Germ Theory and Koch's Postulates

Germ Theory of Disease

• Scientists suspected microbes were responsible for disease soon after their discovery.
• The germ theory replaced the miasma theory of infection.
• Robert Koch was the first to link specific bacteria to specific diseases.
• Koch studied anthrax and tuberculosis, also known as consumption.
• He formulated postulates for determining the microbial cause of infection.

Koch's Postulates (1876)

• The specific organism must be associated with the disease, not present in healthy individuals.
• The specific organism should be isolated.
• The pure isolate should produce disease.
• The organism should be re-isolated.
• Koch demonstrated his postulates with B. anthracis, the causative agent of anthrax.

Koch's Postulates Issues

• Many pathogens are found in asymptomatic carriers.
• Sometimes, disease is due to toxins.
• Many pathogens are unculturable.
• Many pathogens lack a suitable animal model.
• Modern versions of Koch's postulates use genomic-based approaches like DNA sequencing.

Koch's Postulates, Helicobacter pylori, and Gastritis

• Australian scientists Marshall & Warren (1980s) studied gastritis and ulcers.
• Overproduction of stomach acid was believed to cause these conditions.
• They were treated with "bland" diets and antacids.
• H. pylori was discovered/isolated from tissue biopsies.
• Finding an appropriate animal model proved difficult.
• It was also widely believed that microbes could not survive in the stomach.
• Frustration eventually arose from these issues.

What One Does for Science

• Postulate 1: the organism is associated with the disease.
• Postulate 2: H. pylori is isolated in pure culture.
• Postulate 3: Marshall drank a culture of H. pylori and developed gastritis.
• Postulate 4: H. pylori was re-isolated from Marshall's stomach biopsy.

Helicobacter pylori

• Found in 50% of the human population.
• The leading cause of gastric inflammation (gastritis) and ulcers.
• Can lead to gastric cancer in some patients.
• The helical shape and polar flagella is used to penetrate the thick mucous lining of the stomach epithelium.
• Epithelial cells secrete carbonate to reduce acidity.
• H. pylori uses urease to generate ammonia.
• This bacteria produces toxins (VacA) to damage epithelial cells to gain nutrients.
• Can be resolved with antibiotics.

Bacterial Pathogenesis

• Infection is the growth of a pathogenic microbe in/on a host.
• Disease is injury of a host due to infection.
• A pathogen is a microorganism that causes disease.
• An opportunistic pathogen causes disease only under certain circumstances.
• Pathogenicity is the ability of a pathogen to cause disease.
• Pathogenesis is the process by which a pathogen causes disease.

Bacterial Virulence

• Virulence is the relative ability of a pathogen to cause disease and severity of the mechanism.
• Virulence factors are molecules produced or strategies used by a pathogen to cause disease.
• Virulence factors achieve attachment and colonization.
• They also result in evasion of the host immune system.
• They are involved in invasion, dissemination (spread) to other host cells.
• They facilitate acquisition of nutrients and growth.
• Virulence results in release into external environment to find new hosts.

Infection

• 'Portal of entry' examples include skin (wounds or bites), gut (need to survive acidic stomach), and mucous membranes (nose, mouth, eyes, etc).
• Mucous is composed of mucin (glycoproteins and polysaccharides).
• Most microbes cannot penetrate.
• Mucin contains slgA, antibody secreted immunoglobulin A, and defensins, which is a eukaryotic version of bacteria.
• Pathogenic VF includes flagella and enzymes that degrade mucin.

Adherence Molecules

• Microbes are easily removed without adherence.
• Microbial adhesins are extracellular glycoproteins or lipoproteins that bind to host receptors.
• Fimbriae are used for initial attachment.
• Afimbrial structures facilitate close (intimate) contact.
• Host receptors are structural or (essential) functional cell components.
• These include: Fibrinogen, fibronectin, elastin, collagen
• Glycoproteins, glycolipids
• Lectins (carbohydrate-binding proteins)
• Cell receptor proteins

Adherence Secretion Systems

• Pathogens can use T3SS, T6SS, and sometimes T4SS to inject proteins/toxins directly into host cells.
• Examples include pathogenic E. coli, Shigella, Yersinia, and Salmonella.
• Pathogenic E. coli (O157:H7) injects a protein (Tir) into host cells using a T3SS.
• Tir becomes a receptor on the host membrane for an E. coli surface adhesin (intimin) that adheres the cells tightly.

Induced Phagocytosis

• Intracellular pathogens, once attached to a host cell, can induce the cell to internalize the pathogen and it is considered a cell invasion.
• Invasins activate host cell signaling pathways.
• This results in reorganization of cytoskeletal actin to form pseudopod-like structures that engulf the bacterium.
• Once internalized, the pathogen either escapes the phagosome before fusion with a lysosome, or prevents lysosomes from fusing with the phagosome.
• L. monocytogenes uses invasins called internalins to induce endocytosis then release lysteriolysin O to dissolve the phagosome membrane, which have a secretion system.
• Salmonella uses a T3SS to deliver invasins and induce endocytosis and a second T3SS to insert proteins in the phagosome membrane that prevents lysosome fusion.

Pathogens Must Evade the Immune System

• Immunoglobulin Proteases destroy Ig proteins to evade opsonization.
• Mucin is full of sIgA, IgA protease is a major VF of many intestinal pathogens.
• Through antigenic variation pathogens can change the structure of flagella, pili / fimbriae, or other surface antigens.
• Pathogens can change the teichoic acid, LPS, and peptidoglycan layers to evade defensins.
• Pathogens can hide in plain sight by doing some genetic changes.
• Pathogens can stop producing flagella, pili and/or fimbriae.
• Pathogens can use polysaccharides found on host cells to build a capsule.
• Also coat cell surface with host proteins.

Pathogens Must Acquire Nutrients

• Degradative enzymes are used to acquire food.
• Lipases, proteases, glycosidases, etc., are used.
• Phospholipases rupture host cell membranes, releasing nutrients.
• These elicit minor immune responses.
• Furthermore, pathogens damages host cells to release nutrients.
• Iron acquisition occurs with siderophores.
• These also have receptors on them that bind our own iron protein.
• Siderophores bind iron with high affinity.
• Receptors for host iron-binding proteins (or exoenzymes that destroy them) are used.
• Lactoferrin, transferrin, ferritin, heme.
• Iron abstinence is used by B. burgdorferi (Lyme disease).
• This can use manganese as an enzyme cofactor.

Genetics of Virulence Factors

• Genes for chromosomal virulence factors are often clustered together on pathogenicity islands.
• Islands facilitate their spread to other organisms by horizontal gene transfer.
• Genes for many virulence factors are located on plasmids, which also facilitate spread by HGT events.
• Some genes for virulence factors are not bacterial in origin.
• Instead they are found on prophage DNA within the pathogens chromosomes.

Virulence Enzymes and Toxins

• Pathogens cause disease to acquire nutrients for growth, replication, and spread.
• Pathogens that damage cells/tissues have toxicity.
• Three major mechanisms of toxicity:
• Enzymes that break down components of host cells/tissues are involved.
• Exoenzymes are secreted into the surroundings.
• Enzymes can also be delivered directly into host cells.
• Enzymes don't have catalytic activity, but undergo some damage.
• Toxins:
• Are protein molecules that affect host cell/tissue function.
• Exotoxins are secreted into the surroundings.
• Toxins can be delivered directly into host cells.
• Enterotoxins are active in the GI tract.
• Endotoxins
• Are part of the cell wall structure.
• Are a Lipid A portion of LPS in Gram-negative cells.
• Consist of Lipoteichoic acids in Gram-positive cells.

Tissue-Destroying Enzymes

• Invasive pathogens produce enzymes that break down components of the extracellular matrix and open paths for microbes to spread below the epithelium.
• For example Hyaluronidase degrades hyaluronic acid enabling the hyaluronic acid producer to attach to epithelia.
• Enzymes include collagenases, elastases, proteases, lipases, and nucleases.

Clot Forming and Dissolving Enzymes

• Coagulase causes clots to form around the infection site to protect the pathogen from the immune cells.
• Streptokinase (staphylokinase) dissolves clots to allow the pathogen to move into deeper tissue layers.

Pyogenic Infections

• Staph. aureus, Staph. epidermidis, Strep. pyogenese cause opportunistic infections through skin cuts/abrasions.
• Coagulase walls off the infected tissue to slow the immune response.
• Hemolysins and leukocidms destroy RBCs and WBCs, and also result in pus formation.

Staph. aureus α-Toxin

• Cell destruction by Staphylococcus aureus skin infections is mediated by α-hemolysin (S. aureus α-toxin).
• The toxin is a pore-forming cytotoxin to targets surrounding cells, resulting in cell lysis.
• Accumulation of dead cells in the area form pus.
• Treatment is done via antibiotics and drainage.

Digging Deeper

• Multiple adhesins along with hyaluronidase are used to attach to ECM proteins.
  • Allows colonization of subcutaneous tissues.
  • Results in Impetigo, scalded skin syndrome, strep throat.
• Bloodstream spread results in septicemia.
  • It leads to scarlet fever, rheumatic fever, necrotizing fasciitis, and toxic shock syndrome (sepsis).

AB-Type Exotoxins

• "Two-component" toxins include A and B.
  • B subunits bind host cells and facilitate transport and release of the A-component into the host cell.
  • A- subunits have toxic activity.
  • This could include Enzymatic interruption of transcription, translation, cell signaling, etc.

Diphtheria

• Diphtheria, a respiratory disease, happens when Corynebacterium diphtheriae colonizes the throat and URT.
  • It is spread via aerosolized droplets or direct contact with infected individuals.
  • Build up of fibrin clots, dead cells, and an immune response result in typical swelling of the neck and pseudomembrane formation in throat, this is potentially dangerous if difficultes in breathing.
• Fatal if untreated (suffocation) or if the toxin spreads to heart, liver, etc.
• Virtually eliminated in "developed" countries as the Diphtheria toxin has access to the DTaP vaccine.
• Treatment via antibiotics.

Diphtheria Toxin (DT)

• DT is the best studied bacterial toxin.
• Tox gene located on lysogenic bacteriophage (B).
  • Which emedded itself into the bacteria genome.
• DT is an AB-type toxin, is an protein.
• The B-subunit binds a host cell receptor and translocates the A-subunit into the host cell.
• DT ADP-ribosylates EF-2, which stops translation and leads to host cell death.

Botulism

• Botulism is a neuromuscular disease that results when Clostridium botulinum colonizes the GI tract or the toxin is ingested.
• This occurs with unpasteurized milk/honey/juice or improper (home) canning.
• The toxin is absorbed in stomach/small intestine and enters the bloodstream.
• Targets motor neurons at neuromuscular junctions.
• Results in flaccid paralysis, where the host can't initiate muscle contraction.
• Treatment is done via antibiotics and antitoxin.
• No treatment, other than respirator if toxin is bound to its target, damage is permanent if body doesn't replace the neuron.
• Thus, mechanics respirators are required for patients to breathe

Botulism Toxin (BoNT)

• BoNT is the most potent bacterial toxin known.
• Bot genes are located on the chromosome, plasmids, and prophage.
• BONT is an AB-type neurotoxin, where the A subunit = endoprotease.
• B-subunit binds a host cell membrane receptor on motor neurons.
• Translocates the A-subunit into the host cell.
• The various BoNT A-subunits are endoproteases.
• Cleave various SNARE proteins.
• Synaptic vesicles can't merge with the cell membrane to release acetylcholine into the synaptic cleft where they normally initiate muscle contraction.
• Muscle cells are not stimulated by acetylcholine and muscle can't contract.

Tetanus

• Tetanus is a neuromuscular disease where Clostridium tetani colonizes a deep tissue wound. Same Genus for BoNT.
• Tetanus toxin diffuses and enters the bloodstream, where it targets the inhibitory neurons of motor neurons.
  • Causes spastic paralysis.
• Treatment via antibiotics and antitoxin. No treatment, if the body doesn't replace the neuron, because the damage is permanent, once the toxin is bound to its target.
• Tetanus is virtually eliminated in developed countries that have access to the DTaP vaccine.

Tetanus Toxin (TeNT)

• Shares homology with BoNT, but targets different receptors.
  • Tet genes located on a plasmid
• Tent is an AB-type neurotoxin.
• The B-subunit binds a host cell membrane receptor on inhibitory neurons.
  • Translocates the A-subunit into the host cell.

The TeNT A-subunit is an endoprotease, cleaves a SNARE protein synapse, at our um-VAMP.

• Synaptic vesicles can't merge with the cell membrane to release glycine into the synaptic cleft, where they normally inhibit motor neurons.
• Uninhibited motor neurons constantly release acetylcholine.
  • Muscles are locked in a contracted state.

The aP in DTaP

• Pertussis (whooping cough) is a respiratory disease.
• Bordetella pertussis colonize ciliated cells of the LRT.
• Colonized cells secrete:
  • Pertussis toxin (PT), an AB-type toxin, through a T4SS
  • Tracheal cytotoxin (TCT), an endotoxin
• Kills ciliated cells that keep lung tissue free of debris.
• Violent cough lasts 6+ weeks
• Gets its name from the sound patients make when inhaling after a coughing fit
• Hope you didn't skip your last DTaP booster shot!

Cholera

• Cholera is an intestinal disease that results when Vibrio cholerae colonize the small intestine.
  • Exposure occurs via food and/or water contaminated with human feces.
• The toxin targets intestinal epithelial cells.
  • Causes severe diarrhea resulting in life-threatening dehydration and electrolyte loss
• Severe cases can see loss of 20L of water per day.
• Water leaves intestinal cells so violently that the cells rip apart.
• Water loss is often so rapid and constant, and patients are so weak.
  • The army developed "cholera cots" to provide a level of ease for patients and medics.
• Treatment via rehydration therapy and antibiotics.
  • If it is severe, death is inevitable.
• Cholera is virtually eliminated in “developed" countries that have access to clean drinking water.

Cholera Toxin (CT)

• Shares homology with enterotoxins of pathogenic E. coli and Shigella.
  • ctx gene is on a prophage
  • CT is a hexameric AB-type toxin
• The B-subunits bind a host cell membrane receptor on intestinal epithelial cells.
  • Translocates the A-subunit into the host cell.
• The CT A-subunit ADP-ribosylates the alpha subunit of CF proteins.
• The Gs is constitutively activated by adenylates cyclase.
  • Cellular cAMP levels increase.
• cAMP activates cAMP-dependent protein kinase (PKA) signaling pathways.
• Cells stop absorbing Na+ and Cl- from the intestinal lumen, and actively pumps Cl- and HCO3¯ into the lumen.
• Osmosis happens when it loses water.

Superantigens

• Excreted toxin proteins.
• Binds immune cells and indiscriminately activate them.
  • Lots of them are all throughout the body. Normal pathogens/toxins activate ~0.0001-0.001% of immune cells.
  • Superantigens activate up to 20%.
• The massive immune response induces high fever, low blood pressure, organ dysfunction/failure, and system shock/severe multiorgan failure and death.
• Staphylococcus aureus
  • Staphylococcal enterotoxin (SE) – food poisoning
• Streptococcus pyogenes
  • Exotoxin A – scarlet fever, streptococcal TSS

Endotoxins

• Permanent components of the pathogens outer cell surface.
  • Elicit an immune response.
    • Not excreted, but may spread systemically when cells lyse.
• The immune system responds by releasing cytokines, as well as inducing increased heart rate, diarrhea/vomiting, low blood pressure, and inflammation.
• Large amounts of endotoxin can result in blood clotting.
• Also results in organ disfunction/failure, endotoxic shock/septic shock, and death.

Lipopolysaccharide (LPS)

• Components are the O-specific polysaccharide, core polysaccharide and Lipid A.
• The lipid A portion is toxic.
• The polysaccharide portion makes the molecule soluble.
• Induces fever and general inflammation.
• Large doses can cause tachycardia and blood coagulation, where clots form within the vascular system.

Lipoteichoic acid

• Contains a Wall-associated protein, and Peptidoglycan.