic exchange of bot genes among clostridia in the environment occurs through multiple HGT mechanisms.
• Toxins may play roles in bacterial physiology, regulate cellular or phage functions, or bacterial cell-cell signaling.
• Animals other than humans, such as insects or protozoa or plants, may be the intended targets of toxins in the environment.

Toxin Characteristics and Nomenclature

• Bacterial toxins come in various forms, from small lipid-like compounds to peptides and large proteins.
• Exotoxins are toxins secreted from the cell, while endotoxins are components of the bacterial surface released through cell lysis.
• The term "exotoxin" is falling out of use because some protein toxins are not secreted and others are injected directly into host cells.
• Toxin refers to all types of virulence factors of bacteria, whether secreted or not, that are toxic to the human or animal host.
• Some toxins are named for the type of host cells they attack, such as cytotoxins, neurotoxins, leukotoxins, hepatotoxins, cardiotoxins, verotoxins, and enterotoxins.
• Enterotoxin specifically denotes a toxin that attacks the GI tract and causes diarrhea or vomiting.
• Toxins are sometimes named for the bacterial species that produces them or the disease with which they are associated.
• Variants of BoNT are designated by letters (A through G) based on their order of discovery and immunological cross-reactivity.
• Toxins can also be named based on their enzymatic or cellular activities.
• Some toxins have more than one name.
• Toxins are often designated as Types I-III toxins based on their mechanism of action.
• Type I toxins bind to the target cell surface and act extracellularly.
• Type II toxins act extracellularly on eukaryotic cell membranes, destroying their integrity.
• Type III toxins are A-B type toxins, with an enzymatic component (A) and a binding component (B) that delivers the A portion into the cell.

Nonprotein Toxins

• These include endotoxins (LPS) and glycopeptides such as tracheal cytotoxin (TCT).

Endotoxins

• Released into the surrounding medium when bacteria lyse or turn over their cell walls.
• Trigger excessive pro-inflammatory cytokine production, leading to symptoms of shock.
• LPS endotoxin consists of a polysaccharide chain attached to a polysaccharide core, which is attached to lipid A.
• Toxicity is strongly associated with the hydrophobic lipid A component.
• LPS binds to LPS-binding protein (LBP), which interacts with Toll-like receptors (TLRs) on host cells.
• LPS-induced cytokine release causes activation of the alternative complement cascade, increased vascular permeability, and activation of the coagulation cascade.
• Treatment of systemic bacterial infection with antibiotics can worsen the disease due to the release of LPS.
• Treatment of syphilis with penicillin can result in the Jarisch-Herxheimer reaction due to the acute increase in the release of pro-inflammatory cytokines.
• TCT is responsible for the respiratory cytopathology observed during whooping cough.
• TCT triggers the violent paroxysmal coughing episodes symptomatic of pertussis.

Mycolactone Toxins

• Lipid-like toxins with both cytotoxic and immunosuppressive properties.
• Made by Mycobacterium ulcerans, the causative agent of Buruli ulcer.
• M. ulcerans strains make a variety of mycolactones, which are polyketide-derived macrolides that are made as secondary metabolites.
• Infection causes progressive necrotic lesions with very little inflammation or pain.
• The emergence of M. ulcerans as a pathogen most likely reflects the acquisition of a large 174-kilobase plasmid (pMUM001).

Peptide and Protein Exotoxins

• Toxic shock syndrome is caused by a toxin produced by S. aureus that colonized the vagina and entered the bloodstream.
• Toxic shock syndrome toxin (TSST) forces unnatural associations between macrophages and T cells, causing an outpouring of cytokines.
• Superantigens (SAgs) exert their effect by binding to the MHCII of macrophages and the receptors on T cells (TCRs).
• SAgs are not processed by proteolytic digestion inside macrophages, but rather bind directly to MHCII on the macrophage surface.
• SAgs bind TCRs and form many more macrophage-T helper cell pairs than would normally form.
• SAgs can activate up to 20% of the total T cell population.
• T cells release massive amounts of pro-inflammatory cytokines, especially interleukin-2 (IL-2).
• Clinical isolates of S. aureus and Streptococcus pyogenes produce a wide range of other types of SAgs.

Membrane-Disrupting Toxins (Type II Toxins)

• Lyse host cells by disrupting the integrity of the target cell’s plasma membranes.
• Frequently called "hemolysins," but the more general term cytolysin is also frequently used.
• Some disrupt cell membranes to release cellular content for nutrient acquisition and immune evasion.
• Others are used by invasive bacteria to escape from the phagosome and enter the cytoplasm.
• Pore-forming toxins poke holes in membranes by binding to the cell surface, oligomerizing, and inserting into the membrane to form transmembrane channels.
• α-pore-forming toxins form pores in membranes using helices.
• β-pore-forming toxins form a β-barrel in membranes.
• Cytolytic enzymes compromise the integrity of the membrane by hydrolyzing the membrane phospholipids.
• Some phospholipases remove the charged head group from the lipid portion of the molecule.
• Damage caused by α-toxin can be so severe that the limb has to be amputated .

A-B Toxins (Type III Toxins)

• A-B toxins usually contain two types of functional domains or subunits.
• The first component (called the A part) confers the toxic (usually enzymatic) activity, while the second component (called the B part) binds to receptors on host cells and helps the A part cross the plasma membrane into the host cell cytosol.
• The simplest type of A-B toxin is synthesized as a single polypeptide with a single binding domain (B) and a single activity domain (A).
• Another domain, which is distinct from the receptor-binding domain (still called the B domain), is referred to as a T domain for its translocation function.
• A more complex type of A-B toxin, a multi-subunit A-B toxin, has an enzymatic subunit (A) that is expressed as a separate polypeptide from the binding subunit (B), which is composed of a complex of multiple subunits
• Both types of A-B toxins bind to cell surface receptors and enter host cells, but their trafficking once inside the cell and the location where they translocate their catalytic domains vary.
• The receptor-binding domain determines the host cell specificity of the toxin.
• After the B domain/subunit binds to the host cell, the whole toxin is taken up into endocytic vesicles.
• Translocation is a complex process that is only beginning to be understood and appears to be different for different toxins.
• Some multi-subunit A-B toxins have more than one A subunit that can bind to the multimeric B complex.
• Other toxins appear to use alternative routes for gaining entry into the cytosol that do not involve endosomal acidification.
• The activated A domain/subunit exerts its toxic effects.
• The effect can be the same or similar since the A domain/subunit catalyzes the same reaction on its cognate substrate targets.
• The A domain of DT ADP-ribosylates an unusual posttranslationally modified histidine, called diphthamide, of elongation factor-2 (EF -2), a protein that is essential for elongation of the growing peptide chain during host cell protein synthesis, killing the host cell by stopping protein synthesis.
• The A1 subunit of CT ADP-ribosylates an arginine residue of the α subunit of the regulatory heterotrimeric GTP-binding protein, Gs, causing constitutive activation of Gs, which in turn stimulates adenylate cyclase and increases cellular cAMP levels, causing the host cell to lose control of ion flow and causing diarrhea.
• Binary ADP-ribosylating toxin (ex:C2 toxin) modifies actin at arginine-177 which prevents actin polymerization and eventually leads to depolymerization of actin filaments and cell rounding.
• The catalytic A subunit of STx hydrolyzes the adenine base at a specific nucleotide site of the host cell 28S rRNA molecule, resulting in a shutdown of protein synthesis.
• The catalytic domains of the single-chain BoNTs are zinc-dependent metalloproteases that cleave neuron-specific proteins at specific amino acid sequences, which leads to loss of neurotransmitter release and flaccid paralysis.
• The catalytic domain of the cytotoxic necrotizing factors (CNFs) constitutively activates members of the Rho family of small GTP-binding proteins by deamidating a specific active site glutamine, thereby converting it into a glutamic acid, resulting in actin cytoskeletal changes.

Toxic Effector Proteins of Specialized Secretion Systems

• Some toxic bacterial proteins, called effector proteins, are directly secreted from the bacterial cytoplasm and injected into the host cell cytoplasm through specialized bacterial secretion systems.
• These effector proteins were formerly called exoenzymes.
• The toxic activities of these effector proteins are very similar to those of A-B toxins and membrane-disrupting phospholipase exotoxins.
• These effector proteins cause cytopathic or cytotoxic effects on the host cells.
• Toxic effector proteins require the specialized secretion system to deliver them into host cells in order to exhibit their toxicity.

Examples of Toxin-Mediated Diseases

Diphtheria Toxin

• DT is one of the best-studied bacterial toxins.
• Diphtheria provided the first paradigm for a disease in which the symptoms could be explained entirely by the action of a single molecule, DT.
• DT has become a prototype for selective targeting and killing of certain cells, eliminating tumor cells in cancer therapeutics, and for antigen delivery in vaccine development.

Diphtheria

• The disease diphtheria is caused by infection with C. diphtheriae.
• Diphtheria is normally a disease of children, which can be fatal if not treated.
• The diphtheria vaccine is a toxoid version of DT
• Diphtheria starts with colonization of the throat by C. diphtheriae.
• The first symptoms are relatively nonspecific: malaise, low-grade fever, sore throat, and loss of appetite.
• Colonization of the throat causes considerable damage to the mucosal cells due to release of DT that kills the exposed cells.
• A grayish membrane begins to form in the throat called a pseudomembrane, consists of fibrin, bacteria, and inflammatory cells and adheres to underlying tissue.
• DT does enter the bloodstream at lesion sites and causes damage to internal organs, eventually resulting in organ failure.
• Recovery from infection leads to lifelong immunity.

Diphtheria Antitoxin

• Binding of antibodies to the B domain/subunit of the toxin physically interferes with binding of the toxin to its target cell and thus prevents the toxin from entering and exerting its toxic activity.
• Injection with antitoxin (i.e., passive immunization) is an effective means for treating toxin-mediated diseases.

Model for the Mechanism of Action of DT

• DT is secreted as a single 58-kDa polypeptide that is proteolytically cleaved into two fragments, A (21 kDa) and B (37 kDa), which remain attached to each other by a disulfide bond.
• B fragment binds to the cell surface protein receptor called heparin-binding epidermal growth factor-like growth factor (HB-EGF).
• The DT:HB-EGF complex is then internalized via receptor-mediated endocytosis.
• Acidification of the endosome triggers the insertion of the T domain of the B fragment into the membrane.
• A domain is then translocated across the membrane and into the cytosol, where the disulfide bond is reduced.
• A domain is then free to diffuse around the interior of the cell.
• A domain is an enzyme that catalyzes a peculiar reaction: the transfer of the ADP-ribosyl moiety of NAD+ to the diphthamide residue of EF-2.
• ADP-ribosylation of EF-2 inactivated this protein, which is essential for the translocation step of protein synthesis.
• DT enters the host cell, and the host cell kills itself by shutting down protein synthesis.

Regulation of Diphtheria Toxin Production

• Iron is required for synthesis of heme-containing enzymes involved in electron transport.
• In nutrient-rich laboratory media, there are high concentrations of iron, so relatively little DT is produced by C. diphtheriae.
• Humans infected with diphtheria suffer a drastic reduction in the level of free iron in the bloodstream, resulting in iron limitation for the colonizing corynebacteria.
• Expression of the tox gene is regulated by a repressor protein called DtxR,
• When iron is available, DtxR binds iron and then binds to the tox operator (the DNA region upstream of the tox gene), thereby blocking transcription of the tox gene and preventing DT production.
• Iron limitation has the opposite effect, because DtxR cannot bind to the tox operator without iron.
• When iron is limiting, the tox operon is transcribed and DT is produced.
• DT acts to kill host cells, releasing iron that the corynebacteria can then scavenge.

Vaccine Development and Human Therapeutics

• Diphtheria toxoid vaccine is easy to make, because it only requires treatment of the protein with formaldehyde to inactivate its enzymatic activity.
• Diphtheria toxin has been genetically engineered to kill tumor cells.
• A domain of DT was cloned and fused genetically to a single-chain antibody fragment that recognizes and binds CD25, a protein found only on the surface of cancerous T cells.
• Fusion protein is injected intravenously into a patient with T-cell lymphoma who has CD25-positive T cells.
• The fusion protein binds only to the cancerous lymphocytes, delivering the catalytic A domain of DT to those cells and killing them.

Botulinum Toxin

• BoNT is the most acutely toxic substance known.
• It has been estimated that one gram of evenly dispersed and inhaled BoNT could kill more than one million people.
• Botulism is an intoxication resulting from the ingestion of food in which C. botulinum has grown and produced toxin.
• C. botulinum is an obligate anaerobe, whose natural environment is the soil.
• Spores of C. botulinum are highly resistant to heat and can persist in foods that are not adequately sterilized during the canning process.
• If canned foods are underprocessed, C. botulinum spores can germinate in the anaerobic environment inside the can, initiating bacterial growth and toxin production.
• BoNTs are absorbed into the bloodstream through the stomach and small intestine after the canned food is ingested.
• In a few rare cases, botulism results from the spores themselves being ingested in raw foods such as honey, or unpasteurized organic juices, or inadequately washed vegetables.
• Spores cannot grow in an aerobic environment, so these foods are not a problem.
• The only place they can grow is inside an anaerobic environment, namely your body.
• Botulism develops in adults when the spores germinate in the gut and produce colonies of vegetative bacterial cells, which rapidly grow and secrete BoNT.

Food Botulism

• Most often associated with eating improperly home-canned foods.
• Symptoms usually begin 12 to 36 hours after eating, with a range of 6 hours to 10 days.
• The first symptoms are nausea and vomiting, followed by progressive muscle paralysis.
• Double vision is often reported.
• Death results from paralysis of the respiratory muscles.

Other Forms of Botulism

• Two less common forms of botulism involve transient colonization of the body by the bacteria, followed by toxin production: infant botulism and wound botulism.
• Infants less than 1 year old have not yet developed a complete colonic microbiota, C. botulinum can sometimes colonize the infant colon and cause infant botulism.
• Infant botulism has a slower onset than foodborne botulism with an incubation period of 3 to 30 days.
• Special human-derived antitoxin, called BabyBIG (botulism immunoglobulin), is available for infant botulism caused by BoNT/A or BoNT/B.
• A deep wound can become anaerobic because tissue destruction cuts off the blood supply to the area and residual oxygen is rapidly depleted by the surrounding body cells, Botulinum leaks into the bloodstream from the wound area and causes wound botulism.
• Wound botulism is normally only seen during wartime, but civilian cases have occasionally been reported in people with severe, deep wounds that were heavily contaminated with soil.
• Botulism due to inadvertent overdosing of BoNT during injections for medical or cosmetic treatments has shown a marked increase in occurrence.

Bacterial Toxins: Double-Edged Swords that Turn the Bad Guy into the Good Guy

• Eukaryotic cell biologists have found certain toxins to be very useful for elucidating regulatory pathways.
• Botulinum neurotoxin (sold as Botox) has been used as an FDA-approved drug to treat painful, disabling muscle spasms.
• Botox has also found favor with cosmetic surgeons who are using it to reduce the depth of wrinkles.
• Botox has been administered to cerebral palsy patients in an effort to help them control the movement of their limbs, to treat migraines, excessive sweating, urinary incontinence, and hypersalivation (drooling).
• One remarkably successful use for Botox is in the treatment of facial wounds to prevent severe scarring during the wound healing process.

Botulinum Neurotoxin

• BoNT is synthesized and secreted from the bacteria as a single 150-kDa protein, which gets cleaved into two protein fragments, heavy chain (100 kDa) and light chain (50 kDa), which are connected by a disulfide bridge.
• The complex is called progenitor toxin and the toxin itself is called derivative toxin.
• Progenitor toxin help protect the derivative toxin from stomach acid and proteases through the oral route of transmission.
• The specificity of BoNT for peripheral neurons arises both from the specificity of toxin binding due to ganglioside and protein receptors found only on neuronal cells and from the neuron-specific action of the toxin.
• After binding to its receptors through the carboxy terminal region of the heavy chain, the toxin is taken up by endocytosis into neuronal cells.
• Following acidification of endosomes containing the toxin, the amino terminal region of the heavy chain mediates translocation of the catalytic light chain (i.e., the A domain) to enter the cytosol of the cell.
• BoNT serotypes B, D, F, and G, as well as the related TeNT cleave synaptobrevin (also called VAMP).
• BoNT serotypes A and E cleave SNAP25 and BoNT serotype C cleaves syntaxin.
• The net effect of SNARE protein cleavage is that vesicle fusion does not occur, so that ACh neurotransmitter is not released to synapses, causing muscles to stop contracting and flaccid paralysis to set in.

Tetanus

• Tetanus is a neurotoxin-mediated disease caused by another clostridial species, C. tetani.
• In botulism, the patient suffers a flaccid paralysis, whereas in tetanus the patient develops a spastic paralysis, in which the muscles contract and do not relax.
• Tetanus starts when the bacteria colonize a deep puncture wound.
• TeNT acts on neurons that control the neural feedback that tells flexed muscles to relax after having performed a task, preventing these neurons from signaling the relaxation after a muscle contraction.
• In developed countries today, tetanus is virtually unknown because of the tetanus vaccine.
• Worldwide, tetanus remains a major cause of infant deaths.
• BoNTs target peripheral neurons, TeNT acts on the central nervous system and causes spastic paralysis.
• TeNT targets different neuronal receptors from that of the BoNTs and undergoes retrograde transport to the cell body of spinal cord motor neurons and cleaves synaptobrevin at inhibitory synapses.

Cholera Toxin

• The multi-subunit AB5 CT is secreted by Gram-negative V. cholerae that harbor the prophage CTXϕ.
• ctxAB gene cluster encoding CT is located on this prophage.
• CTXϕ phage are located on a second prophage, VPIϕ.
• Frequent HGT events among Vibrio in the environment have contributed to the emergence of new epidemic strains of toxin-producing V. cholerae.
• V. cholerae are primarily found associated with zooplankton, phytoplankton, or marine snow in marine environments.
• Seasonal outbreaks of cholera in endemic regions of the world are strongly correlated with abundances (blooms) of zooplankton or phytoplankton.
• When ingested through contaminated water, the V. cholerae uses flagellar motility to penetrate through the viscous mucus layer and attach to epithelial cells in the small intestine.
• The bacteria are noninvasive, so little or no inflammatory response results.
• CT triggers a massive diarrheal response, such that a person can lose up to 20 liters of fluid per day
• Without rehydration therapy, death can result from dehydration.

Mechanism of Cholera Toxin-Mediated Diarrhea

• The B subunits of CT form a pentameric complex that binds to GM1 gangliosides on intestinal epithelial cells.
• After internalization via receptor-mediated endocytosis into recycling endosomes, the toxin is processed by furin and transported through retrograde trafficking to the Golgi apparatus and then the ER.
• The A1 subunit is dissociated from the A2B5 complex with the assistance of protein disulfide isomerase (PDI) and ER oxidoreductin (Ero1).
• A1 subunit into the cytosol, where it avoids ubiquitination and proteasome degradation and refolds.
• A1 subunit's ADP-ribosyltransferase activity covalently modifies the GTPase α-subunit of the stimulatory heterotrimeric Gs protein that constitutively up-regulates the cell’s adenylate cyclase (AC).
• The stimulated AC converts ATP into cAMP, greatly increasing cellular cAMP levels, which in turn stimulates cAMP-dependent protein kinase (PKA).
• PKA is a key regulator of various cellular signaling processes, including Na+ and Cl– ion transporters.
• CT-mediated activation of PKA leads to phosphorylation and inhibition of the Na+/H+ exchanger and blockade of Na+ adsorption by villus absorptive enterocytes.
• In crypt cells, CT-mediated activation of PKA leads to phosphorylation and stimulation of the major Cl– ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR).
• This causes a net efflux of Na+ and Cl – into the lumen of the gut.
• Since water will follow the osmotic gradient, this results in rapid loss of water into the lumen, causing massive intestinal distention and diarrhea.

Oral Rehydration Therapy (ORT)

• The mainstay for preventing dehydration and death in cholera patients is oral rehydration therapy (ORT).
• The current WHO ORT formula recommended for children consists of: 2.6 g NaCl, 1.5 g KCl, 2.9 g Na+3-citrate (or 2.5 g NaHCO3), and 13.5 g anhydrous glucose per liter of clean water.
• The key aspect of ORT is the inclusion of a mixture of NaCl plus glucose to bypass the inhibited Na+/H+ exchanger and force the Na+-glucose cotransporter to bring Na+ into cells, thereby reversing the water flow direction caused by CT action.
• ORT has proven to be over 90% effective in preventing dehydration and death from cholera, thereby saving millions of lives.

Toxin-Based Therapeutics and Research Tools

• Toxins are very useful research tools for cell biologists and immunologists.
• Researchers have sufficient insights into how toxins work allowing scientist to use them for their own benefit.
• Immunotoxins have been used in developmental biology to knock out certain cell types.
• The toxin is expressed under cell type-specific mammalian promoters that turn on only during certain stages of development.
• The elimination of a specific cell type can provide information about its role and importance in development.
• BoNT application to treat neurological conditions, aid in wound healing, reduce pain, or reduce facial wrinkles.
• Toxins are efficient cargo delivery vehicles and have been used for intracellular delivery of peptide antigens to immune cells in novel vaccine strategies.
• Not all bacterial toxins are directed at human cells, some are used for beneficial agricultural purposes.
• Cell-killing toxins produced by Bacillus thuringiensis, the Bt toxins (crystal or Cry toxins), are widely used as insecticides in agriculture.
• Bt toxins selectively attack a small subset of harmful insects (moths, flies, mosquitoes, beetles, ants, and nematodes), but not beneficial insects (bees and butterflies).

Immunotoxins

• Cell-killing toxins such as the catalytic domain of DT, P. aeruginosa exotoxin A, or ricin can be used to selectively ablate or kill certain cells.
• This approach has been applied toward the killing of cancer cells by coupling the cell-killing portion of the toxin with selective cancer cell-targeting antibodies or ligands.
• These new immunotoxin therapeutic agents have revolutionized treatment options for prostate, breast, bladder, ovarian, colorectal, and pancreatic cancers, as well as leukemia and melanoma.
• DT was used as the prototype for new therapeutic approaches against cancer cells or viral-infected cells due to its potent cell-killing ability.
• Early immunotoxin approaches used hybrid fusion proteins between the catalytic domain (the A fragment) of DT and cell-targeting, receptor-binding proteins, such as antibodies or receptor ligands.
• Which could selectively target the fusion protein to particular cancer cells or viral-infected cells.
• These hybrid proteins are referred to as immunotoxins because they contain a cell-killing part (the A fragment of DT) and a cell-targeting part (an antibody or receptor ligand).
• The A fragment of DT fused with IL-2 recognizes, enters, and kills IL-2 receptor-expressing lymphoma and leukemia cells, and showed good response in phase I and II clinical trials.
• A conjugate of human transferrin with the A fragment of DT showed good clinical efficacy when it was administered intratumorally or when it was infused in the brain tissue surrounding a malignant glioma (brain tumor).
• Leukemia cells in the blood are exposed to high immunotoxin concentrations
• The immune system is impaired in many hematologic malignancies leading to impaired antibody responses against the immunotoxin.
• Immunotoxins are more effective against leukemia-like diseases, since many cycles of immunotoxin therapy can be given without antibody formation against the therapeutic agent.
• Immunotoxins can reach the cells within solid tumors only by slow diffusion and in low concentrations.
• Antibody formation against the DT-based immunotoxins prevents multiple treatments for solid tumor cases due to an intact immune system.
• Direct injection into the center of the tumor is the only way for immunotoxins to work against solid tumors.
• Researchers have turned to other cell-killing toxins as alternatives because most people are immunized against DT and have antibodies that can neutralize its activity.
• Other cell-killing toxins include the ADP-ribosylating domain of Pseudomonas exotoxin A and ricin (a plant toxin with catalytic activity like STx).