Bacterial Toxins Objectives: Be familiar with different classes of bacterial toxins

1. Bacterial Toxins Objectives: Be familiar with different classes of bacterial toxins Understand the concept of the A-B toxin model. Be able to explain how different toxins can damage the host. Toxins in detail: Cholera toxin, Pertussis toxin, Anthax toxin, Shiga toxin, Botulism toxin

2. Exotoxinsare typically soluble proteins secreted by living bacteria. • Both Gram-positive and Gram-negative bacteria produce soluble protein toxins. • A specific toxin is generally specific to a particular bacterial species • e.g. only Clostridium tetani produces tetanus toxin; • Only Corynebacterium diphtheriae produces the diphtheria toxin. • Usually, virulent strains of the bacterium produce the toxin while non-virulent strains do not Effector proteins are considered to be a novel class of toxins

3. Exotoxin = extracellular protein toxin • Endotoxin = name that the immunologists use for LPS • Enterotoxin = toxin that acts on gastrointestinal tract, producing typical food poisoning symptoms • Nomenclature • Named for host cell attacked: cytotoxin, neurotoxin • Named for producer or disease: cholera, Shiga • Named for activity: lecithinase, adenylate cyclase

4. Figure 9-23 Many diseases are caused by bacterial toxins- proteins secreted by bacteria.

6. ….plus all the type III and type IV effectors!!

7. The toxin is made of two elements: • One having enzymatic activity (called “A”, for activity) • One binding at the cell surface, which leads to internalization (called “B” for binding) I. II. Early Endosome H+ H+ B B H+ B B B B B B B B A A A Effect Nucleus Nucleus Host cell The A-B toxin concept RME Direct entry

8. Attachment and Entry of Toxins Three mechanisms of toxin entry into target cells. • direct entry, the B subunit of the native (A+B) toxin binds to a specific receptor on the target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into the cell. • receptor-mediated endocytosis (RME). The A+B structure toxin is internalized in the cell in a membrane-enclosed vesicle called an endosome. H+ ions enter the endosome lowering the internal pH, which causes the A and B subunits to separate. ● Injected toxins (effectors) mediated by type III, IV or VI secretion systems

9. There are at least two mechanisms of toxin entry into target cells, In one mechanism called direct entry, the B subunit of the native (A+B) toxin binds to a specific receptor on the target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into the cell cytoplasm. In an alternative mechanism, the native toxin binds to the target cell and the A+B structure is taken into the cell by the process of receptor-mediated endocytosis (RME). The toxin is internalized in the cell in a membrane-enclosed vesicle called an endosome. H+ ions enter the endosome lowering the internal pH which causes the A+B subunits to separate. Somehow, the B subunit affects the release of the A subunit from the endosome so that it will reach its target in the cell cytoplasm. The B subunit remains in the endosome and is recycled to the cell surface. In both cases (above) a large protein molecule must insert into and cross a membrane lipid bilayer (either the cell membrane or the endosome membrane). This activity is reflected in the ability of most A+B or A/B toxins, or their B components, to insert into artificial lipid bilayers, creating ion permeable pathways. A few bacterial toxins(e.g. diphtheria) are known to utilize both direct entry and RME to enter into host cells, which is not surprising since both mechanisms are variations on a theme. Bacterial toxins with similar enzymatic mechanisms may enter their target cells by different mechanisms. Thus, the diphtheria toxin and Pseudomonas exotoxin A, which have identical mechanisms of enzymatic activity, enter their host cells in slightly different ways. The adenylate cyclase toxin of Bordetella pertussis and the anthrax toxin (Edema Factor) of Bacillus anthracis act similarly to catalyze the production of cAMP from host cell intracellular ATP reserves. However, the anthrax toxin enters cells by receptor mediated endocytosis, whereas the pertussis adenylate cyclase traverses the cell membrane directly.

10. RME Direct entry Pertussis toxin

11. The pertussis toxin ptx The normal activity of the eucaryotic adenylate cyclase complex The mechanism of action of ptx: ADP ribosylation of the regualtory proteins of the eucaryotic adenylate cyclase complex

12. ADP ribosylation of target proteins is the way of action of: • pertussis toxin • diphtheria toxin • cholera toxin ADP ribosylation is also the activity of several type III secretion system effectors (like ExoT from Pseudomonas) and some type IV secretion systems (like RalFfrom Legionella)

13. Target cell intoxication route of C. diphtheriae diphtheria toxin (DT) and P. aeruginosa exotoxin A (ETA). Diphtheria toxin is nicked before or during its association with the HB–EGF protein; this complex then binds to the surface of target eukaryotic cells and is internalized in the process of receptor-mediated endocytosis (RME). On acidification of the early endosome (EE), the catalytic domain (A fragment) translocates to the cytoplasm and inhibits protein synthesis by ADP-ribosylation of eEF2. By contrast, ETA binds to the α2-macroglobulin (also known as low density lipoprotein receptor-related) protein on target eukaryotic cells, is internalized by RME and then nicked to form the A and B fragments. The A fragment of ETA follows the retrograde pathway to the ER, where it is thought to translocate into the cytoplasm; it also inhibits protein synthesis by modifying eEF2. Furin-like enzyme is an endogenous protease that is thought to be responsible for nicking ETA during its intoxication mechanism.

14. Cholera toxin is secreted by the type II secretion system. A neuraminidase also secreted by the T2SS eliminates the sialic acid from the glycolipid GM1, making it accessible for binding.

15. Bacterial toxins subvert transport pathways and endoplasmic reticulum (ER) functions for delivery into the host cytosol. For cholera toxin (CT), the KDEL sequence at the carboxyl terminus of the CT A subunit binds to Erd2 in the Golgi. CT is packaged into retrograde transport vesicles and delivered to the ER. CT is unfolded by protein disulfide isomerase (PDI) in the ER lumen. The dissociated CT A subunit is delivered into the cytosol by reverse translocation through the Sec61 complex. Shiga toxin (ST) is transported from the Golgi to the ER inside retrograde transport tubules. Upon delivery to the ER, the protein HEDJ in conjunction with Bip might assist in unfolding. The dissociated ST A subunit is delivered into the cytosol by reverse translocation through the Sec61 complex.

16. Shiga toxin-producing Escherichia coli (STEC) are important in human disease. Healthy dairy and beef cattle are a major reservoir of a diverse group of STEC that infects humans through contamination of food and water, as well as through direct contact. Infection of humans by STEC may result in combinations of watery diarrhea, bloody diarrhea, and hemolytic uremic syndrome. Severe disease and outbreaks of disease are most commonly due to serotype O157:H7 Severe disease in the form of bloody diarrhea and the hemolytic uremic syndrome is attributable to Shiga toxin (Stx), which exists as 2 major types, Stx1 and Stx2. A major public health goal is to prevent STEC-induced disease in humans. Studies aimed at understanding factors that affect carriage and shedding of STEC by cattle and factors that contribute to development of disease in humans are considered to be important in achieving this objective.

17. The anthrax toxin Anthrax toxin consists of three nontoxic proteins that self-assemble at the surface of receptor-bearing mammalian cells or in solution, yielding a series of toxic complexes. Two of the proteins, called Lethal Factor (LF) and Edema Factor (EF), are enzymes that act on cytosolic substrates. The third, termed Protective Antigen (PA), is a multifunctional protein that binds to receptors, orchestrates the assembly and internalization of the complexes, and delivers them to the endosome. There, the PA moiety forms a pore in the endosomal membrane and promotes translocation of LF and EF to the cytosol. Recent advances in understanding the entry process include insights into how PA recognizes its two known receptors and its ligands, LF and EF; how the PA:receptor interaction influences the pH-dependence of pore formation; and how the pore functions in promoting translocation of LF and EF across the endosomal membrane Annual Review of BiochemistryVol. 76: 243-265 (Volume publication date July 2007)

18. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (or OF) (edema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. OF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes

19. Botulism is a neuromuscular disease caused by the bacterium Clostridium botulinum. There are several different types of botulism. Type C and type E are responsible for extensive waterfowl die-offs and some fish kills. Type E is more prevalent in the Great Lakes. Botulism in humans is usually caused by type A or B and results from consuming improperly home-canned foods. In the Great Lakes, botulism spores (the resting stage of the bacteria) are abundant in anaerobic habitats, such as soils, and aquatic sediments of many lakes. When the correct environmental factors are present, the spores germinate and begin vegetative growth of the toxin-producing bacterial cells. The nearly 2,900 waterbirds that died on Lake Michigan in November 2006 were poisoned by eating fish that carried the toxin. One theory is that infected fish, partially paralyzed by the toxin, became easy prey for flocks of migrating waterbirds

20. There was no case of botulism contamination in commercially canned products in USA since the 1970's, but it looks like we were overdue. The FDA just released a warning that applies to 10-ounce cans of Castleberry's, Austex, and Kroger brands of hot dog chili sauce with "best by" dates from April 30, 2009, through May 22, 2009. Four people — two from Texas and two from Indiana — have already been hospitalized, described as "seriously ill but expected to survive." The products were made by the Castleberry Food Co., owned by Bumble Bee Seafoods LLC. They are cooperating with the Food and Drug Administration officials and are voluntarily recalling all potentially contaminated products. Botulism is a serious and potentially life-threatening disease caused by a bacterium called Clostridium botulinum. Symptoms include double or blurred vision, drooping eyelids, slurred speech, difficulty swallowing, dry mouth and muscle weakness that moves down the body, according to the CDC.

22. Mechanism of action of botulinum toxin: the L-chain cleaves a component of synaptobrevin, thereby preventing the release of acetyly choline across the synaptic cleft. The result is flaccid paralysis.

23. Type II (membrane disrupting exotoxins) Examples of membrane disrupting toxins: large pores induced by PFO, an example of a CDC (cholesterol-dependent cytolysins) and small pores resulting from heptamerization of Staphylococcus-toxin

24. Haemolysin is pore forming toxin secreted by many different species

25. Superantigens have the ability to bind the exterior surface of the MHC class II protein on the surface of antigen-presenting cells and link it to T-cell receptors on the surface of a T helper cell (FIG. 4). Binding occurs without the requirement for the MHC class II molecule to present an antigenic peptide to a suitable T-cell receptor. Each type of toxin recognizes a specific subset of variable Vβ chains of T-cell receptors and therefore has characteristic Vβ signatures. Up to 30% of T cells can become activated, leading to proliferation, with the high levels of cytokines expressed causing a toxic shock syndrome.