Bacterial Pathogenesis Lecture Notes 2024 PDF

BIOM 611G, Medical Microbiology PCOM Georgia MECHANISMS OF BACTERIAL PATHOGENESIS Valerie E. Cadet, PhD Assistant Dean of Health Equity Integration Professor of Microbiology and Immunology...

BIOM 611G, Medical Microbiology PCOM Georgia MECHANISMS OF BACTERIAL PATHOGENESIS Valerie E. Cadet, PhD Assistant Dean of Health Equity Integration Professor of Microbiology and Immunology BMS1 & BMS2 Department of Biomedical Sciences November 26, 2024  Reading: Medical Microbiology by Patrick R. Murray, 9th Edition, Ch. 14 LEARNING OBJECTIVES Through the study of this content and recommended reading, the successful student will be able to: 1. Explain how the following types of virulence factors contribute to pathogenicity: a. bacterial products that allow attachment to host cells b. different types of enzymes (for example, proteases, catalase, hemolysins) c. glycocalyx (capsules & slime layers) & biofilms d. endotoxins & exotoxins 2. Define pathogenicity islands and secretion systems. 3. Describe the mechanisms that bacteria use to avoid being killed/cleared a. defense against antibodies b. antigenic masking c. antiphagocytic mechanisms d. inhibition of chemotaxis 4. Describe intracellular invasion by bacterial pathogens & how intracellular bacteria evade degradation and cell killing. 2  Define pathogenicity islands and secretion DEFINITIONS systems. 3 Virulence Factors: intrinsic characteristics of bacteria that contribute to pathogenicity, often encoded on pathogenicity island Pathogenicity islands Large segments of DNA carrying virulence genes acquired by horizontal gene transfer ▪ May be turned on by single stimulus ▪ Ex: pH of GI tract or vesicle; gut temp ▪ Ex: SPI-2 turned on to produce ~25 proteins that make Type III secretion system of Salmonella activated by acidity of phagocytic vesicle within macrophage ▪ May be turned on by quorum ▪ Sufficient bacteria producing sufficient amounts of particular substance ▪ Ex: polysaccharide produced to promote biofilm production of Pseudomonas aeruginosa when quorum makes enough N-acyl homoserine lactone (AHL) ▪ Not present in nonpathogenic members of same genus or species 4  1. Capsule and Biofilm BACTERIAL VIRULENCE 2. Adherence 3. Invasion MECHANISMS 4. Growth byproducts (ex: gas, acid, ammonia) 5. Degradative enzymes 6. Cytotoxic proteins 7. Evasion of phagocytic and immune clearance 8. Antibiotic resistance 9. Intracellular growth 10. Toxins ▪ Exotoxins ▪ Endotoxins ▪ Superantigen 11. Induction of excess inflammation 12. Evasion of phagocytic and immune clearance 13. Resistance to antibiotics 14. Intracellular growth 5  Explain how the following COLONIZATION, types of virulence factors contribute to pathogenicity: ADHESION, a. bacterial products that allow attachment to host INVASION, cells b. different types of TISSUE enzymes (for example, proteases, catalase, DESTRUCTION, hemolysins) c. glycocalyx (capsules & EXO- & slime layers) & biofilms ENDOTOXINS d. endotoxins & exotoxins 6  GLYCOCALYX VS CAPSULES VS SLIME LAYER/BIOFILM 2 types of Glycocalyx (sugar coat) exist 1. Capsule 2. Slime Layer ▪ Thick, discrete detectable layer of ▪ Thin, less discrete, unorganized structure polysaccharides deposited outside the or extracellular matrix which surrounds cell wall and embeds the cells ▪ Tightly bound on the outside of the cell ▪ consists mostly of exopolysaccharides, ▪ Virulence factor that assists in evading glycoproteins, and glycolipids phagocytosis ▪ Irregularly shaped and loosely bound, layer of material that bacteria cells ▪ Easily removable ▪ Mainly aids in adherence, protection from dehydration and nutrient loss (a) Capsules are a type of glycocalyx composed of an organized layer of polysaccharides. b) A capsule stain of Pseudomonas aeruginosa, a bacterial pathogen capable of causing many different types of infections in humans. 7 (credit b: modification of work by American Society for Microbiology) COLONIZATION: BIOFILMS Biofilms provide attachment and resistance ▪ Aggregate of microorganisms in which cells adhere to each other on a surface and resist clearance ▪ Reduce efficacy of antibiotics 🡪 🡪 antibiotic resistance ▪ May form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings ▪ Contain water channels that help distribute nutrients and signaling molecules Staphylococcus aureus biofilm on an indwelling catheter Quorum sensing bacteria produce signaling compounds which allow for inter-bacterial communication that occurs on the surface of the biofilm When in sufficient concentration, transcription of specific response genes (e.g. for biofilm production, etc.) is initiated Allows for populations to multiply 8  HOW DO BIOFILMS FORM? 1. Free-floating, or planktonic, bacteria encounter a submerged surface and within minutes can become attached. ▪ Held together and protected by exopolysaccharide (EPS) in slime layer ▪ Alginate, an EPS component, inhibits innate immune defenses 2. EPS production allows the emerging biofilm community to develop a complex, 3-D structure within hours. 3. Biofilms propagate through detachment of small or large clumps of cells, or by a "seeding dispersal" that releases individual cells. Pseudomonas aeruginosa produces and secrets EPS that helps it bind to host mucus membranes 🡪 forms 3D microcolonies 🡪 biofilms in lungs 9 Lungs at autopsy from cystic fibrosis patient who succumbed to Biofilm of Pseudomonas aeruginosa from pus chronic lung infection caused by Pseudomonas aeruginosa in bronchi of the lungs in figure on the left. 10  Explain how the following COLONIZATION, types of virulence factors contribute to pathogenicity: ADHESION, a. bacterial products that allow attachment to host INVASION, cells b. different types of TISSUE enzymes (for example, proteases, catalase, DESTRUCTION, hemolysins) c. glycocalyx (capsules & EXO- & slime layers) & biofilms ENDOTOXINS d. endotoxins & exotoxins 11  see Table 14-2 ADHERENCE: ADHESINS ▪ Adhesins: surface projections/ specialized molecules that bind specific receptors on host cell surface (typically sugars) ▪ Promote attachment and colonization but not necessarily invasion of host cells and tissue ▪ Compete with normal microbiota for essential nutrients ▪ Promote avoidance of host defense ▪ Stimulate inflammatory response (+/-) ▪ Adhesins can be part of ▪ Glycocalyx: Capsule (Streptococcus mutans) or slime layer ▪ Ex: Fimbriae/pili: Smaller than flagella, but with similar structure (Ex: E. coli) 12  INVASIVE BACTERIA CROSS MEMBRANES Utilizing ▪ flagella to swim through ▪ proteases to digest the mucous layer to approach the epithelial lining ▪ destroy the membrane barrier, induce inflammation to permeabilize the barrier, or penetrate into the cells of the barrier Examples ▪ Salmonella and Yersinia organisms use fimbriae to bind to M (microfold) cells of the colon ▪ then inject proteins into the M cell that stimulate the cell membrane to surround and take in the bacteria ▪ Enteropathogenic E. coli secretes proteins into host cell that creates portable docking system ▪ Salmonella uses the device to promote its uptake into a vesicle and live intracellularly within the macrophage. https://www.biointeractive.org/classroom-resources/how-pathogenic-e-coli-infection-begins 13 and https://www.biointeractive.org/classroom-resources/how-salmonella-infection-begins  Define pathogenicity islands and secretion DEFINITIONS systems. 14 SECRETION SYSTEMS ▪ Enables bacterium to directly inject effector molecules into cytoplasm of the host cell Alters host cellular machinery, function or communication to benefit the bacteria Most common: Type III secretion systems (T3SS) injects secretory proteins into host cell A type 4 secretion system can transfer effector proteins and/or DNA into the host cell Helicobacter pylori 15  TYPE III SECRETION SYSTEMS (T3SS) ▪ Involves formation of a ~20 protein complex, that reside within the cytoplasmic membrane of the bacterial cell ▪ Additional examples of production of type III secretion devices ▪ Shigella flexneri and Salmonella typhimurium triggered by oxygen tension and pH, respectively, to ensure proximity to appropriate host membranes 16  Explain how the following COLONIZATION, types of virulence factors contribute to pathogenicity: ADHESION, a. bacterial products that allow attachment to host INVASION, cells b. different types of TISSUE enzymes (for example, proteases, catalase, DESTRUCTION, hemolysins) c. glycocalyx (capsules & EXO- & slime layers) & biofilms ENDOTOXINS d. endotoxins & exotoxins 17 MAJOR GROUPS OF BACTERIAL ENZYMES ▪ By-products of bacterial ▪ Degradative enzymes and growth spreading factors ▪ Fermentation ▪ Break down tissue ▪ Acids ▪ Provide food for the organisms’ ▪ Gas growth ▪ Other substances ▪ Promote bacterial spread 18 DEGRADATIVE ENZYMES 1. Lecithinase: destroy lecithin (phosphatidylcholine) in cell membrane (Clostridium perfringens) 2. Phospholipase: breaks down phospholipids in cell membrane (Clostridium perfringens alpha toxin) 3. Leukocidin/ streptolysin: lyse phagocytes and their granules (Staphylococcus spp./ Streptococcus spp. exotoxins) 4. Hemolysins: Lyses erythrocytes (Streptococcus spp. streptolysin O, Staphylococcus spp. alpha toxin) 19 SPREADING FACTORS Beta-hemolytic Streptococcus 1. Hyaluronidase: cleaves hyaluronic acid, component of e/cell matrix 🡪 tissue penetration (Streptococcus spp.) 2. Collagenase: breaks down collagen network in connective tissues (Clostridium spp.) 3. Streptokinase/ Staphylokinase: converts plasminogen (inactive) to plasmin 🡪 fibrin digestion, preventing clotting 🡪 spread (group A Streptococcus/ Streptococcus spp.) 4. Neuraminidase: degrades neuraminic acid (aka sialic acid), intercellular cement of epithelial cells of intestinal mucosa (Vibrio cholerae and Shigella20 dysenteriae)  Explain how the following COLONIZATION, types of virulence factors contribute to pathogenicity: ADHESION, a. bacterial products that allow attachment to host INVASION, cells b. different types of TISSUE enzymes (for example, proteases, catalase, DESTRUCTION, hemolysins) c. glycocalyx (capsules & EXO- & slime layers) & biofilms ENDOTOXINS d. endotoxins & exotoxins 21 TOXINS Bacterial products that directly cause harm to tissue or trigger destructive biologic activities ▪ Attack the ground substance and basement membranes of integuments and intestinal linings ▪ Degrade carbohydrate-protein complexes between cells or on cell surfaces ▪ Penetration of the host’s epithelial cells or tissues ▪ Continued avoidance of host defenses ▪ Can be exotoxins (G+ or G-) or endotoxins (G-) ▪ Many toxins are secreted by bacteria in the stationary phase. This timing may be advantageous to the bacteria because the toxins may contribute to bacterial nutrition. 22  COMPARISON OF TYPES OF TOXINS Exotoxin Endotoxin Present on outer membrane of Location in Excreted by G+ and G- G-; liberated when bacteria die bacteria and cell wall is disrupted Chemical Proteins; usually heat-labile Lipid A of LPS; heat-stable Properties Specific depending on type Innate immunity; systemic Mode of action (enterotoxin, cytotoxin or neurotoxin) response; pyrogenic Toxicity Extremely toxic; can be fatal Weakly toxic; rarely fatal Poor immunogen; not able to Highly immunogenic; stimulates Immunogenicity neutralize though produces neutralizing Ab proper immune response Inactivating destroys toxicity; Potential for Toxoid None maintains immunogenicity 23  EXOTOXINS TRUCTURE AND DELIVERY OF A-B Receptor-mediated endocytosis (RME): B subunit of native (A+B) toxin binds to a specific receptor on the target cell and the A+B structure is internalized in an endosome H+ ions enter endosome, lowering internal pH causing A+B subunits to separate. B subunit affects the release of A subunit from the endosome so it can reach its target in the cytoplasm. B subunit remains in the endosome and is recycled to the cell surface. Direct entry: B subunit of native (A+B) toxin binds to a specific receptor on the target cell Binding induces formation of pore in the membrane through which the A subunit is transferred into the cell through a needle-like structure (type III secretion system) Yersinia spp., salmonella spp. 24  see Table 14-3 Properties of A-B–Type Bacterial Toxins 5 Main Mechanisms 1. Inhibiting protein EXOTOXIN synthesis 2. Hyperactivation of cellular proteins S 3. Forming pores in cellular membranes 4. Interfering with nerve transmission 5. Non-specific activation of T cells 25  EXOTOXINS (ENTEROTOXIN): 1. PROTEIN SYNTHESIS INHIBITION ▪ Act at elongation factor-2 (EF-2) ▪ Toxin🡪 A attaches ADP ribosyl to EF2 🡪inhibition of protein elongation >> cell death ▪ Diptheria toxin: Corynebacterium diphtheriae - pharyngitis and pseudomembane in throat ▪ Exotoxin A: Pseudomonas aeruginosa - cell death ▪ Inactivate 60s subunit of ribosome by cleaving rRNA to inhibit protein synthesis. ▪ Shiga toxin (ST): Shigella spp. - damage intestinal epithelium 🡪dysentery; enhances cytokine release and damage of glomerular endothelial cells 🡪kidney failure (HUS) ▪ Shiga-like toxin (SLT): Enterohemorrhagic E. coli (EHEC) - enhances cytokine release and damage of glomerular endothelial cells 🡪kidney failure (HUS) http://highered.mheducation.com/sites/0072556781/student_view0/chapter34/animation_quiz.html 26  EXOTOXINS (ENTEROTOXIN): 2. CELLULAR PROTEIN HYPERACTIVATION Cholera toxin ADP ribosylation of G protein overactivates adenylate cyclase Increased concentration of cAMP 🡪 excretion of Cl- and H20 from gut Results in life-threatening diarrhea. Other examples of bacteria with toxins with similar mechanism Enterotoxigenic E. coli (ETEC): Heat-labile toxin (LT) and Heat-stable toxin (ST) Yersinia enterocolitica: Heat-stable toxin (ST) Bacillus anthracis: Edema factor 27 EXOTOXINS (CYTOTOXIN) 3. PORE FORMING Staphylococcus aureus alpha toxin ▪ Membrane-disrupting ▪ Creates pores causing hemolysis and tissue damage ▪ Abscesses Clostridium perfringens alpha toxin ▪ Phospholipase ▪ Targets cholesterol ▪ Directly responsible for gas gangrene and myonecrosis 28 EXOTOXINS (NEUROTOXINS) 4. INHIBITING NERVE-MUSCLE TRANSMISSION Clostridium tetani tetanospasmin Cleaves SNARE protein required for neurotransmitter release Prevents release of inhibitory neurotransmitters GABA and glycine in spinal cord Result is spastic paralysis, muscle rigidity and lock jaw Clostridium botulinum toxin Cleaves SNARE protein required for neurotransmitter release Prevents release of stimulatory neurotransmitters ACh at musculoskeletal junction Result is flaccid paralysis, floppy baby 29  EXOTOXINS (ENTEROTOXIN): 5. SUPERANTIGENS-NON-SPECIFIC T CELL ACTIVATION Overrides the specificity of T cell response ▪ Non-specifically binding first to MHC-II on antigen presenting cells (APCs) followed by the TCR of T cells ▪ A single SAg can crosslink > 30% of T cells ▪ T cells are activated 🡪 Overwhelming cytokine secretion and proliferation ▪ IFNg and IL-2 ▪ APCs are induced to secrete huge amount of pro-inflamamtory cytokines ▪ Secrete IL-12, IL-1, IL-6, IL-8, TNFa ▪ If in high enough concentration in blood 🡪 systemic shock and Examples multi-system organ failure (MSOF) Staphylococcus aureus: Enterotoxin B (SEB) 🡪 food poisoning: shock and MSOF (potential biological weapon) TSST-1 🡪 toxic shock syndrome: fever, rash and shock Toxin A and B 🡪 scalded skin syndrome and food poisoning Streptococcus pyogenes 30 Exotoxin A 🡪 Toxic shock syndrome: fever, rash and shock  N-dotoxin is an integral part ENDOTOXI of gram-Negative outer membrane ❖ Lipid A stimulates inflammatory NS response 1. Lysis of G- bacteria 🡪 release LPS or LOS from outer membrane of their cell wall 2. LPS binds LPS-binding protein in blood 3. LPS+LPS-binding protein bind CD14 (macrophages); other molecules (B cells, other innate LPS: lipopolysaccharide cells) LOS: lipooligosaccharide Results 4. Macrophages release cytokines ▪ IL-1, IL-6, TNF-a, PAF and prostaglandins 🡪 fever (IL-1); shock (TNF-induced decrease in blood pressure if high enough concentration); bacteria cross BBB (TNF); acute-phase proteins to be secreted from liver 🡪 hypogycemia 5. Cytokines stimulate production of inflammatory mediators ▪ B cells stimulated to secrete antibodies; mast cells stimulated; complement pathways and coagulation pathway activated 🡪 Prostaglandins, leukotrienes and complement molecules damage vascular endothelium 6. Excessive production of clotting factors leads to ARDS and DIC 7. Overproduction of prostaglandins, leukotrienes, and complement proteins damages vascular endothelium 🡪 hypotension, shock and multiple organ system failure ▪ Gram (+) cell wall products with endotoxin-like activity: ▪ Peptidoglycan, lipoteichoic acid, teichoic acid 32 SUMMARY: ENDOTOXIN-MEDIATED TOXICITY 1. Fever 2. Leukopenia followed by leukocytosis 3. Activation of complement 4. Thrombocytopenia 5. Disseminated intravascular coagulation (DIC) 6. Decreased peripheral circulation and perfusion to major organs 7. Shock 8. Death 33  Describe the mechanisms that bacteria use to avoid VIRULENCE being killed/cleared a. defense against FACTORS: antibodies HOST IMMUNE b. antigenic masking c. inhibition of chemotaxis EVASION d. antiphagocytic mechanisms BACTERIAL PATHOGENESIS: Bacteria choose to fight and flee Nature Reviews Microbiology 12, 460–461 (2014) 34  MECHANISMS OF HOST 1. EVASION 1 Defense against antibodies i. Production of anti-IgA proteases: Neisseria gonorrhoeae ii. Prevention of opsonization via binding Fc region of IgG: Staphylococcus aureus protein A 2. Antigenic masking i. O antigen (Lipid O) on LPS of E. coli ii. Staphylococcus aureus: produces cell-bound coagulases converts (soluble) fibrinogen/FG to (insoluble) fibrin in plasma 🡪 clot and covers in fibrin/FG pseudo-capsule iii. Treponema pallidum: binds fibronectin to its surface iv. Group B Streptococcus (GBS), Neisseria meningitidis capsules contain sialic acid 🡪 prevention of opsonization by complement v. Group A Streptococcus (GAS): M protein binds factor H and fibrinogen is bound on surface (inhibits complement activation) vi. Polysaccharide capsule: Streptococcus pneumoniae vii. Hyaluronic acid capsule: GAS, Haemophilus influenzae viii. Slime layer of Pseudomonas aeruginosa 35  Describe the mechanisms that bacteria use to avoid being killed/cleared INTRACELLULAR a. defense against antibodies BACTERIA HAVE b. antigenic masking inhibition of chemotaxis FIGURED OUT A c. d. antiphagocytic mechanisms PLAN Describe intracellular invasion by bacterial pathogens & how intracellular bacteria evade degradation and cell killing. 36  MECHANISMS OF HOST EVASION 2 3. Inhibition of chemotaxis (suppresses neutrophils) i. Streptococcus spp. streptolysin ii. Clostridium perfringes α-toxin 4. Antiphagocytic mechanisms Method Example i. Inhibition of phagolysosome fusion Mycobacterium tuberculosis, Legionella and Chlamydia spp. ii. Resistance to lysosomal enzymes Salmonella typhimurium, M. leprae, Coxiella, Ehrlichia and Leishmania spp. iii. Adaptation to cytoplasmic replication Listeria, Francisella, and Rickettsia spp. 37  BACTERIAL MECHANISMS FOR ESCAPING PHAGOCYTIC CLEARANCE ▪ Produce enzymes capable of lysing phagocytic cells ▪ Streptococcus pyogenes (GAS) streptolysin or Clostridium perfringens α-toxin ▪ Inhibit uptake by phagocytosis ▪ S. pyogenes (GAS) effects of the capsule and M protein ▪ Block intracellular killing ▪ Mycobacterium spp. blocks fusion of lysosome and phagosome to prevent contact with its bactericidal contents ▪ Having protective capsule or lipid-rich waxy cell wall (Mycobacteria and Nocardia spp.) ▪ Produce catalase (Staphylococcus spp.) to break down hydrogen peroxide produced by myeloperoxidase system; or other means to resist the bactericidal lysosomal enzymes or substances ▪ Listeria monocytogenes lyse phagosome with a toxin and enter the cell’s cytoplasm before being exposed to lysosomal enzymes 38  “MUST-KNOWS” FOR BACTERIAL VIRULENCE MECHANISMS: EAT RICE Enzyme-mediated tissue damage (e.g., hyaluronidase, hemolysin, streptokinase) Adherence (pili, other adhesins) Toxin-induced localized and systemic effects (exotoxins, endotoxin) Resistance to antibiotics Invasion and growth in normally sterile sites Circulation via the blood or other means of spreading from primary infection site Evasion of host immune response by capsule, catalase, intracellular growth, and other mechanisms 39  STUDY QUESTIONS TO CONSIDER 1. 2. Describe potential drawbacks for having normal flora? List terms that refer to both the presence of microbes and the categories that cause disease. Be sure you can define/recognize each term. 3. What terms are associated with pathogen transmission? What are the different ways in which pathogens can be transmitted? What is involved for each mode of transmission? 4. Describe factors that determine if disease will occur. 5. What are the different terms associated with bacterial pathogenesis? How do they differ and what to they refer to? 6. What are virulence factors? What role does pathogenicity islands play in the dispersal of virulence factors? 7. Explain how capsules and cell wall components contribute to pathogenicity. 8. What are the components that play a role in the host-pathogen interaction? 9. Why are adherence and colonization important to a bacterial pathogen? How does invasion differ? 10. What types of toxins are made by bacterial pathogens? What characteristics do they have? Suggestions: ▪ Make a list (or notecard) of all the bacteria we have discussed. For each bacteria, find every reference of virulence factor that we went over. Keep this list (or notecards) with you as you learn the G+ and G- bacteria to add more detail and/or refer back to. ▪ As you go on to discuss specific bacteria of medical importance, note the types of virulence factors utilized (including toxins) and the mechanism of transmission used. ▪ Use the 7 questions you need to ask for each pathogen of medical importance 40