Toipc Number Seven Mechanism of Bacterial Damage and Bacterial Toxins

Microbial Damage Pathogenicity = ability to cause disease Virulence = degree of pathogenicity ID50 (Infectious Dose) = number of microbes required to cause infection in half the hosts. It depends on the virulence factors of the pathogen and the portal of entry For example, Shigella and Salmonella both cause diarrhea by infecting the gastrointestinal tract, but the infectious dose of Shigella is less than 100 organisms, whereas the infectious dose of Salmonella is on the order of 100,000 organisms LD50 (Lethal Dose) amount of toxin or pathogen necessary to kill half the hosts Microbes cause damage to host cells by three major mechanisms: 1. Direct damage of host cells 2. Inflammation 3. Bacterial Toxins

1. Direct damage of host cells - Siderophores Iron required for electron transport chain in both host and pathogen. Host usually does not have free iron available (free iron leads to easy colonization by pathogens) Humans bind unused iron to transport proteins by transferrin or lactoferrin Pathogens can produce siderophores: secreted by bacteria to compete iron from host proteins, siderophore iron complex then absorbed by bacteria

2. Inflammation In most cases, focal infections are eradicated by an intense, localized inflammatory response. By contrast, severe sepsis is characterized by dissemination of inflammatory mediators (e.g. circulating cytokines) resulting in widespread activation of the immune system referred to as the systemic inflammatory response syndrome (SIRS). SIRS is often complicated by systemic hypotension and tissue hypo- perfusion (shock), and direct (e.g. TNFα-mediated) cell injury, which ultimately leads to multiple organ dysfunction syndrome (MODS), and in many cases death

Cont

3. Bacterial Toxins The table on the right compares the main features of exotoxins and endotoxins

Cont Exotoxins Exotoxins are toxic proteins released from the pathogen cell as it grows. Exotoxins fall into three categories: the cytolytic toxins, the AB toxins, and the superantigen toxins. Endotoxins Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. Endotoxins are released in large amounts only when the cells lyse. Endotoxins consist of a core polysaccharide chain, O-specific polysaccharide side chains (O-antigen) and a lipid component, Lipid A, which is responsible for the toxic effects

Endotoxins and Exotoxins

Types of exotoxins 1. Cytolytic Toxins Cytolytic toxins damage the host cytoplasmic membrane, causing cell lysis and death. Because the activity of these toxins is most easily observed with assays involving the lysis of red blood cells (erythrocytes), the toxins are often called hemolysins Some hemolysins attack the phospholipid lecithin (phosphatidyl choline) of the host cytoplasmic membrane, these enzymes are called lecithinases or phospholipases. An example is the α-toxin of Clostridium perfringens Streptolysin O, a hemolysin produced by streptococci, affects the sterols of the host cytoplasmic membrane. Staphylococcal α-toxin is a pore-forming. It is released as a monomer, seven identical protein subunits oligomerize in the cytoplasmic membrane of target cells. The oligomer forms a pore, releasing the contents of the cell and allowing the influx of extracellular material and the efflux of intracellular material.

Staphylococcal α-toxin

Conti 2. A-B toxins A-B toxins are so named because they consist of two parts, an A (catalytic) domain and a B (receptor binding) domain. The A domains of most A-B toxins 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 AB toxin enters cells via: 1) Receptor mediated endocytosis 2) Fusion of vesicle with lysosome 3) Acid environment of lysosome reduces disulfide bonds and releases A into cell 4) A has various cellular activities

Cont 3. Superantigens Superantigens are unusual bacterial toxins that activate very large numbers of T-lymphocytes results in the secretion of excessive amounts of cytokines. Excessive cytokine production leads to a number of symptoms, including fever, nausea, vomiting, diarrhea, and sometimes shock and even death. Bacterial superantigens include the staphylococcal toxins that cause food poisoning and toxic shock syndrome

Diphtheria toxin Diphtheria: Infection of upper respiratory tract by Corynebacterium diphtheria bacteria grow on throat tissues Characterized by the formation of pseudomembrane (greyish membrane of bacteria, damaged host cells) as a result of host’s inflammatory response Diphtheria toxin is encoded by the tox gene in a lysogenic bacteriophage called phage β. Toxigenic, pathogenic strains of C. diphtheriae are infected with phage β and encode the toxin. Nontoxigenic, nonpathogenic strains of C. diphtheriae can be converted to pathogenic strains by infection with phage β, a process called phage conversion

Mechanism of Action of diphtheriae toxin

Cholerae toxin Cholera toxin is released from bacteria in the gut lumen and binds via the B subunit to GM1 receptors on enterocytes, triggering endocytosis. The A subunit enzymatically activates a G protein and locks it into its GTP-bound form through an ADP- ribosylation reaction. G protein activity leads to activation of adenylyl cyclase and increased cAMP levels. High cAMP levels then go on to activate the membrane-bound CFTR protein, leading to dramatic efflux of chloride, sodium, and water from the intestinal epithelium.

Anthrax toxin Bacillus anthracis, the causative agent of anthrax, secretes three monomeric, plasmid-encoded proteins that are collectively called anthrax toxin. Two are enzymes: Lethal Factor, a Zinc protease that specifically cleaves and inactivate MAP kinase kinases, and Edema Factor (EF), a Calcium and calmodulin dependent adenylyl cyclase. The third, Protective Antigen (PA83), named for its effectiveness in inducing protective immunity against anthrax. It is also binds to receptors and promotes translocation of LF and EF to the cytosol.

Mechanism of Action of anthrax Edema F Lethal F EDEMA Increased expression of pro-inflammatory mediators B LF B EF B EF Endosome Acidic Environment cAMP B MAPK Mitogen activated protein kinase LF IMMUNE SUPPRESSION WBCs do not divide in the presence of pathogens; overall decrease in phagocytosis

Tetanus and Botulinum toxins Clostridium tetani and Clostridium botulinum are endospore forming bacteria commonly found in soil. These organisms occasionally cause disease in animals through potent AB exotoxins that are neurotoxins—they affect nervous tissue. C. botulinum sometimes grows directly in the body, causing infant or wound botulism Death from botulism is usually from respiratory failure due to flacid muscle paralysis. C. tetani grows in the body in deep wounds that become anoxic, such as punctures. Although C. tetani does not invade the body from the initial site of infection, the toxin can spread via the neural cells and cause spastic paralysis Botulinum toxins, the most potent biological toxins known, are seven related AB toxins. One milligram of botulinum toxin is enough to kill more than 1 million guinea pigs.

Mechanism of Action of botulinum toxin A-Upon stimulation of peripheral and cranial nerves, acetylcholine is normally released from vesicles at the neural side of the motor end plate. Acetylcholine then binds to specific receptors on the muscle, inducing contraction. B-Botulinum toxin acts at the motor end plate to prevent release of acetylcholine from vesicles, resulting in a lack of stimulus to the muscle fibers, irreversible relaxation of the muscles, and flaccid paralysis.

Mechanism of Action of tetanus toxin (a) Muscle relaxation is normally induced by glycine (G) release from inhibitory interneurons. Glycine acts on the motor neurons to block excitation and release of acetylcholine (A) at the motor end plate. (b) Tetanus toxin (tetanospasmin) binds to the interneuron to prevent release of glycine from vesicles, resulting in a lack of inhibitory signals to the motor neurons Blockage of release of the inhibitory transmitter leads to convulsive contractions of the voluntary muscles best exemplified by spasm of the jaw and neck muscles ("lockjaw").

Mechanism of Action of tetanus toxin

Mechanism of the endotoxin Begins with CD14 binding of receptors on Macrophages that: 1. Induces cytokine production: IL-1, IL-6, IL-8, TNF, PAF, PG 2. Activation of complement cascade (C3a, C5a or alternate pathway) 3. Activation of coagulation cascade (Hageman factor; Factor XII) The clinical effects of endotoxin

Mechanism of the endotoxin

Endotoxins and the pyrogenic response The cytokines induce the hypothalamus to release lipids called prostaglandins, which reset the thermostat in the hypothalamus at a higher temperature