Testing of Toxins and Toxin Inactivating Compounds

Figure 1: Several of the toxins used in prior EP projects. From left to right: α-hemolysin toxin from Staphylococcus aureus, enterotoxin type B from Staphylococcus aureus, and streptolysin O from Streptococcus pyogenes.

Toxins are naturally occurring, poisonous substances produced in living cells. Toxins are also commonly referred to as biotoxins, while man-made chemicals with toxic activity are referred to as toxicants. Biotoxins can be found in a wide variety of organisms including honey bees, which release apitoxin when they sting, and snake venom, which can contain a variety of different protein toxins. Many single celled organisms, including bacteria and yeast, also produce various toxins. Although toxins are technically poisons, many have potential therapeutic value. For example, warfarin is a toxicant that was synthetically derived from a naturally occurring fungal toxin, dicoumarol, which was used as a rat poison. Warfarin is now one of the most commonly prescribed anticoagulants.

	Figure 3: Amanita phalloides, commonly referred to as “deathcap” mushrooms, contain many different toxins and account for the majority of the deaths associated with mushroom poisoning.

Microorganism toxins can be classified as endotoxins or exotoxins. Endotoxins are lipopolysaccharides that are bound to the cell membrane and are released upon lysis.  In contrast, exotoxins are released from the cell as the organism grows. Endotoxins are only produced by Gram-negative bacteria, while exotoxins can be produced and released by both Gram-positive and Gram-negative bacteria. The most lethal toxin currently known is botulinum toxin, which is an exotoxin produced by Clorstridium botulinum that can be lethal at several ng/kg. Table 1 summarizes some of the key differences between endotoxins and exotoxins.

Table 1: Endotoxins vs. Exotoxins

Figure 4 (CDC/ Christina Nelson): A patient infected with Yersinia pestis, the bacteria responsible for the bubonic plague. Yersinia murine toxin (Ymt) and endotoxins play crucial roles in the disease state.	Figure 5 (Bobjgalindo): A rattlesnake bite on a foot. The inflammation observed is a result of protein toxins in rattlesnake venom.

Different species of microorganisms produce different types of toxins. These toxins can directly damage host tissue and can affect the immune response. As a result, specific toxins play a large role in the disease states associated with infections. Some examples of microorganisms that produce toxins associated with disease include Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes.  E. coli produce Shiga toxins which inhibit protein synthesis by hydrolyzing RNA at specific adenine nucleotides. In extreme cases Shiga toxin can cause hemolytic-uremic syndrome, which is characterized by hemolytic anemia (destruction of red blood cells) and kidney failure.

Staphylococcus aureus can produce a wide variety of toxins, including superantigens and exfoliative toxins. Superantigens, such as enterotoxin type A/B, function by non-specifically binding to T lymphocyte receptors, which can lead to toxic shock syndrome. Exfoliative toxins can cause staphylococcal scalded skin syndrome (bullous impetigo), a dermatological condition that is most common in children. These toxins cause the scabs and fluid filled sores that are associated with impetigo. Other Staphylococcus aureus toxins include alpha (α), beta (β), and delta (δ) toxins, each of which play a different role in infectious diseases. For example, α-hemolysin toxin causes the lung inflammation associated with pneumonia.

Streptococcus pyogenes also produces a variety of toxins, including Streptolysin O/S and Streptococcal pyrogenic exotoxin A/C (SpecA and SpecC). SpecA and SpecC cause the rash associated with scarlet fever and can cause streptococcal toxic shock syndrome. Both staphylococcal and streptococcal toxins play a role in necrotizing fasciitis, or flesh-eating bacteria, a serious disease which often results in limb amputation or death. Necrotizing fasciitis causes tissue destruction due to the release of bacterial toxins. In severe cases, the infection enters the bloodstream and causes septic shock, resulting in organ failure.

Figure 6 (CDC): A lesion and cutaneous reaction caused by anthrax toxin, a protein exotoxin released from Bacillus anthracis. Cutaneous anthrax, Hide-porter’s disease, is not a lethal as pulmonary anthrax	Figure 7: A patient with necrotizing fasciitis, or flesh-eating bacteria. Tissue damage to the leg is a result of bacterial exotoxins.

Different isolates of the same bacterial species can produce different toxins. For example, while S. aureus can often be found on healthy human skin, toxin production can make this organism highly pathogenic. Many companies and academic groups are developing therapies to inactivate toxins. One application of toxin inactivating compounds is that the presence of toxins in wounds and lesions may prolong the healing process. This is why it is important to test for toxin activity.

One way to test for toxin activity is through cytotoxicity testing by cell culture. CT50, concentration toxic to 50% of cells, can be used to determine activity of a toxin. In order to test the efficacy of a toxin inactivating compound, toxin can be incubated with serial dilutions of the compound for a predetermined incubation period. Cells can then be treated with the compound/toxin mixture and later assayed for cell viability. The EC50, half maximal effective concentration, can then be determined to evaluate the efficacy of the toxin inactivating compound towards specific toxins. EP has experience culturing and conducting studies with a wide variety of cell lines. Please read our blog on cytotoxicity testing for additional information on cell biology techniques that can be applied to the testing of toxins.

Emery Pharma has experience and expertise in testing the activity of toxins and toxin-inactivating compounds. In addition, EP can help you design and conduct a custom toxin related study. Contact EP for consultation or additional information on our services.

References:

1. Smitt CK, Meysick KC, O’Brien AD. Bacterial Toxins: Friends or Foes? Emerging Infectious Diseases. 1999;5(2):224-34.

2. Wong C, Kurup A, Tan KC. Group B Streptococcus necrotizing fasciitis: an emerging disease? European Journal of Clinical Microbiology. 2004;23:573-575.

3. Todar K. Online Textbook of Bacteriology. Principles of Bacterial Pathogenisis. Bacterial Pathogens and Diseases of Humans. University of Washington.
http://www.textbookofbacteriology.net, 2012.

4. Center for Disease Control and Prevention. National Center for Immunization and Respiratory Diseases. Division of Bacterial Diseases.
http://www.cdc.gov/ncird/dbd.html, August 2014.