Unit 11-Pathogenicity; Lesson 2- Bacterial Pathogenicity

Enzymes

• 3. Local infections can be protected in a fibrin clot caused by the bacterial enzyme coagulase. • 4. Bacteria can spread from a focal infection by means of kinases (which destroy blood clots), hyaluronidase (which destroys a mucopolysaccharide that holds cells together), and collagenase (which hydrolyzes connective tissue collagen). • 5. IgA proteases destroy IgA antibodies.

List diseases or clinical manifestations caused by endotoxins and exotoxins.

see previous slides

Example of an Exotoxin

Bacterial Source: Gram POSITIVE Chemistry: Proteins Toxigenicity: High Pharmacology: Destroy certain cell parts or physiological functions Example: Botulinum toxin

Example of an Endotoxin

Bacterial Source; Gram NEG Chemistry: Lipid A Toxigenicity: Low Pharmacology: Systemic, fever, weakness, aches, and shock Example: Salmonellosis

SUMMARY!

How Microorganisms Enter a Host (pp. 430-433) • 1. The specific route by which a particular pathogen gains access to the body is called its portal of entry. Portals of Entry (p. 430) • 2. Many microorganisms can penetrate mucous membranes of the conjunctiva and the respiratory, gastrointestinal, and genitourinary tracts. • 3. Most microorganisms cannot penetrate intact skin; they enter hair follicles and sweat ducts. • 4. Some microorganisms can gain access to tissues by inoculation through the skin and mucous membranes in bites, injections, and other wounds. This route of penetration is called the parenteral route. The Preferred Portal of Entry (pp. 430-431) • 5. Many microorganisms can cause infections only when they gain access through their specific portal of entry. Numbers of Invading Microbes (p. 432) • 6. Virulence can be expressed as LD50 (lethal dose for 50% of the inoculated hosts) or ID50 (infectious dose for 50% of the inoculated hosts). Adherence (pp. 432-433) • 7. Surface projections on a pathogen called adhesins (ligands) adhere to complementary receptors on the host cells. • 8. Adhesins can be glycoproteins or lipoproteins and are frequently associated with fimbriae. • 9. Mannose is the most common receptor. • 10. Biofilms provide attachment and resistance to antimicrobial agents.

Food poisoning

Staphylococcus aureus -Superantigen -Enterotoxin causes secretion of fluids and electrolytes that results in diarrhea

Cholera

Vibrio cholerae A-B exotoxin -enterotoxin- causes secretion of large amounts of fluids and electroyltes that result in diarrhea

How Bacterial Pathogens Damage Host Cells Using the Host's Nutrients: Siderophores

• 1. Bacteria get iron from the host using siderophores.

Capsules

• 1. Some pathogens have capsules that prevent them from being phagocytized.

Plasmids, Lysogeny, and Pathogenicity

• 14. Plasmids may carry genes for antibiotic resistance, toxins, capsules, and fimbriae. • 15. Lysogenic conversion can result in bacteria with virulence factors, such as toxins or capsules.

Direct Damage

• 2. Host cells can be destroyed when pathogens metabolize and multiply inside the host cells.

Cell Wall Components

• 2. Proteins in the cell wall can facilitate adherence or prevent a pathogen from being phagocytized.

Antigenic Variation

• 6. Some microbes vary expression of antigens, thus avoiding the host's antibodies.

Anthrax

-Bacillus anthracis -A-B exotoxin Two A components enter the cell via the same B. The A proteins cause shock and reduce the immune response

Botulism

-Clostridium botulinum -A-B exotoxin -Neurotoxin prevents the transmission of nerve impulses; faccid paralysis results

Antibiotic-associated diarrhea

-Clostridium difficile -Membrane-disrupting exotoxin -Enterotoxin causes secretion of fluids and electrolytes that results in diarrhea; cytotoxin disrupts host cytoskeleton

Gas Gangrene and food poisoning

-Clostridium perfringens and other species of Clostridium -Membrane disrupting exotoxin -One exotoxin (cytotoxin) causes massive RBC destruction (hemolysis); antoher exotoxin (enterotoxin) is related to food poisoning and causes diarrhea.

Tetanus

-Clostridium tetani -A-B exotoxin -Neurotoxin blocks nerve impulses to muscle relaxation pathway; results in uncontrollable muscle contractions

Traveler's diarrhea

-Enterotoxigenic Escherichia coli and Shigella spp. A-B exotoxin -Enterotoxin causes secretion of large amounts of fluids and electrolytes that result in diarrhea

Describe how hemolysins, leukocidins, coagulase, kinases, hyaluronidase, siderophores, and IgA proteases might contribute to pathogenicity.

-Hemolysins lyse red blood cells; hemolysis might supply nutrients for bacterial growth. -Leukocidins destroy neutrophils and macrophages that are active in phagocytosis; this decreases host resistance to infection. -Coagulase causes fibrinogen in blood to clot; the clot may protect the bacterium from phagocytosis and other host defenses. -Bacterial kinases break down fibrin; kinases can destroy a clot that was made to isolate the bacteria, thus allowing the bacteria to spread. -Hyaluronidase hydrolyzes the hyaluronic acid that binds cells together; this could allow the bacteria to spread through tissues. -Siderophores take iron from host iron-transport proteins, thus allowing bacteria to get iron for growth. - IgA proteases destroy IgA antibodies; IgA antibodies protect mucosal surfaces.

Leukocidins, Hemolysins, Streptolysins

-Leukocidins- membrane-disrupting toxins that kill phagocytis leukocytes (WBCs) -act by forming channels -are also active against macrophages, phagocytes present in tissues. -most are produced by staphylococci and streptococci -damage to phagocytes decreases host resistance -Hemolysins- membrane-disrupting toxins that destroy erythrocytes (RBCs)- also by forming protein channels (produced by staphylococci and streptococci) -Streptolysins- hemolysins produced by streptococci. -streptolysin O (SLO)-it is inactivated by atmospheric oxygen -streptolysin S (SLS)- stable in an oxygen environment. -both streptolysins cause lysis not only of RBCs but also of WBCs (whose function is to kill the streptococci) and other body cells

Plasmids, Lysogeny and Pathogenicity

-Recall: plasmids are small, circular DNA molecules that are not connected to main bacterial chromosome and are capable of independent replication. -one group called R (resistant) factors- responsible for resistance of some microbes to antibiotics. -a plasmid may carry the information that determines a microbes pathogenicity. -ex of virulence factors that are encoded by plasmid genes are tetanus neurotoxin, heat-labile enterotoxin, and staphylococcal enterotoxin. -other examples: dextransucrase- an enzyme produced by Streptococcus mutans- involved in tooth decay -adhesins and coagulase produced by S. aureus -a type of fimbria specific to enteropathogenic strains of E. coli

Toxic Shock Syndrome (TSS)

-S. aureus Superantigen -Toxin causes secretion of luids and elecrolytes from capillaries that decreases blood volume and lowers blood pressure

Endotoxins Cont'd

-Shock- refers to any life-threatening decrease in blood pressure. -Septic Shock- shock caused by bacteria -gram-negative bacteria cause ENDOTOXIC SHOCK -like fever, shock produced by endotoxins is related to secretion of acytokine by macrophages -phagocytosis of gram-neg bacteria cause the phagocytes to secrete tumor necrosis factor (TNT)- sometimes called cachectin -TNF-binds to many tissues in body and alters their metabolism in a number of ways 1 effect of TNF- damage to blood capillaries; their permeability is increased- lose large amount of fluid drop in B.P. that results in shock -low B.P. has serious effects on kidneys, lungs, and GI tract -presence of Gram-Neg bacteria such as Hemophilus influenza type b in cerebrospinal fluid causes the release of IL-1 and TNF -these, in turn, cause a weakening of the blood-brain barrier that normally protects the CNS from infection. -weakened barrier lets phagocytes in, but also lets more bacteria enter from the bloodstream. -endotoxins- do not promote formation of effective antitoxins against the carbohydrate component of the endotoxin -antibodies are produced, but they tend not to encounter the effect of the toxin; sometimes they actually enhance its effect. -microbes that produce endotoxins: Salmonella typhi (causes typhoid fever) Proteus spp. (causative agents of UTIs) Neisseria meningitidis (causative agent of meningococcal meningitis)

Production of Toxins

-Toxins- poisonous substances that are produced by certain microbes -often primary factor contributing to pathogenic properties of those microbes -Toxigenicity- capacity of microbes to produce toxins. -toxins- transported by blood or lymph- can be serious and sometimes fatal -some produce fever, cardiovascular disturbances, diarrhea, and shock -can also inhibit protein synthesis, destroy blood cells and vessels, and disrupt the nervous system by causing spasms. -nearly 40% cause disease by damaging eukaryotic cell membranes. -Toxemia- refers to presence of toxins in the blood. -toxins are of 2 general types, based on their position relative to microbial cell: exotoxins and endotoxins.

Direct Damage

-after pathogen attaches, they can cause direct damage as pathogen uses host cell for nutrients and prouduces waste products -as pathogens metabolize and multiply in cells, cell usually ruptures -many viruses and intracellular bacteria & protozoa are released when cell ruptures -following release- pathogens can spread to other tissues in even greater numbers -E. coli, Shigella, Salmonella, Neisseria gonorrhoeae- can induce host epithelial cells to engulf them by process that resembles phagocytosis. -pathogens can disrupt host cells as they pass through and can then be extruded from host cell by a reverse phagocytosis process- enabling them to enter host cells. -some can also penetrate host cells by excreting enzymes and by their own motility- such penetration can itself damage the host cell. -most damage by bacteria is done by toxins

Endotoxins

-are the lipid portions of lipopolysaccharides (LPS) that are part of the outer membrane of the cell wall of gram-NEGATIVE bacteria (Lipid A is the endotoxin) -endotoxins are lipopolysaccharides whereas exotoxins are proteins -endotoxins are liberated when the bacteria die and the cell wall breaks apart. -the endotoxins are just toxins composed of lipids that are part of the cell membrane --Antibiotics to treat diseases caused by gram-neg bacteria can lyse the bacterial cells- releasing endotoxins- leading to immediate worsening of symptoms, but conditions usually improve as endotoxin breaks down -endotoxins- exert their effects by stimulating macrophages to release cytokines in very high concentrations. -at these levels, cytokines are toxic -all endotoxins produce same signs and symptoms regardless of species of microbe, although not to the same degree. -chills, fever, weakness, generalized aches, shock, death, can also induce miscarriage -another consequence of endotoxins- activation of blood-clotting proteins, causing formation of small blood clots -blood clots obstruct capillaries- resulting in decreased blood supply - induces death of tissues -condition is referred to disseminated intravascular coagulation (DIC) -the FEVER (pyrogenic response) is caused by endotoxins 1. macrophage ingests a gram-Negative bacterium 2. bacterium is degraded in a vacuole, releasing endotoxins that induce the macrophages to produce cytokines IL-1 and TNF-alpha 3. The cytokines are released into the blood-stream by the macrophages through which they travel to the hypothalamus of the brain 4. The cytokines induce the hypothalamus to produce prostaglandins, which resets the body's "thermostat" to a higher temp, producing a fever

Bacteriophage genes that contribute to pathogenicity are genes for

-bacteriophage genes that contribute to pathogenicity are genes for : Diphtheria toxin Erythrogenic toxin Staphy-lococcal enterotoxin -pyrogenic toxin -botulinum neurotoxin -and the capsule produced by Streptococcus pneumoniae -gene for Shiga toxin in E. coliO157 is encoded by phage genes -Pathogenic strains of Vibrio cholera carry lysogenic phages -these phages can transmit the cholera toxin gene to non-pathogenic V. cholera strains, increasing the number of pathogenic bacteria

Antitoxins and Taxoids

-body produces antibodies called antitoxins that provide immunity to exotoxins. -when exotoxins are inactivated by heat or by formaldehyde, iodine, or other chemicals, they no longer cause the disease but can still stimulate the body to produce antitoxins. -altered exotoxins are called Taxoids -when toxoids are injected into body as a vaccine, they stimulate antitoxin production (so immunity is produced) -Diptheria and tetanus can be prevented by taxoid vaccination

Litmus amebocyte lysate (LAL) assay

-important to have a sensitive test to identify the presence of endotoxins in drugs, medical devices, and body fluids. -sterilized materials may contain endotoxins, even though no bacteria can be cultured from them. -one such lab test: called the Litmus amebocyte lysate (LAL) assay -can detect even minute amounts of endotoxin -The hemolymph (blood) of the Atlantic coast horseshoe crab, Limulus Polyphemus contains WBCs called amebocytes which have large amounts of a protein (lysate) that causes clotting. -in presence of endotoxin, amebocytes in crab hemolymph lyse and liberate their clotting protein. -resulting gel-clot (precipitate) is a positive test for the presence of endotoxin -degree of reaction is measured using a spectrophotometer

Using the Host's Nutrients: Siderophores

-iron- required for growth of most pathogenic bacteria -conc of free iron in body is low b/c most iron is tightly bound to iron-transport proteins (lactoferrin, transferrin, ferritin, hemoglobin) -to obtain free iron, some pathogens secrete proteins called siderophores siderophore Bacterial iron-binding proteins. -when pathogens need iron- siderophores are released into medium where they take iron away from iron-transport proteins by binding the iron even more tightly. -iron-siderophore complex is formed- then its taken up by siderophore receptors on bacterial surface. -iron is then brought into bacterium. -some cases: iron is released from complex to enter bacterium- other cases: iron enters as part of the complex. -alternative to iron acquisition by siderophores, some pathogens have receptors that bind directly to iron-transport proteins and hemoglobin. -taken into bacterium directly along w/ iron -also possible that some bacteria produce toxins when iron levels are low. -toxins kill host cells, releasing their iron- making it available to bacteria.

Naming Exotoxins

-named on basis of several characteristic: -one is the type of host cell that is attacked -neurotoxin-attacks nerve cells -Cardiotoxins-attack heart cells -hepatotoxins- attack liver cells -leukotoxins- attack leukocytes -enterotoxins- attack lining of GI tract -cytotoxins-attack a wide variety of cells. -some are named after diseases with which they are associated: -diptheria toxin (causes diphtheria) -tetanus toxin (causes tetanus) -some are named for specific bacterium that produces them. -botulinum toxin (Clostridium botulinum) -Vibrio enterotoxin (Vibrio cholera)

Explain how direct damage of host cells by pathogenic bacteria occurs. How can other tissues also be damaged?

-once microbe invades body tissue- initially encounters phagocytes of host -if phagocytes destroy invader- no further damage done to host -if pathogen overcomes host's defenses- microbes can damage host cell in 4 basic ways: 1. By using host's nutrients 2. By causing direct damage in the immediate vicinity of the invasion 3. Producing toxins, transported by blood and lymph that damage sites far removed from original site of invasion 4. By inducing hypersensitivity reactions (discussed in Ch. 19- we will only discuss first 3 mechanisms)

Exotoxins

-proteins produced inside pathogenic bacteria, most commonly- gram-POSITIVE bacteria, as part of their growth and metabolism. -Exotoxins are then secreted into the surrounding medium during log phase -TOXIC SUBSTANCE IS RELEASED OUTSIDE THE CELL! (EXITS) -may be released following lysis -many exotoxin proteins are enzymes that catalyze only certain biochemical reactions -the genes for most exotoxins are carried on bacterial plasmids or phages -exotoxins- soluble in body fluids- can easily diffuse into blood and are rapidly transported throughout body -work by destroying particular parts of the host\s cell or by inhibiting certain metabolic functions -highly specific in effects on body tissues -among most lethal substances known -exotoxins- produce specific signs and symptoms of the disease -exotoxins are disease-specific. -ex. Botulism- usually do to ingestion of the exotoxin- not a bacterial infection -staphylococcal food poisoning- an intoxication, not an infection. -Examples: Diptheria Toxin Erythrogenic Toxins Botulinum Toxin Tetanus Toxin Vibrio Enterotoxin Staphylococcal Enterotoxin

-divided into 3 principal types on basis of their structure and function:

1) A-B toxins 2) membrane-disrupting toxins 3.) superantigens

A-B exotoxin

A-B Toxins- consist of 2 parts designated A and B, both of which are polypeptides. -Most exotoxins are A-B toxins. -A part is the active (enzyme) component -B part is the binding component -example of A-B toxin- diptheriatoxin • In the first step, the A-B toxin is released from the bacterium. • The B componentattaches to a host cell receptor. • The plasma membrane of the host cell invaginates (folds inward) at the point where the A-B exotoxin and plasma receptor make contact, and the exotoxin enters the cell by receptor-mediated endocytosis. • The A-B exotoxin and receptor are enclosed by a pinched-off portion of the membrane. • The A-B components of the exotoxin separate. The A component alters the function of the host cell, often by inhibiting protein synthesis. The B component is released from the host cell, and the receptor is inserted into the plasma membrane for reuse. (FIG 15.5)

exotoxins vs endotoxins

Bacterial source: Exotoxin: gram-positive Endotoxin:gram-negative Relation to microorganisms: Exotoxin: metabolic product of growing cell Endotoxin: Present in LPS of outer membrane of cell wall and released w/ destruction of cell or during cell division Chemistry: Exotoxin: Proteins, usually with 2 parts (A-B) Endotoxin- Lipid portion (Lipid A) of LPS of outer membrane Pharmacology (effect on body) Exotoxin: specific for a particular cell structure or function in the host (mainly affects cell functions, nerves, and GI tract) Endotoxin: General, such as fever, weaknesses, aches, and shock; all produce the same effects Heat Stability Exotoxin: Unstable; can usually be destroyed at 60-80 degrees C (except staphylococcal enterotoxin) Endotoxin: Stable; can withstand autoclaving (121 degrees C for 1 hr) Toxicity(ability to cause disease) Exotoxin: High Endotoxin: Low Fever Producing Exotoxin: No Endotoxin: Yes Immunology (relation to antibodies) Exotoxin: can be converted to toxoidsto immunize against toxin; neutralized by antitoxin Endotoxin: not easily neutralized by antitoxin; therefore, effective toxoids cannot be made to immunize against toxin Lethal Dose Exotoxin: Small Endotoxin: Considerably Larger Representative Diseases: Exotoxin: Gas Gangrene, tetanus, botulism, diptheria, scarlet fever Endotoxin: Typhoid Fever, UTI, and meningococcal meningitis

Diptheria

Corynebacterium diptheriae A-B exotoxin -Cytotoxin inhibits protein synthesis, especially in nerves, heart and kidney cells

How does toxigenicity differ from direct damage?

Direct damage is the damage done by pathogens to the host as by using the host cell for nutrients and excreting waste. Toxigenicity is the ability of a bacteria to produce toxin.

How are capsules and cell wall components related to pathogenicity? Give specific examples.

Encapsulated bacteria can resist phagocytosis and continue growing.Streptococcus pneumoniae and Klebsiella pneumoniae produce capsules that are related to their virulence. M protein found in the cell walls of Streptococcus pyogenes and A protein in the cell walls of Staphylococcus aureus help these bacteria resist phagocytosis. -capsule resists host's defenses by impairing phagocytosis- process by which cells of body engulf and destroy microbes -chemical nature of capsule- prevents phagocytic cell from adhering to bacterium. -Streptococcus pneumoniae- owes its virulence to polysaccharide capsule -Other bacteria that produce capsules related to virulence: -Klebsiella pneumonia- causes bacterial pneumonia -Haemophilus influenza- causes pneumonia & meningitis in children -Bacillus anthracis- cause of anthrax -Yersinia pestis- causative agent of plague Cell wall components: -cell walls of certain bacteria contain chemical substances that contribute to virulence. -ex Streptococcus pyogenes- produces heat-resistant and acid-resistant protein called M Protein -protein is found on both cell surface and fimbriae -mediates attachment of the bacterium to epithelial cells of the host and -helps the bacterium resist phagocytosis by white blood cells. -protein- increases virulence -immunity of S. pyogenes- depends on body's production of antibodies specific to M protein. -Neisseria gonorrhoeae- grows inside human epithelial cells & leukocytes -these bacteria- use fimbriae and an outer membrane protein called Opa to attach to host cells. -following attachment by both Opa and fimbriae- host cells take in bacteria -Waxy lipid (mycolic acid) that makes up cell wall of Mycobacterium tuberculosis- also increases virulence- resists digestion by phagocytes. Can even multiply inside phagocytes.

Explain whether each of the following examples is a food infection or intoxication. What is the probable etiological agent in each case? • a. Eighty-two people who ate shrimp in Louisiana developed diarrhea, nausea, headache, and fever from 4 hours to 2 days after eating. • b. Two people in Vermont who ate barracuda caught in Florida developed malaise, nausea, blurred vision, breathing difficulty, and numbness 3 to 6 hours after eating.

a. Intoxication-An intoxication results when a person eats food containing toxins that cause illness. Toxins are produced by harmful microorganisms, the result of a chemical contamination, or are naturally part of a plant or seafood (THE SHRIMP). Some bacteria cause an intoxication. Viruses and parasites do not cause foodborne intoxication. Probably etiological agent- vibrio spp. -V. parahaemolyticus (prefer warm coastal areas) b. Infection-Foodborne infection is caused by the ingestion of food containing live bacteria which grow and establish themselves in the human intestinal tract. Ciguatera is a foodborne illness caused by eating certain reef fish whose flesh is contaminated with a toxin made by dinoflagellates such as Gambierdiscus toxicus which live in tropical and subtropical waters.

Superantigens

antigens that provoke a very intense immune response -bacterial proteins -nonspecifically stimulate proliferation of immune cells called T cells. -these cells are types of WBCs that act against foreign organisms and tissues (transplants) and regulate the activation and proliferation of other cells of the immune system. -In response to superantigens, T cells are stimulated to release enormous amounts of chemicals called cytokines. -Cytokines- small protein molecules produced by various body cells, especially T cells, that regulate immune responses and mediate cell- to cell communication -excessively high levels of cytokines released by T cells enter bloodstream and give rise to a number of symptoms- fever, nausea, vomiting, diarrhea, and sometimes shock and even death -include staphylococcal toxins that cause food poisoning and toxic shock syndrome.

The Production of Toxins

• 3. Poisonous substances produced by microorganisms are called toxins; toxemia refers to the presence of toxins in the blood. The ability to produce toxins is called toxigenicity. • 4. Exotoxins are produced by bacteria and released into the surrounding medium. Exotoxins, not the bacteria, produce the disease symptoms. • 5. Antibodies produced against exotoxins are called antitoxins. • 6. A-B toxins consist of an active component that inhibits a cellular process and a binding component that attaches the two portions to the target cell, e.g., diphtheria toxin. • 7. Membrane-disrupting toxins cause cell lysis, e.g., hemolysins. • 8. Superantigens cause release of cytokines, which cause fever, nausea, and other symptoms; e.g., toxic shock syndrome toxin. • 9. Endotoxins are lipopolysaccharides (LPS), the lipid A component of the cell wall of gram-negative bacteria. • 10. Bacterial cell death, antibiotics, and antibodies may cause the release of endotoxins. • 11. Endotoxins cause fever (by inducing the release of interleukin-1) and shock (because of a TNF-induced decrease in blood pressure). • 12. Endotoxins allow bacteria to cross the blood-brain barrier. • 13. The Limulus amebocyte lysate (LAL) assay is used to detect endotoxins in drugs and on medical devices.

Penetration into the Host Cell Cytoskeleton

• 7. Bacteria may produce proteins that alter the actin of the host cell's cytoskeleton allowing bacteria into the cell.