Weapons of Mass Infections - BIO3124 Lecture Notes PDF

Weapons of mass infections Norton Chap. 25 Rice Uni chap 15 Picture from NewScientist Chapter Overview § Host-pathogen interactions § How microbes attach to host cells § How toxins subvert host functions § How toxins and effectors are deploye...

Weapons of mass infections Norton Chap. 25 Rice Uni chap 15 Picture from NewScientist Chapter Overview § Host-pathogen interactions § How microbes attach to host cells § How toxins subvert host functions § How toxins and effectors are deployed § How pathogens survive within their hosts § Tools used to probe pathogenesis 2 Introduction How can an organism too small to be seen with the naked eye kill a human a million times larger? 3 Let’s start with some vocabulary 4 The Language of Pathogenesis § Primary pathogens: cause disease in healthy hosts For example: Shigella flexneri, the cause of bacillary dysentery § Opportunistic pathogens: cause disease only in compromised hosts or following entry into unprotected sites For example: Pneumocystis jirovecii, the cause of life-threatening infections in AIDS patients § An infection occurs when a pathogen or parasite enters or begins to grow on a host. However, the term “infection” does not necessarily imply overt disease. Some microbes even enter a latent state during infection, in which the infectious organism cannot be found by culture. For example: herpesvirus, the cause of cold sores 5 The Language of Pathogenesis § Pathogenicity refers to an organism’s ability to cause disease. It is defined in terms of... how easily an organism causes disease (infectivity) how severe that disease is (virulence) the specific genetic makeup of the pathogen § For example, Ebola is highly virulent, whereas rhinoviruses are not. 6 The Language of Pathogenesis § Pathogens use various strategies to establish virulence. Virulence is the relative ability of a pathogen to cause disease. § Measuring virulence Virulence can be estimated from experimental studies of the ID50 (infectious dose) LD50 (lethal dose50). ― ID50 :number of pathogen cells or virions required to cause active infection in 50% of inoculated animals ― LD50 :the amount of an agent that kills 50 percent of the animals in a test group 7 Example of ID50 for selected foodborne diseases Table 15.5, Chapter 15, Rice University, Openstax, Microbiology, 2nd ed. 8 Immunopathogenesis § It is often “friendly fire” by our immune system reacting to a pathogen that causes major tissue and organ damage. § The term immunopathogenesis applies when the immune response to a pathogen is a contributing cause of pathology and disease. § To fully understand any infectious disease, researchers must study both the pathogenic mechanisms of the pathogen and the disease symptoms caused by immunopathogenesis. 9 Infection Cycles § The infection cycle describes the route of transmission of an infectious organism. Horizontal transmission: passage from one person or animal to another within the same generation ― Can be direct (e.g., handshaking, kissing) or indirect (e.g., sharing contaminated objects, sharing an office space) ― Fomites: inanimate objects (e.g., doorknobs, hand towels, utensils) ― Vehicles: ingested or inhaled materials (e.g., food, water, air) Vertical transmission: passage from a mother to her fetus during pregnancy (transplacental) or birth (parturition) 10 Steps to pathogenesis What does a bug have to do to cause disease? 11 Steps to pathogenesis Multiple events, each a limiting barrier for infection, need to occur before a full-fledged bacterial infections translate into symptoms and pathology. Break in barrier Brock, Biology of Microorganisms, 15th ed., Pearson. Portals of Entry § Infectious agents enter the body through one or more portals of entry that are best suited to their mechanism of pathogenesis. Mouth Respiratory tract Conjunctiva and mucous membranes Wounds, injuries, and skin lesions Parenteral route: direct injection into bloodstream (e.g., tick and mosquito bites, needle punctures) 13 Virulence Factors and How to Find Them § To cause disease, all pathogens must... § Attach to our surfaces and tissues § Damage tissues to obtain nutrients and replicate § Avoid host immune responses § Pathogens employ virulence factors, encoded by virulence genes, to accomplish these goals. § Virulence factors include toxins, attachment proteins, capsules, and other devices. 14 Genetics of Virulence (weapon manufacturing) Virulence in Salmonella: Pathogenicity Islands and Plasmids Salmonella species encode a large number of virulence factors. § Several genes that direct invasion are clustered together on the chromosome as pathogenicity islands. § Another Salmonella pathogenicity island contains genes that promote a more systemic disease. § Salmonella also contains resistance plasmids (R plasmids). Brock, Biology of Microorganisms, 15th ed., Pearson. Pathogenicity Islands § Most pathogenicity islands appear to have been horizontally transmitted via conjugation or transduction Unique GC/AT ratio Codon bias Flanked by inverted repeats Found in only certain strains of a species 16  Pathogenicity Islands DR: direct repeats IS: insertion sequence 17 Pathogenicity Islands (examples) 18  Human-Microbial Interactions 25.2 Microbial Attachment Attachment Adhesins, capsules, fimbriae and pili, flagella 25.3 Colonization and Invasion Hyaluronidase, coagulase, streptokinase and other proteases Tissue and nucleases damage and colonization 25.3 Pathogenicity, Virulence, and Attenuation All of the above, exotoxins/endotoxins, superantigens, T3SS and others Microbial Adherence: Adhesins § There are many different receptors coating both the pathogen and tissues where the bacteria or virus binds. § Adhesins are glycoproteins or lipoproteins found on the pathogen’s surface that enable it to bind to host cells. Source: Biology LibreTextsTM Microbial Adherence: Pili or fimbriae Fimbriae, Flagella, and pili are bacterial cell surface protein structures that function in attachment. https://veteriankey.com/ mechanisms-of-microbial- infections/ Pili Many bacteria typically attach to specific host cells using hairlike appendages called pili (fimbriae). [Note that fimbriae pili are not the same as conjugation, or sex, pili used for gene transfer.] Type I: adhere to carbohydrates on host membranes ― Produce a static attachment to host cell ― Grow from outer membrane of certain Gram-negative bacteria Type IV: involved in “twitching motility” ― Produce a dynamic attachment via assembly and disassembly ― Grow from inner membrane of many Gram-negative bacteria 22 Pili – 2 Example of Type I pili 23 Example of Type IV Pili 24 Microbial Adherence: non-pilus adhesins Fimbriae, Flagella, and pili are bacterial cell surface protein structures that function in attachment. https://veteriankey.com/ mechanisms-of-microbial- infections/ Nonpilus Adhesins § Bacteria also possess adhesins that are not pili. Streptococcus pyogenes: M protein ― Binds to fibronectin Bordetella pertussis: pertactin ― Binds to host cell integrin Pseudomonas aeruginosa: multivalent adhesion molecule 7 (MAM7) ― Binds to phospholipids and fibronectin in host cell membranes 26 Nonpilus Adhesins 27 Nonpilus Adhesins § Why are some people susceptible to certain infections, whereas others are not? Immunocompetence (capacity to see and eliminate danger) Receptor availability (capacity to adhere to host cell) § Pathogens rely on very specific surface structures (receptors) to recognize and attach to appropriate host cells. Person-to-person differences in receptor structures are possible. Example: HIV binds C-C chemokine receptor type 5 (CCR5); individuals with a CCR5 mutation are resistant to HIV infection! 28 Microbial Adherence: capsules The bacterial capsule forms a thick coating outside the plasma membrane and cell wall and serves two important functions in bacterial pathogenicity. § Often, without a capsule, the bacterial strain is not pathogenic. § The capsule is both sticky and contains specific receptors to facilitate attachment on host tissues. § Capsules, such as those found in Streptococcus pneumoniae, protect the bacteria phagocytosis: Inhibits opsonization. Zhensong Wen, Jing-Ren Zhang, Chapter 3 - Bacterial Capsules, Molecular Medical Microbiology (Second Edition), Academic Press, 2015, Pages 33-53. https://doi.org/10.1016/B978-0-12-397169-2.00003-2 Capsules prevent phagocytosis (and hides the bacteria) § Blocks opsonization, interfering with phagocytosis. § Blocks antibody/complement. § Reduced entry into endocytic pathway lessens antigen presentation. § Mimics “self” molecules, preventing stimulation of antibody/complement responses. A. Non-encapsulated bacterium B. Encapsulated bacterium Wessner, Microbiology, 3rd ed., Chapter 21, Copyright ©2018 John Wiley & Sons, Inc. 30 Biofilms and Infections § Bacteria can attach to surfaces as a population, forming a biofilm. § Biofilms play an important role in chronic infections by enabling persistent adherence, resistance to host defenses, and tolerance to antimicrobial agents. C. jejuni adhering to gut mucosa Mucosa of pediatric adenoids (tonsils) Biofilm Lymphocytes 31 25.3 Colonization and Invasion Colonization is the growth of microorganisms after they’ve gained access to host tissues. Break in barrier Brock, Biology of Microorganisms, 15th ed., Pearson. Colonization and Invasion Invasion and Systemic Infection Invasiveness ― ability of a pathogen to grow in host tissue at densities that inhibit host function Bacteremia: the presence of bacteria in the bloodstream, not always harmful, a vigorous toothbrushing can cause bacteremia Septicemia: bloodborne systemic infection ― may lead to massive inflammation, septic shock, and death Infection: any situation in which a microorganism (not a member of the local flora) is established and growing in a host Enzymes and Toxins of Pathogenesis As the microorganisms gain a foothold (adhere and invade) and proliferate, some microorganisms have features that will cause disease in the host. Those features can be beneficial to the microorganisms and facilitate their invasion and propagation, or enhance their capacity of evading the immune system. § Enzymes involved in tissue invasion § AB-Type Exotoxins § Cytolytic (cytolysins) and Superantigen Exotoxins § Endotoxins § Effector proteins (Secretion systems)  Enzymes involved in tissue invasion Invasiveness requires a pathogen break down host tissues. This is often done with enzymes that attack host cells. Tissue-Destroying Enzymes § Hyaluronidase breaks down host tissues. Brock, Biology of Microorganisms, 15th ed., Pearson. Enzymes involved in tissue invasion Invasiveness requires a pathogen break down host tissues. This is often done with enzymes that attack host cells. Tissue-Destroying Enzymes § Hyaluronidase breaks down host tissues. § Coagulase and streptokinase manipulate clotting. Coagulase forms clots, while streptokinase breaks them down. Brock, Biology of Microorganisms, 15th ed., Pearson. Other Enzymes used as Virulence Factors Rice Uni Chap 15.3 25.3 Toxins Subvert Host Functions § Bacterial toxins can be divided into two main types. 1. Exotoxins ― Proteins produced and secreted by various types of bacteria ― Kill host cells and unlock their nutrients ― Typical examples: AB toxins, cytolysins and superantigens 2. Endotoxin ― A part of lipopolysaccharide (LPS) of Gram-negative bacteria ― Lipoteichoic acid on Gram-positive cells ― Hyperactivate host immune systems to harmful levels 38 25.3 Toxins Subvert Host Functions 39 Categories of Microbial Exotoxins § Microbial exotoxins fall into several categories based on their mechanisms of action. Plasma membrane Signal transduction disruption disruption Cytoskeleton alterations Cell-cell adherence Protein synthesis Vesicular traffic disruption Inhibit exocytosis Cell cycle disruption 40 Categories of Microbial Exotoxins Membrane disruptors AB-type toxins Transport disrupting toxin (cytolysins) (two subunits) 41 Membrane Disruption (cytolysins) Two types of exotoxins disrupt host cell membranes. § Pore-forming proteins insert themselves into membranes by binding cholesterol and membrane receptors Alpha toxin of Staphylococcus aureus (see figure) Panton-Valentine toxin of MRSA (see Special Topic 25.1 Norton textbook) Listeriolysin O of Listeria monocytogenes § Phospholipase enzymes hydrolyze phospholipids into fatty acids Wessner, Microbiology, 3rd ed., Chapter 21, Copyright ©2018 John Wiley & Sons, Inc. Membrane Disruption (cytolysins) Two types of exotoxins disrupt host cell membranes. § Pore-forming proteins insert themselves into membranes by binding cholesterol and membrane receptors § Phospholipase enzymes hydrolyze phospholipids into fatty acids Phospholipase C of Clostridium perfringens C. perfringens α-toxin is a lecithinase associated with gas gangrene. Perfringolysin (also produced in gas Wessner, Microbiology, 3rd ed., Chapter 21, Copyright ©2018 John Wiley & Sons, Inc. gangrene) is a pore-forming cytolysin, however. 43 Membrane Disruption (cytolysins) § Some exotoxins disrupt host cell membranes by forming pores that cause leakage of cell constituents (host cell lysis). Hemolysins lyse red blood cells (and sometimes other cells). Leukocidins lyse white blood cells (leukocytes). Some membrane-disrupting exotoxins function as both hemolysins and leukocidins. ― Streptolysin S of Streptococcus pyogenes 44 Two-Subunit AB Exotoxins § AB exotoxins consist of two subunits, usually called A and B, that work together to disrupt host cell functions. “A” subunit: toxicity-associated factor “B” subunit: binds host cell, delivers “A” subunit AB5 exotoxins consist of five “B” subunits arranged in a ring with a single “A” subunit nestled in the center. § One major subclass of AB exotoxins includes an “A” subunit that has ADP-ribosyltransferase enzymatic activity 45 Two-Subunit AB Exotoxins § Cholera toxin is an AB5 exotoxin made by Vibrio cholerae that disrupts the signaling functions of host cells. The “B” subunits bind to intestinal cell membranes and trigger endocytosis of cholera toxin complex. The “A” subunit ADP-ribosylates a host cell target that leads to a sharp increase in cAMP levels. cAMP activates ion transporters that ultimately cause water to leave the cell, leading to watery stools (diarrhea). 46 Two-Subunit AB Exotoxins 47 Two-Subunit AB Exotoxins Diphtheria Exotoxin: § Diphtheria toxin is an exotoxin produced by Corynebacterium diphtheriae that attacks protein synthesis in a different manner. § Blockage of Protein Synthesis § The toxin destroys healthy tissues in the respiratory system. § Within two to three days, the dead tissue forms a thick, gray coating that can build up in the throat or nose. This thick gray coating is called a “pseudomembrane.” It can cover tissues in the nose, tonsils, voice box, and throat, making it very hard to breathe and swallow. https://www.omicsonline.org/norway/diphtheria-peer-reviewed-pdf- ppt-articles/ § The toxin may also get into the blood stream and cause damage to the heart, kidneys, and nerves. Two-Subunit AB Exotoxins § Neurological Exotoxins: Botulinum toxins Tetanus Toxins § Both toxins are also transport disrupters § Clostridium tetani and Clostridium botulinum produce potent AB exotoxins that affect nervous tissue. The virulence advantages of producing these toxins is not clear to me. Botulinum toxin This video was scripted by Dr Lisa D'Ambrosio and Dr Elaine Beaulieu and animated by the talented students from the Algonquin College Interactive Multimedia Design program. Two-Subunit AB Exotoxins § Mechanism of action of the Tetanus Toxins Tetanus toxin is also an AB protein neurotoxin. § Its actions are opposite to the botulinum toxin, where muscles will be kept in a constant state of contraction. Tetanus, or Lockjaw disease Endotoxin (LPS) Is Made Only by Gram-Negative Bacteria § Lipopolysaccharide (LPS) is composed of lipid A (endotoxin), core glycolipid, and a polysaccharide chain called O antigen. § LPS molecules form the outer leaflet of the Gram- O-antigen negative outer membrane. As bacteria die, they release endotoxin, a microbe- associated molecular pattern (MAMP) molecule that binds to receptors on macrophages or B cells. Receptor binding triggers a massive cytokine release that can trigger fever, inflammation, shock, and death. 52 Endotoxin (LPS) Is Made Only by Gram-Negative Bacteria § Lipid A is the endotoxin part of LPS § Generally, less toxic than exotoxins § Less immunogenic (can’t use for vaccines) § Generally local infections, effects include diarrhea, vomiting and fever § Immunopathogenic: sends the immune system in a spiral § Can lead to toxic shock if systemic infections occur. Sepsis-related endotoxin release causes a massive depletion of clotting factors, which leads to a petechial rash.  Other PAMPs Endotoxin-mediated septic shock Septic shock can be induced by PAMPs (pathogen- associated molecular patterns) other then endotoxins: Some exotoxins Superantigens Secreted enzymes Highly immunogenic PAMPs End result is the same: Overactivation of coagulation (DIC) Overaction of inflammation (fever, cytokine/chemokine production, cell death) Vasodilation leading to hypovolaemia Organ failure Potentially death DIC: Disseminated intravascular coagulation Important Infectious Agents Review of Medical Microbiology and Immunology, 14e Warren Levinson, McGraw-Hill Education, 2016 Causing Sepsis and Septic Shock Type of Infectious Name of Infectious Agent Agent Bacteria Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus 1. Gram-positive cocci pyogenes, Streptococcus agalactiae, Enterococcus faecalis 2. Gram-positive rods Listeria monocytogenes, Bacillus anthracis 3. Gram-negative cocci Neisseria meningitidis Enterobacteriaceae (such as Escherichia coli, Enterobacter, Klebsiella, 4. Gram-negative rods Serratia, and Proteus), Pseudomonas, Salmonella typhi, Vibrio vulnificus, Yersinia pestis, Francisella tularensis 5. Rickettsia Rickettsia rickettsiae Viruses Ebola virus, influenza virus, hantavirus, yellow fever virus, and dengue virus Fungi Candida albicans Protozoa Plasmodium falciparum Don’t memorize this, simply remember that Gram +, Disseminated intravascular coagulation. Note purpuric lesions on Gram -, viruses, fungi and protozoa infections can all leg caused by endotoxin-mediated disseminated intravascular result in septic shock under the right conditions. coagulation (DIC) in a patient with meningococcemia. Limulus amoebocyte lysate (LAL) to detect LPS contamination (4min) ~ 4min https://www.youtube.com/watch?v=5bcveNe68xg Exotoxin and endotoxin comparison (Means you need more to get sick) Rice UniChap15.3  Bacteria Diseases Virulence Factors Mode of Action Staphylococcus aureus Depending on what virulence factors they possess, the different 1. Exotoxin mediated Toxic shock syndrome Toxic shock syndrome toxin Superantigen strains of a same species will cause Food poisoning Enterotoxin Superantigen (gastroenteritis) different diseases. Scalded skin syndrome Exfoliatin Protease cleaves desmoglein Coagulase, hyaluronidase, The different virulence factors are 2. Pyogenic Skin abscess, osteomyelitis, Enzymes causing leukocidin, lipase, and encoded on plasmids, on transposons, and endocarditis inflammation and necrosis nuclease on the genome of temperate (lysogenic) Streptococcus pyogenes phages, and on pathogenicity islands. 1. Exotoxin mediated Scarlet fever Erythrogenic toxin Superantigen These transferable genetic elements may Streptococcal toxic shock or may not be present in any single Toxic shock syndrome toxin Superantigen syndrome bacterium, which accounts for the ability Pharyngitis, cellulitis, and Enzymes causing Hyaluronidase (spreading 2. Pyogenic (suppurative) to cause different diseases. This table necrotizing fasciitis inflammation and necrosis factor) describes the different virulence factors Antibody to M protein cross- 3. Nonsuppurative Rheumatic fever Certain M proteins on pilus reacts with cardiac, joint, and for three of the most important bacterial (immunopathogenic) brain tissue pathogens: S. aureus, S. pyogenes, and Immune complexes deposit Acute glomerulonephritis Certain M proteins on pilus E. coli. on glomeruli Escherichia coli (Don’t memorize all this, but I want Activation of adenylate Watery, nonbloody diarrhea 1. Exotoxin mediated Labile toxin cyclase increases cyclic AMP; you to be able to explain why 2 (traveler’s diarrhea) no cell death strains of the same bacteria can cause Bloody diarrhea (associated Cytotoxin inhibits protein 2 different diseases) with undercooked Shiga-like toxin (verotoxin) synthesis; cell death occurs hamburger); O157:H7 strain Pili attach to Gal–Gal Review of Medical Microbiology and Immunology, 14e 2. Pyogenic Urinary tract infection Uropathic pili receptors on bladder Warren Levinson, McGraw-Hill Education, 2016 epithelium Neonatal meningitis K-1 capsule Antiphagocytic 25.4 Deploying Toxins and Effectors § We will look at several secretion systems and the molecular processes with which they share an evolutionary history. Type II secretion (T2SS): homologous to type IV pilus biogenesis Type III secretion (T3SS): homologous to flagellar synthesis Type IV secretion (T4SS): homologous to conjugation § Other secretion system exist, but we will concentrate on these. 59  Secretions systems Outside bacteria Types I through VI secretion systems § facilitate cellular activities including symbiosis, biofilm formation, enzyme secretion, DNA transfer, antibiotic release, protein delivery § Each is a large complex of proteins that forms channels through membranes. § Secretes proteins directly outside the cells into the environment or injects proteins into a host cell Schematic overview of the different secretion systems of Gram-negative airway The Role of Bacterial Secretion Systems in the Virulence of pathogens associated with cystic fibrosis (CF). Gram-Negative Airway Pathogens Associated with Cystic Fibrosis, Depluverez S. et al, Front. Microbiol., 30 August 2016. https://doi.org/10.3389/fmicb.2016.01336 25.4 Deploying Toxins and Effectors 61 Type II Secretion Resembles Type IV Pilus Assembly § The type II secretion system (T2SS) is a modification of the same system used for type IV pilus biogenesis. Secretion structures extend and retract, just like pili. Proteins to be secreted first enter the periplasm, then they get folded and secreted via an outer membrane pore. 62  Type III Secretion Is an Injection Machine § Some microbes do not rely solely on the natural array of host receptors for attachment. § Instead, these bacterial pathogens use a T3SS to insert their own receptors into target cells. § The type III secretion system (T3SS) is a reengineered flagellar synthesis mechanism that uses a molecular syringe to inject proteins from the bacterial cytoplasm directly into the host cell. Secretion is normally triggered by cell-cell contact between host and bacterium. T3SS genes usually are located within pathogenicity islands inherited via horizontal gene transfer. Inject toxins (effectors) into eukaryotic host cells. Found in Salmonella, Yersinia, Shigella, and Escherichia species. 63 Type III Secretion Is an Injection Machine 64 Secreted effector proteins § T3SS directly inject effector proteins inside the host cell. § Effector proteins: proteins that have a function that will help the bacteria manipulate the host to its advantage (invasion, immune evasion) Vibrio parahaemolyticus effectors in animal pathogenesis. V. parahaemolyticus translocates T3SS1 effectors (VopQ, Macrophages Colon epithelial cells VopR, VopS, and VPA0450) into host cells to cause cytotoxicity in different cell types such as macrophages and HeLa cells (left panel). T3SS2 effectors (VopA, VopC, VopL, and VopT) are translocated into host cells to cause cytotoxicity of colon epithelial cells or enterotoxicity within Modified from: Vibrio parahaemolyticus cell biology and pathogenicity the host (right panel). determinants. Broberg C.A. et al. Microbes Infect. 2011, Nov; 13(12-13): 992–1001. Published online 2011 Jul 7. doi: 10.1016/j.micinf.2011.06.013 Functions of effector proteins Type III Secretion Systems and Disease, Coburn et al, Clinical Microbiology Reviews 20(4):535- 49 · November 2007. Type IV Secretion Resembles Conjugation Systems § The type IV secretion system (T4SS) is an evolutionary modification of a conjugation pilus that secretes proteins only, or proteins plus DNA. The T4SS allows bacterial pathogens to secrete proteins directly from their cytoplasms or from their periplasms. Found in Agrobacterium tumefaciens and Bordetella pertussis 67 Nice recap video by Vincent Racaniello Bacteria toxins: exotoxins, endotoxins and membrane-damaging toxins § https://www.youtube.com/watch?v=mjm5mjBVceo § 13:31 min Case study #6 Secretion systems with François-Xavier Campbell-Valois That’s all folks! 69