Pathogens: general features. Specific host defenses.
Last revised: Wednesday, April 16, 2003
Reading: Ch. 26 in text

Pathogens

• Pathogens are parasitic organisms with specialized adaptations that allow them to interact directly or indirectly with hosts so they can grow, reproduce, and spread. In the process they cause host damage ranging from mild annoyance to death.
• To understand pathogens, we must understand how they can overcome host obstacles (including the presence of other microbes already present), and how they can cause disease through specific virulence factors.
• Disease is a process (verb), not a state (noun). Every disease is a race between pathogen trying to gain a foothold and host defenses trying to defeat pathogen. Many factors involved: virulence and numbers of pathogen, health and age of host, etc.
• Wrong to equate "one pathogen" = "every infection produces similar disease". Much more complex.
• Terminology
• Disease = state of being not in good health (not at ease, "dis-ease").
• Parasite often used to refer to protozoans or worms; the term "Pathogen" is typically used when referring to bacteria, virus, or fungus that causes disease. All are parasitic.
• Pathogen = organism with potential to cause disease
• Infection = pathogen is growing in or on host
• Virulence = degree or intensity of pathogenicity
• Invasiveness = ability of pathogen to spread to other tissues in body
• Infectivity = ability of pathogen to establish infection
• Toxigenicity = ability of pathogen to secrete toxins
• Septicemia = infection in which pathogen grows massively in the body, being found in blood and throughout organs. Usually leads to death.

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Intracellular vs Extracellular Life: choices & consequences

• Most pathogens have evolved to live either inside or outside of host cells, rarely in both habitats.
• Intracellular life
• Poses special problems for host; can't easily attack pathogen without harming its own tissues. Many pathogens are adapted for intracellular life, including all viruses, certain bacteria (e.g. TB, plague),
• Since white blood cells (macrophages, lymphocytes) are major components of defense system, many successful pathogens target these cells specifically for intracellular growth.
• Problem: to be successful, pathogen at some point must leave cells, exit host. Best chance to prevent infection is sometime during exit -- transmission -- entry to new host, before it has a chance to hide in new cells.
• Some intracellular parasites are so highly evolved that they can't survive at all outside their host's cells. Ex: Chlamydia, Rickettsia. To be successful, these must rely on mechanisms such as sexual contact or animal bites to transmit them to new hosts.
• Extracellular life
• Pathogen must deal with host's defensive strategies: white blood cells, immune system, etc.
• But does provide greater opportunities for grown, reproduction, and spreading than living inside cells.
• Can rapidly colonize a habitat; Ex. when cholera invades intestine, can quickly multiply, spread to cover large surface area
• Typical bacterial pathogens that act extracellularly: E. coli, Pseudomonas sp., Vibrio cholera
• Facultative intracellular pathogens
• Some bacteria can grow either inside host cells or outside, depending on circumstances.
• Examples: Shigella, Salmonella sp.

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Bacterial Disease Case Study: Staph infections

Staphylococcus aureus is so common that it is hard to appreciate the full range of its virulence. S. aureus is an opportunist, a common member of normal flora that rarely afflicts healthy individuals, but can seize on sudden opportunity (such as a break in the skin, a burn, or a superabsorbent tampon) to turn vicious, activating an extraordinary range of virulence factors. Over all of human history, S. aureus may be responsible for more collective suffering and pain than any other single bacterium! S. aureus infections are especially common in hospitals. About 1/3 of strains isolated from hospitalized infections are now resistant to all antibiotics except vancomycin, and resistant strains to that drug are beginning to show up! Diseases caused by S. aureus include: boils and abcesses cellulitis furuncles and carbuncles impetigo and "scaled skin" syndrome pneumonia, endocarditis, and septicemia toxic shock syndrome: many strains of Staph. aureus release "superantigens", act as activators of T-cells. View mechanism of action of superantigen. Normally, a foreign antigen might cause 0.001% of T-cell population to divide; superantigen can cause 2-20% to divide. Result = upset delicate balance that normally keeps T-cell response very local and specific; instead, many T-cells are killed, much energy is wasted in ways that don't harm pathogen. Responsible for "Toxic shock syndrome". S. aureus carries a number of virulence factors, including: an exotoxin that causes skin layers to separate and slough off (scalded skin syndrome) an enterotoxin that causes vomiting and diarrhea an exotoxin that causes toxic shock syndrome, leading to death the enzyme coagulase, that coagulates blood and prevents phagocytes from reaching the site of infection enzymes called leukocidins that kill phagocytes, resulting in pus and many more ..... Recently, scientists have begun to understand how virulence factors in S. aureus are regulated. RAP (RNAIII activating protein) controls production of toxins and other virulence proteins. Naomi Balaban discovered RAP a few years ago, and suggested that this protein shifts S. aureus from early stage of infection (attachment) into its later stage of infection (virulence), turning on toxins and other enzymes. If animals could be immunized against RAP, might be possible to "short-circuit" the expression of Staph virulence factors. A report in the journal Science, 280:438-440 (1998) published April 17, 1998 suggests this possibility.

Virulence Factors

• Virulence Factors are specific adaptations that allow pathogen to:
  1. attach selectively to host tissues
  2. gain access to nutrients by invading or destroying host tissues
  3. avoid host defenses
• Many examples of virulence factors:
  1. Specific attachment & entry factors
     • Pathogen must be able to bind to some receptor molecule on cell surfaces. These typically have necessary functions for cell.
     • Most diseases are tissue specific, because only certain tissues have receptor molecule needed. Ex: HIV binds to cells that have CD4 receptor (only certain lymphocytes).
     • Fimbriae or pili are used by some bacteria to attach selectively to certain tissues. Ex: Neisseria gonorrhaea binds to genital epithelium by fimbriae. In mutant cells w/o fimbriae, infectivity and pathogenicity are lost.
  2. Invasive enzymes
     • Many bacteria have specific enzymes that allow cells to penetrate host tissues
     • Example 1: collagenase produced by Clostridium perfringens. Enzyme degrades collagen, the primary structural fiber of connective tissue (25% of body's protein), allows penetration deeper into tissues fulminating gangrene. Strictly anaerobic process, only occurs when tissue is damaged so blood can't supply oxygen (e.g. serious wounds, frostbite).
     • Example 2: hemolysin enzymes produced by Streptococcus pyogenes dissolves cell membranes of tissues, produces typical symptoms of "strep throat".
  3. Tricks to avoid host defenses.
     1. Capsules
        • Many pathogens have thick extracellular polysaccharide capsules. Capsules inhibit phagocytosis, prevent quick disposal of bacterium by WBCs. Loss of capsule typically causes loss of infectivity.
     2. "Nasty Enzymes"
        1. Leukocidins
           • Some pathogens secrete chemicals that specifically kill WBCs.
           • Ex: Staphylococcus aureus. Accumulation of pus at infected site is caused by dead WBCs.
        2. Coagulase
           • Staph. aureus produces enzyme that coagulates blood.
           • Result: WBCs, other body defenses can't reach site of infection. Staph typically remains localized, "walled off" from defenses, produces many nasty types of localized infections such as boils, abcesses, etc.
             
     3. Exotoxins
        • Most exotoxins are proteins, secreted from cell, often damaging tissues at some distance. Very potent, small amounts are very toxic.
        • Often coded by plasmid DNA (ex. E. coli) or lysogenic phage DNA (ex. botulism, diphtheria)
        • Almost always inactivated by heat. Most are good antigens when inactive, can make toxoids (antigens without poison activity) = strong immune response.
        • Ex. 1: Diphtheria toxin. From Corynebacterium diphtheriae. Enters cell, inactivates elongation factor needed for protein synthesis. Result = cell gradually loses ability to make proteins (same toxin molecule keeps inactivating more and more factors), shuts down.
        • Ex. 2: Botulin toxin, a neurotoxin (attacks nervous system). From Clostridium botulinum, anerobic soil bacterium, endospore former. Most potent toxin known --- 1 gram could kill 10 million people. Toxin interferes with synaptic transmission at nerve-muscle junctions flaccid paralysis. Occurs most typically in home canned foods that are not cooked long enough to kill endospores.
          • View movie showing botulin toxin activity (requires free Shockwave plug-in)
        • Ex. 3: Tetanus toxin, another neurotoxin. FromClostridium tetanus, anerobic soil bacterium, endospore former. Blocks synaptic transmission to inhibitory neurons needed to relax one muscle when the other in paired muscle contracts (e.g. biceps must relax when triceps contracts, vice versa), leads to rigid paralysis. Common from deep wounds, pulled teeth. (But in 20% of cases, no history of injury!). Kills about 1 million infants/year by infecting umbilical stump. Treatment: antitoxin. Prevention: toxoid immunization (lasts 5-10 yrs).
        • Ex. 4: Cholera toxin = an enterotoxin (attacks enteric tract). From Vibrio cholerae. Binds to receptors on intestinal cells, chemically alters molecule involved in c-AMP production, leaves cAMP stuck in the "on" position. Causes massive outflow of water (chasing outflow of Na+/Cl-). Similar mode of action for other enterotoxin. Epidemic in S. America currently. Pathogen is free-living in fresh water, only causes infection in humans. Can be spread by drinking water, food (shellfish common). Untreated, mortality is ~50%. With fluid replacement, <1%. Prevention: clean drinking water.
          • Visit cholera (Vibrio cholerae) web page from Bacteriology 330, by Kenneth Todar, University of Wisconsin Department of Bacteriology
     4. Endotoxins
        • Endotoxins are integral parts of Gram-negative outer membrane (= LPS, lipolysaccharide). Unlike Exotoxins, they are typically heat resistant, active only in sizable amounts, and remain bound to cells.
        • Mechanism of action is very diverse. "When we sense LPS, we are likely to turn on every defence at our disposal" (Lewis Thomas), including fever, decrease in iron, inflammation, blood clotting, reduced sugar in blood, etc. Most important clinical problems are fever and shock.
        • Typical scenario: Gram-negative bacteria (e.g. E. coli, Pseudomonas) enter body via clinical procedure (improperly sterilized kidney dialysis tubing, catheter, etc.), cause sudden decrease in blood pressure (hypotension) = "septic shock". Can be lethal.
     5. Siderophores
        • Iron plays special role in control of infection. Most bacteria require iron to synthesize cytochromes. Iron in human body is tightly bound, either in hemoglobin (blood cells), on transferrin proteins (serum and lymph), or lactoferrin (milk, tears, saliva, mucus, etc.).
        • Some bacteria (Streptococci) do not require iron -- metabolism is strictly fermentative, no respiratory system. But most bacteria have to find way to get iron or cannot grow.
        • Siderophores = iron-binding factors that allow some bacteria to compete with the host for iron. Ex: Enterochelin produced by enteric bacteria (E. coli, Salmonella). Mutants that cannot synthesize enterochelin lose virulence. These mutants can regain virulence if pure enterochelin is injected along with mutant bacteria.

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Nonspecific Host Defenses

Tissue barriers

• Skin : skin associated lymphoid tissue (SALT) underlies epidermis, contains Langerhans cells, phagocytes that destroy invading microbes. Skin defenses. ("protected" image.) Why is this protected?
• Mucosal surfaces: all inner channels (respiratory, GI tract, vagina, bladder) are lined with mucus-producing cells = mucosal cells. Mucosal membrane defenses. ("protected" image.) Why is this protected? Mucus secretion is slimy polysaccharide, traps microbes and prevents access to epithelial cell surfaces. Mucus contains protective enzymes such aslysozyme (attacks peptidoglycan cell walls), lactoferrin (iron-binding protein), defensins (proteins that attack cell membranes). Mucosal membranes also have phagocytes, called mucosal-associated lymphoid tissue (MALT).
• Respiratory tract
• Intestinal tract
• Genitourinary tract

Chemical barriers

• Include a variety of enzymes; lysozyme, fibronectin, hormones (such as corticosteroids), etc.

Phagocytes

• Blood consists of both red blood cells (carry O₂ and CO₂) and white cells (major components of immune system). View picture of blood cells.
• Certain white blood cells (granulocytes (aka neutrophils), macrophages, others) are highly mobile, carry out phagocytic ("cell eating") activity
• White blood cells are chemically attracted to foci of disease or tissue damage by process of chemotaxis = chemical signals stimulate motion towards the attractant. See phagocyte migration. ("protected" image.) Why is this protected?
• View images and movie demonstrating phagocytosis (from Cells Alive, by James A. Sullivan)
• Phagocytosis begins with engulfment of particulate matter (can be bacteria, clumps of virions, cell debris, etc.) into phagosome.
• Phagosome fuses with lysosomes phagolysosome. Two kinds of lysosomes:
  1. acid hydrolases, lysozyme, neutral proteases, myeloperoxidase;
  2. lactoferrin, lysozyme, phospholipase A.
• These enzymes can degrade biomolecules; but many pathogenic bacteria have walls that are resistant to lysozyme.
• View stages of phagocytosis ("protected" image.) Why is this protected?
• View animation of stages of phagocytosis
• View Dr. Terry's animation of phagocytosis (requires Flash plug-in)
• Another feature of phagocytes: "Respiratory burst" (not associated with energy production in mitochondria). During phagocytosis, see dramatic increase in oxygen uptake. Produces superoxide (O₂^-), hydrogen peroxide (H₂O₂).
• View respiratory burst. Note: certain people genetically lack capacity for respiratory burst; they cannot kill many pathogens that ordinary people can, are at much greater risk from infections.
• Many pathogens withstand phagocytosis because they have special tricks to avoid phagocytosis. See Phagocytosis and Bacterial Pathogens for examples of how plague and tuberculosis bacteria avoid being killed by phagocytes.

Inflammation

• A nonspecific reaction to tissue damage. Stimulated by complex series of steps, initiated by cell damage.
• Effects include:
  1. vasodilation (opening junctions between capillary cells, allowing fluid and WBCs to leave blood and enter surrounding tissues) swelling of afflicted tissues
  2. redness (from heightened blood flow)
  3. pain (from prostaglandins released by tissues binding to nerve receptors)
  4. heat (produced by pyrogens liberated at site of inflammation); may inhibit microbial growth
  5. a variety of altered functions at site of inflammation; fibrin clotting, platelet aggregation, chemotactic signaling to attract WBCs, activation of complement factor C3.
• View QT animation of inflammation
• Inflammation is triggered by several chemicals called inflammation mediators, which include: complement, prostaglandins, and cytokines. These attract phagocytes to the locus of inflammation.
• Inflammation mediators also act on mast cells, found throughout connective tissue and mucous membranes. These release further chemicals: histamine and heparin. View animated cartoon of mast cell.
• Histamine binds to capillary receptors and causes dilation. (Antihistamines are drugs used to counteract this effect when inflammation is triggered by allergies.)
• Heparin binds to a clot-inhibiting protein (AT3) and reduces the chance that leakage from blood vessels will produce a clot.
• View diagram summarizing effects of inflammation (scroll down the page to the first diagram).
• Septic Shock: Inflammation gone wild. If significant numbers of microbes get into body (e.g. by contaminated catheters), can trigger a widespread systemic inflammatory response = septic shock. See stages of septic shock. ("protected" image.) Why is this protected? Phagocytes are stimulated to migrate out of bloodstream throughout body, leading to rapid swelling, loss of fluid, loss of blood pressure.

Complement and Opsonization

• Complement = complex of 17 proteins present in normal serum. Heat labile, destroyed @ 56 deg. C. Named C1-C9, also Factor B, D, H, I etc. Complement is used up (fixed) in antibody-antigen reactions, as a result of series of reactions called classical complement cascade. Alternatively, complement C3 can interact directly with certain chemicals (teichoic acids, LPS) found in bacterial cell walls, activate alternate pathway. See pathways that activate complement. ("protected" image.) Why is this protected?
• View movie showing complement activation on bacterial surface, resulting in lysis. (overdramatized, but fun)
• Classical cascade reaction: When Ab-Ag complex forms, base of Ab (constant region) changes shape. This activates Complement C1, which acquires esterase activity. Activated C1 not activates C2 and C4; generates a new activity which activates C3, etc. (Don't need to memorize steps in this course). After several more steps, activate C8 and C9 membrane attack complex, creates pores in membrane of target cell lysis.
• View animation of classical complement cascade
• Opsonization = enhanced phagocytic activity. Stimulated by complement bound to Ab-Ag targets. Especially important in binding to capsules, triggering effective phagocytic uptake of capsulated bacteria.

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