By: Mary N. Wessling, Ph.D. ELS

In this blog we will unravel the terminology describing bacterial toxins. In general, there are at least three ways that bacterial toxins are described in the literature:

• by their biological designation—the genus and/or species from which they come
• by the origin of the toxin, either as innate to the bacterial structure or released by the bacterium into surrounding body fluids
• by the body part that is damaged by the toxin

Below are examples of each:

Biological designation

When described by their biological designation a part of the genus or species name is used for the toxin. For example: Clostridium tetani produces Tetanus toxin and Corynebacterium diphtheriae produces Diphtheria toxin.

Origin of the toxin

Exotoxins (e.g. polypeptides) are toxins released by a cell, whereas endotoxins (e.g. lipopolysaccharides) are an integral part of the bacterial cell wall.

Body part damaged by the toxin

Bacteria may cause disease through their toxins that enter the body via the respiratory tract, gastrointestinal tract, genital tract, and the skin. Enterotoxins mostly affect the gastrointestinal tract. “Entero” comes from the Greek word “enteron” meaning intestine.

Bacterial enterotoxins include examples of exotoxins produced by some strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli).Staphylococcal enterotoxin acts on intestinal neurons to induce vomiting; E. coli producing Shiga toxin causes serious dysentery and can lead to hemorrhagic diarrhea and kidney failure.

You will also see other terms used to designate toxins…

Superantigens: toxins that cause over-reaction

Antigens are characterized by their ability to activate T-cells and other immune system cells; while the T-cell response is a normal part of the immune process, over-activation of T-cells can cause an inflammatory response that can result in shock and multiple organ failure.

Pore-forming toxins that open host cell membranes

Pore-forming toxins (PFT) are toxin proteins with the ability to spontaneously self-assemble forming transmembrane pores in the membrane of target cells. Staphylococcal alpha toxin, also known as alpha-haemolysin, makes specific pores in target cells which are part of the pathology of infection and a valuable tool in construction of nanopores. Tetanolysin is another pore forming toxin produced by C. tetani which can make cells permeable to materials for experimentation.

Intracellular toxins

These toxins have two-part structures and are termed AB toxins. The A stands for “active”, the B for “binding”, for the ways that the two structures cooperatively cause cell damage. In most cases, the B structural element attaches to the cell membrane and provides an entry point for the other part, the A-enzyme component that causes damage to the inside of the cell through its enzymatic activity.

Some AB toxins have more than one B moiety: for example, the cholera toxin has five B proteins that provide entry for the A moiety, so it is designated AB₅. The A moiety is initially a coiled chain but once inside the cell it uncoils, where its enzymatic activity kills the enteric cell.

Ligand-receptor interactions

The actions of exotoxins and endotoxins depend on a process whereby a part of their molecular structure, a ligand, can bind or otherwise interact with a structure on the host cell being attacked, a receptor. Thus, this ligand–receptor interaction is crucial to most diseases produced by bacterial toxins.

Lethal dose 50%

Bacteria cause disease by toxin production, invasion and inflammation. All toxins damage or disrupt the functions of the host cells. The term that describes the level of danger presented to the host by a toxin is “Lethal Dose 50%”, abbreviated LD₅₀; the lower the LD₅₀, the lower the amount of toxin to cause death.

 

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

 

Three anthrax toxin components—Protective antigen (PA), edema factor (EF) and lethal factor (LF) are available for research purposes from LIST Biological Laboratories, separately at a high level of purification. At least two out of three of these components are necessary to enter a mammalian cell and exert a toxic effect.

With the aim of developing antitoxin therapies, scientists have been investigating the structure of PA, EF, and LF, and the complexes that they form with mammalian cell surface receptors, in hopes of finding the best way to disrupt or block the toxicity. Previously, NIAID-supported scientists have shown that protective antigen can bind edema factor and lethal factor at the same time, forming a greater variety of toxin complexes than were formerly known.¹ They also had produced a three-dimensional molecular structure of the anthrax protective antigen protein bound to one of the receptors (CMG2) it uses to enter cells.² More recently, a group of students in Kansas used Jmol and 3D printing technology to model and Anthrax toxin heterotrimer (PA, EF and LF) which forms a pore in the mammalian cell surface.

In an in vitro disease model, researchers constructed an artificial membrane bilayer using lipid and demonstrated that the blood of animals carrying anthrax infections was able to disrupt this membrane, a model of the cell membrane.  Membrane disruption requires acidification, and therefore the membrane remains intact until the pH is lowered.  When the pH is lowered to the required level for toxin complex binding, the membrane is disrupted by the anthrax toxin already embedded in it.⁴

Anthrax researchers have explored ways to protect healthcare workers and others who may have been exposed or are likely to be infected. One group of scientists has investigated the feasibility of RNA silencing technology (siRNA) to block expression of the anthrax toxin PA receptors on the cell surface, two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2).  Blocking expression of the receptors was reported to provide almost complete protection against the LF intoxication in mice, and also protected against LF effects in human kidney cells as well as macrophage-like cells.⁵ 

Methods of vaccination have been under investigation for some time, as one of the most likely methods to provide lasting protection against anthrax infection.  In another 2014 publication, researchers at the University of Texas have reported success in vaccinating guinea pigs against anthrax infection using vaccines based on DNA-protein antigen components as well as another based on recombinant protein components.  After immunization, the animals were challenged with a lethal dose of B. cereus G9241 aerosol.  Complete protection against lethal challenge was observed in all guinea pigs that had a detectable pre-challenge serum titer of toxin neutralizing antibodies.⁶

List Biological Laboratories, Inc. offers EF (Product number 167A), LF (176), and PA (171) as well as the antibodies for their detection (773, 769 and 772, and 771, respectively). Refer to the website: https://listlabs.com/product-information/anthrax-toxins/ for more information.

 

References

1. NIAID website: http://www.niaid.nih.gov/topics/anthrax/pages/default.aspx
2. CDC website: http://www.cdc.gov/anthrax/
3. Andrews J, Chick A, Chao T, Chogada V, Douglas A, Florack A, McCormick S, Kessler E, Tuel K, Whalen J and Fisher M (2014) The Anthrax Toxin Heterotrimer: Explorations of the Protective Antigen and Edema and Lethal Factors (LB98). FASEB J 28:LB98 
4. Nablo BJ, Panchal RG, Bavari S, Nguyen TL, Gussio R, Ribot W, Friedlander A, Chabot D, Reiner JE, Robertson JW, Balijepalli A, Halverson KM, Kasianowicz JJ (2013) Anthrax toxin-induced rupture of artificial lipid bilayer membranes J Chem Phys 139(6):065101. PMID: 23947891
5. Arévalo MT, Navarro A, Arico CD, Li J, Alkhatib O, Chen S, Diaz-Arévalo D, Zeng M  (2014) Targeted silencing of anthrax toxin receptors protects against anthrax toxins.  J Biol Chem 289(22):15730-8. PMID: 24742682
6. Palmer J, Bell M, Darko C, Barnewall R, Keane-Myers A. (2014) Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241. Pathog Dis 72(2):138-42. PMID: 25044336

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

 

Vaccines have been used to help control diseases for more than 200 years and are the common practice for children and adults. Childhood vaccination has substantially reduced the morbidity and mortality from infectious diseases in much of the developed world, and influenza vaccinations have reduced the impact of seasonal influenza infections.¹ However, medical researchers are constantly looking for ways to improve the vaccines that are already used, and develop new ones.

Opportunities for improvement of vaccines abound. For example, although much attention is given to child vaccinations, a reservoir of infection could be eliminated through promotion of adult booster shots such as pertussis booster shots for expectant mothers and close family members, to help protect susceptible newborns. In addition, some diseases that have vaccines currently available still flourish in areas of the world where infrastructures for vaccination are poor and are too costly or cannot be delivered in their current forms.¹ Researchers are still trying to develop vaccines for other important diseases, such as HIV/AIDS, malaria and leishmaniasis. Vaccines are also being developed for bacterial pathogens, such as Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) that are responsible for a high proportion of diarrheal disease and death in adults and children in many countries in Africa and Asia.²

By modifying the factors included in the vaccine, researchers balance the effectiveness of the immune response with the side effects. Previously, whole cell vaccines containing whole organisms that had been chemically inactivated were the norm, but the side effects of fever and discomfort following injections were much more common. Many of the vaccines used today, including those for measles and some influenza vaccines, use live, attenuated viruses. Others use killed forms of viruses, pieces of bacteria (lipopolysaccharides), or inactivated forms of bacterial toxins, known as “toxoids.” Killed viruses, lipopolysaccharides and toxoids can evoke an immune response that protects against future infection.³ Acellular vaccines were introduced in the late 1990’s that contain either three or five key bacterial proteins and have been quite effective in protecting infants and children under four with a much lower rate of side effects.

List Labs offers several virulence factors which are used in vaccine testing.  For testing C. difficile vaccines; available reagents are C. difficile Toxin A (Product #152), C. difficile Toxin B (Product #155), C. difficile Toxoid A (Product #153), C. difficile Toxoid B (Product #154), and both subunits of the Binary Toxin (Products #157 and #158).  Numerous Bordetella pertussis virulence factors are available for use in testing including: Products #179, #180 or #181 Pertussis Toxin, Product #170 FHA, Product #186 Fimbriae, Product #187 Pertactin, Product #188 and #189 Adenylate Cyclase and Product #400 Highly Purified B. pertussis LPS.  Anthrax vaccine testing maybe carried out using Protective Antigen (Product #171) with Lethal Factor (Product #172) in a toxin neutralization assay.  Although these factors are not suitable for testing on humans, they are excellent research tools.

Inactive toxins are quite useful in making antibodies or in capturing antibodies from a vaccinated population on ELISA plates.  Three of our inactivated toxins, which carry mutations in the toxin active site, are B. pertussis Adenylate Cyclase Toxoid, Pertussis Toxin Mutant, Product #184 and CRM197, a non-toxic Mutant Diphtheria Toxin, Product #149.  Toxoids made by formaldehyde treatment of toxins include versions of C difficile Toxins (Products #153 and #154), Diphtheria Toxoid (Product #151), Staphylococcus aureus Enterotoxoid B (Product #123), Tetanus Toxoid (Product #191) and Toxoids of Botulinum Neurotoxins A and B (Product #133 and #139, respectively).

 

1. Hammond B., Sipics M., Youngdhal K., (2013). From the History of Vaccines, a project of the college of physician of Philadelphia. ISBN: 9780988623101
2. Svennerholm AM., (2011) From Cholera to Enterotoxigenic Escherichia coli (ETEC) vaccine development.  Indian J Med Res. 133(2): 188–194. PMID: 21415493
3. Leitner DR., Feichter S., Schild-Prüfert K., Rechberger GN., Reidl J., Schild S., (2013) Lipopolysaccharide modifications of a cholera vaccine candidate based on outer membrane vesicles reduce endotoxicity and reveal the major protective antigen. Infect Immun 81(7):2379-93. PMID: 23630951

 

List Biological Laboratories, Inc. (List) actively supports and participates in the BabyBIG^® project.

What is BabyBIG^® ?

BabyBIG^® (Human Botulism Immune Globulin; BIG-IV) is a public service, not-for-profit orphan drug manufactured and distributed by the California Department of Public Health.It is the only therapy available for infants who are infected with the organism that causes botulism, a life-threatening disease.

List Labs Volunteers Donate Plasma to Support Orphan Drug BabyBIG^® 

Because List produces the botulinum toxin for research use, employees are vaccinated against the toxin, thereby producing antibodies which circulate in their plasma.  This puts List Laboratories in a rare position to help with this project.  These antibodies are donated by volunteer employees via plasmapheresis, a procedure similar to a blood donation, for a period of up to 12 weeks.  Life-saving plasma is blended and processed into the final BabyBIG^® product.  We are proud of being able to be a big part of this amazing product and effort.  There are only a handful of organizations and entities who would be able to participate at any level and over 1/3 of our employees are active donors.  We salute them and support them in their time commitment to a worthy cause.

Infant Botulism Patients Helped in a Big Way by BabyBIG^® 

Since licensure of BabyBIG^® in 2003, approximately 1100 infant botulism patients nationwide have been treated with it, thereby shortening each hospital stay by almost one month and reducing the negative impact of this disease on these young patients.  In the aggregate since licensure, treatment with BabyBIG^® has resulted in more than 65 years of avoided hospital stay and more than $100 million in avoided hospital costs.

More information about BabyBIG^® may be found on their web site www.infantbotulism.org