Introduction to Pathogens PDF

Introduction to Pathogens Session 2 David Everly Associate Professor [email protected]  Objectives Define the general characteristics of bacterial cells and how they are classified. Describe the structural components of b...

Introduction to Pathogens Session 2 David Everly Associate Professor [email protected]  Objectives Define the general characteristics of bacterial cells and how they are classified. Describe the structural components of bacteria that are important for immune regulation and growth. Describe the general characteristics of viruses and the prototypical viral life-cycle. Describe how viruses are sensed by the immune system. Describe the general characteristics of fungi and parasites and how they cause infections.  Microbiology Resources on D2L Supplemental Lectures – Basic Microbiology I and II – Introduction to Virology I and II – Microbiology Lab Techniques I and II Pathogen Review Slides (right) – All the pathogens in the course going forward. – Reviews the 8 questions for each pathogen.  Symptoms and Pathogenesis of Infectious Disease Pathogen Growth – Anti-infective Agents Therapeutic antibodies Immune Response – Bolster/Shift Immune Response Vaccination Increase immune response Cell Death and Tissue Destruction – Decrease Inflammation – Manage pain  Medically Important Qualities of Bacteria Cell Wall and Beyond – Attachment to body surfaces and sensing by the immune system. Pathogenesis Factors – Qualities that induce disease. Targets of Antibiotics (Sessions 31-32, (Pharmacology P2 Course) – Metabolic and biochemical pathways that can be inhibited therapeutically.  Introduction to Bacteria Size varies by species. – 0.1-3 μ long or more – 1 μ can be seen at 100x magnification via light microscopy. http://academic.pgcc.edu/~kroberts/Lecture/Chapter%2013/size.html  Eukaryotic vs. Prokaryotic Cells* Eukaryotic (animals) Prokaryotic (bacteria) Membrane-bound No membrane-bound compartments. compartments. – Nucleus, mitochondria, – Oxidative phosphorylation endoplasmic reticulum, Golgi occurs on the cell membrane. No cell wall. Cell wall Cholesterol in the cell No sterols in the cell membrane. membrane. 80S ribosome 70S ribosome – 60S and 40S subunits – 50S and 30S subunits – 16S rRNA sequence is used for classification. (*There are some exceptions in some species)  Characteristics of Bacteria that are Important for Classification Shape – Cocci, bacilli, spiral, or other Cell wall (Gram stain) – Gram positive, negative, or neither Other factors. Growth properties. – Aerobic/anaerobic. – Positive or negative selection on specific media. Antibiotic sensitivity. Characteristic growth in the presence or absence of nutrients or minerals.  Bacterial Shape and Morphology Cocci (Round) Bacilli (Rod) https://upload.wikimedia.org/wikipedia/commons/6/69/Bacterial_morphology_diagram.svg  Bacterial Cell Wall Provides structure and protection from osmotic pressure. Key distinguishing factor for different bacteria. Unique enzymes that synthesize the cell wall are inhibited by antibiotics. Contains unique factors that allow for binding and adhesion to cells and surfaces. Induces innate immune reactions and can act as immune evasion factors.  Cell Wall, Gram Stain* Gram stain is the most widely used diagnostic stain to help differentiate most bacteria. – Gram + (PURPLE) Cells retain the primary stain following destain. – Gram – (PINK) Cells are destained and stained with a counter-stain. – Some species do not gram stain however. Gram stain is used in conjunction with other diagnostics to identify specific species. – Shape, growth, etc. will identify specific species. *Specific discussion in Micro Lab Techniques supplement lecture Bacteria Cell Wall https://microbewiki.kenyon.edu/images/0/05/Cell_wall.jpeg  Gram Positive/Negative: Cell Wall Structures Gram Positive Gram Negative 20-30 nm thick 8-12 nm thick Thick peptidoglycan Thin peptidoglycan layer – Few layers – Multiple layers – No teichoic acid – Teichoic acids often present Two membranes One membrane – Periplasmic space between membranes Virtually no LPS Lipopolysaccharide (LPS) No porins Endotoxin Exotoxins Porins Exotoxins  Gram Positive/Negative: General Qualities Gram Positive Gram Negative Resistant to physical Sensitive to physical disruption disruption Sensitive to lysozyme Resistant to lysozyme Sensitive to β-lactams and Sensitive to tetracyclines sulfonamides antibiotics aminoglycosides antibiotics Susceptible to anionic Resistant to anionic detergents detergents Resistant to drying Sensitive to drying  Gram Positive: Peptidoglycan Provides structure and rigidity. Alternating glycan backbone. – N-acetyl muramic acid – N-acetyl glucosamine Tetrapeptide cross- bridges. – D- and L- amino acids Several components not synthesized by animal cells. – Muramic acid and D- amino acids https://en.wikipedia.org/wiki/Peptidoglycan  Gram Positive: Teichoic Acids and Cell Wall Proteins Teichoic and teichuronic acids and cell wall- associated proteins. – Mediate binding and invasion of host cells. – Regulate immune function. Activate PAMP receptors – Aid in metal scavenging. – Direct autolytic enzymes Cell wall digestion and synthesis. Nature Reviews Microbiology 12, 49–62 (2014) S. aureus MSCRAMMs In addition to LTA or WTA, S. aureus and many other pathogens have numerous proteins that bind cellular receptors and/or extracellular matrix proteins to facilitate colonization and infections. These act as major class of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), collagen-binding adhesin (CNA), pathogenesis factors. fibronectin-binding proteins (FnBPs), lipoteichoic acid (LTA), wall teichoic acid (WTA). Nature Reviews Microbiology volume 16, pages397–409(2018)  Gram Negative: LPS Lipopolysaccharide (LPS) is an outer membrane lipid anchored polysaccharide. – Membrane anchoring lipid tails – Inner and outer core Oligosaccharides – O-antigen Major antigenic determinant. Repeating oligosaccharide units. Variable composition between species and sometimes strains. Important for structural integrity. Wikipedia, Mike Jones  Gram Negative: LPS Induces very strong innate immune signaling and activation. – Intracellular and extracellular immune sensors. LPS is also called endotoxin. Nature Reviews Immunology 9, 692-703 (October 2009)  Pili and Fimbriae Filamentous protein structures. flagella Hair-like projections. Important for bacterial sexual conjugation. Mediate adhesion to host cells. – Specific molecular interactions with cellular proteins. – Induce signaling changes in the host cells. fimbriae – Especially epithelium. Major class of pathogenesis factors. http://proteus-mirabilis.com/ – Critical for mucosal infections. – GI, urinary tract, and lung.  Flagella Large macromolecular motors which give bacteria the ability to swim. – Embedded in the cell membrane and wall. – Spinning protein filaments. Virulence determinants. – Urinary tract infections require bacteria to swim against the flow of urine. – Components of the flagella induce innate immune http://phys.org/news/2010-03-molecular- reactions. bacterial-flagellar-nano-motor.html © FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]  Beyond the (Cell) Wall A number of bacteria have specific structures beyond their cell wall. – Glycocalyx, “slime” Not well-defined. Loosely adherent. Non-uniform in density and thickness. – Capsule Well-defined. Condensed layer. Closely surrounds cell wall. Usually polysaccharides. Pathogenesis factors and immune regulators. – Blocks phagocytosis. – Targets of vaccination.  Glycocalyx and Biofilms Biofilms are composed of many bacteria living together on a surface within a matrix, e.g. glycocalyx. – Occur in tissues, instruments, environment, or industrial settings. Durable and resistant to antibiotics, disinfectants, and host immune responses. Biofilms go through a cycle of development and maturation. – Attachment, growth, maturation, and dispersal. Important for recurrent infections. Can undergo intercellular communication and coordinated gene expression. – Quorum sensing. – Induce coordinated secreted signals. Staphylococcus aureus biofilm – Regulation of pathogenesis genes. on an indwelling catheter En.wikipedia.org  Capsules Can be visualized by negative stain. – Non-polar and doesn’t bind most stains. – Halo around the bacteria. Protective against phagocytosis. Generally non-immunogenic. – Some attempts to induce immunity using capsule components. Several common bacteria produce capsules. – Streptococcus pneumoniae – Klebsiella pneumoniae – Haemophilus influenzae – Pseudomonas aeruginosa – Neisseria meningitidis – Cryptococcus neoformans http://academic.missouriwestern.edu/jcbaker/bio251sec01/DSC00537.JPG  Tissue Injury from Infection Pathogen Host Degradative Enzymes Innate immunity – DNases – Cytokines can stress and – RNases damage cells. – Collagenases – Enzymes/reactive chemicals – Proteases (many substrates) e.g. reactive oxygen – Hyaluronidases Adaptive immunity Exotoxins – Antibodies and T cells can induce cell damage. – Secreted proteins that damage cells Chronic inflammation and – Superantigens induce resulting mechanisms can polyclonal T cell activation lead to loss of function. Type 3 Secretion Systems Apparatus that bacteria use to directly inject toxic proteins into cells. eLife 2018;7:e39514 DOI: 10.7554/eLife.39514 Pathogens with Type-3 Secretion Systems (T3SS) -Yersinia spp. (Yop proteins) -Salmonella spp. (many proteins) -EPEC/EHEC (many proteins) -Bordetella spp. (BopC) -Shigella spp. (Ipa proteins and others) -Pseudomonas aeruginosa (Exo proteins) -Burkholderia pseudomallei (Bop proteins) -Vibrio spp. (Vop proteins) -Chlamydia spp. (Inc proteins) Clin Microbiol Rev. 2007 Oct; 20(4): 535–549. eLife 2018;7:e39514 DOI: 10.7554/eLife.39514 A-B Toxins Produced and secreted by bacteria and have effects at distant sites. “A” is the active component Normally an enzymatic activity “B” is the binding component Binds to the cell surface via a cellular receptor and facilitates the entry of A, at the surface or internal. Neutralizing antibodies and/or antiserum are critical to neutralize A-B toxins to inhibit the toxin activity in circulation. Copyright © Gary E. Kaiser  A-B Toxins TOXIN (subunit arrangement*) ENZYMATIC ACTIVITY BIOLOGICAL EFFECTS Activates adenylate cyclase; ADP ribosylates eukaryotic increased level of intracellular Cholera toxin (A-5B) adenylate cyclase Gs regulatory cAMP promote secretion of fluid protein and electrolytes in intestinal epithelium leading to diarrhea Inhibits protein synthesis in animal Diphtheria toxin (A/B) ADP ribosylates elongation factor 2 cells resulting in death of the cells Blocks inhibition of adenylate ADP ribosylates adenylate cyclase cyclase; increased levels of cAMP Pertussis toxin (A-5B) Gi regulatory protein affect hormone activity and reduce phagocytic activity ADP ribosylates adenylate cyclase E. coli heat-labile toxin LT (A-5B) Similar or identical to cholera toxin Gs regulatory protein Inactivates the mammalian 60S ribosomal subunit and leads to Glycosidase cleavage of ribosomal inhibition of protein synthesis and Shiga toxin (A/5B) RNA (cleaves a single Adenine base death of the susceptible cells; from the 28S rRNA) pathology is diarrhea, hemorrhagic colitis (HC) and/or hemolytic uremic syndrome (HUS) *Subunit arrangement: note many of the toxins have different ratios of A and B subunits, e.g. A-5B has one A subunit and 5 B subunits.  A-B Toxins (cont.) TOXIN (subunit arrangement) ENZYMATIC ACTIVITY BIOLOGICAL EFFECTS Inhibits protein synthesis in ADP ribosylates elongation factor-2 Pseudomonas Exotoxin A (A/B) susceptible cells, resulting in death analogous to diphtheria toxin of the cells Inhibits presynaptic acetylycholine Zn++ dependent protease acts on release from peripheral cholinergic Botulinum toxin (A/B) synaptobrevin at motor neuron neurons resulting in flaccid ganglioside paralysis Zn++ dependent protease acts on Inhibits neurotransmitter release Tetanus toxin (A/B) synaptobrevin in central nervous from inhibitory neurons in the CNS system resulting in spastic paralysis Combined with the B subunit (PA), Metallo protease that cleaves LF induces cytokine release and Anthrax toxin LF (A2+B) MAPKK (mitogen-activated protein death of target cells or kinase kinase) enzymes experimental animals Calmodulin-regulated adenylate Increases cAMP in phagocytes cyclases that catalyze the leading to inhibition of Bordetella pertussis AC toxin (A/B) formation of cyclic AMP from ATP phagocytosis by neutrophils and and Bacillus anthracis EF (A1+B) in susceptible cells, as well as the macrophages; also causes formation of ion-permeable pores hemolysis and leukolysis in cell membranes Cleaves desmoglein 1, a cadherin Separation of the stratum found in desmosomes in the granulosum of the epidermis, Staphylococcus aureus Exfoliatin B epidermis between the living layers and the (also a superantigen) superficial dead layers.  Membrane Damaging Toxins Proteases Hemolysin Phospholipases – Lyse RBC – e.g. C. perfringens – Sometimes can lyse other phospholipase cells too. a.k.a. alpha factor Leukocidins Destroys blood vessels. – Attack and kill WBC Stops influx inflammatory Prevent phagocytosis cells. Release and rupture Creates anaerobic lysosomes environment. – Hydrolytic enzymes can Allows growth of this strict do further tissue damage anaerobe. Pore forming toxins  Pore-Forming Toxins Toxin Bacteria Target Disease Perfringolysin O Clostridium Gas gangrene Cholesterol perfringens UTI (urinary tract Hemolysin Escherichia coli Cell membrane infection) Listeria Systemic; Listeriolysin Cholesterol monocytogenes meningitis Anthrax EF Bacillus anthracis Cell membrane Anthrax (edema) Alpha toxin Staphylococcus Cell membrane Abscesses aureus Streptococcus pneumonia; otitis Pneumolysin Cholesterol pneumoniae media Streptococcus Streptolysin O Cholesterol Strep throat pyogenes Staphylococcus Leukocidin WBC membrane Pyogenic infections aureus  Hemolysis Blood agar plates determine the ability of bacteria to lyse red blood cells (RBC). – Normally sheep or horse blood. Alpha Hemolysis: – Partial lysis of RBC but membrane remains intact. – Colonies appear green or brown. Beta Hemolysis: – Complete lysis and digestion of RBC. Y tambe, Wikipedia – Zone of lysed RBC surrounding colonies. Gamma Hemolysis: – Non-hemolytic. – Colonies have normal appearance.  Commensal versus Pathogenic Bacteria Commensal (Benefical) Pathogenic (Harmful) Beneficial for the host Harmful to the host – Digestion. – Structural or metabolic Breakdown and/or products that induce damage absorption of specific to cells. nutrients. Immune reactions, – e.g. inflammation, etc. – Immune development and function. Bacterial endotoxins and exotoxins. Mucosal and systemic affects. – Growth of a commensal bacteria in an abnormal site – Protection from pathogenic can induce pathology. strains. – Bacteria grow to abnormal Actively or passively. levels. – Many other benefits are Proper immune response to being explored. control bacterial growth is not induced.  Endospores Some bacteria have an alternative life cycle in which they become dormant. – Can be dormant for many, many years. Up to 25-40 million years has been claimed. – Science (1995) 268:5213, pp. 1060-1064 – Bacillus and Clostridium species are medically relevant. Spores can be visualized By Y tambe (original uploader) - Own work, CC BY-SA 3.0, using a modified gram stain. https://commons.wikimedia.org/w/index.php?curid=49530 – Green structures (right)  Endospore Structure and Stability Highly stable structure. – Double membrane, cortex (peptidoglycan), and coat. Resistant to adverse conditions, including many of those used for disinfection. – Resistant to High/low temperatures, solvents, dry conditions, detergents, UV light, etc. – Can be inactivated by bleach, sterilization (autoclaving), and alkylating agents.  Introduction to Viruses Varies by virus. – 15 nm to 300 nm – Too small to be seen via light microscopy. http://academic.pgcc.edu/~kroberts/Lecture/Chapter%2013/size.html  What is a Virus? Obligate intracellular parasites All viruses utilize mRNA (+sense RNA) to DNA(+/-) translate proteins Dependent on host cells for most processes – Metabolism and energy production (ATP) – Protein synthesis, modification, and trafficking mRNA(+) – Anabolic pathways to produce or modify molecular building blocks Amino acids, lipids, sugars, nucleotides*, etc. Viruses must have mechanisms to – Replicate their genetic material.* Proteins – Assemble and release new viral particles.* *Virus encoded enzymes have been successfully targeted pharmacologically with antiviral compounds  Virus Structure Core – DNA/RNA double-/ single-stranded, positive/negative sense – Associated proteins Capsid – Protein shell Helical or icosahedral Tegument or matrix proteins* – Protein layer between capsid and http://www.virology.ws/2009/03/06/virus-images-at-viperdb/ envelope – Facilitates entry and assembly Envelope* – Lipid bilayer derived from infected cells – Contains viral glycoproteins for cell binding and entry *lacking in some groups Linda M Stannard, 1995.  Viral Life Cycle Reservoir Transmission Transmission Binding Entry Replication Assembly Release Gao H et al. PNAS 2005;102:9469-9474 Sensing of Viral Replication by the Immune System Viral Viral dsRNA dsDNA Cell Stress Innate Immune Danger/Damage Signals Activation Membrane Perturbations  Alternative Infection Outcomes Productive Infection One-step growth curves of viral isolates – One or more rounds of infection that produces bursts of infectious progeny virions. Chronic Infection* – Low level of productive infection over an extended period of time. Latent Infection* – Non-productive infection where genome persists within the infected cell without replicating. – After variable times the virus may initiate productive infection from the latently infected cells. Abortive Infection – Non-productive infection where the infected cell fails to produce progeny virions and the infected cell often undergoes apoptosis. *Both chronic and latent infection are sometimes referred to as persistent infections. PLoS Pathog 5:5 (2009) e1000412  Chronic Infection Chronic Infection (e.g. HCV, HBV) – Productive infection can lead to chronic infection. – Virus replicates at a low but detectable level over many years or decades despite the development of an immune reaction. Chronic infection can be difficult to target therapeutically. – Activate anti-viral immunity to eliminate the chronically infected cells. – Current approaches also include replication inhibitors. Nature Medicine 19, 859–868 (2013)  Latent Infection Latent Infection – Herpesvirus latency Distinct viral gene expression to maintain viral genome and infected cell Latency occurs in a specific cell type for each herpesvirus. – Dormant latency Infected cells that harbor the virus without viral gene expresion, e.g. HIV and HPV. Underlying mechanisms are unclear. Latent infection is difficult to treat. – Preclinical strategies induce viral replication in the presence of anti-viral drugs. – HIV and herpesvirus drugs can be prescribed to suppress of viral replication. http://classes.midlandstech.edu/carterp/Courses/bio225/chap19/lecture8.htm  Fungi not Fun Guy What/who is a fungi Eukaryotic Divides by mitosis or meiosis No chlorophyll Has cell wall – Chitin and glucans – No peptidoglycan Filamentous structures Spore producing – Haploid and diploid. – Differ from bacterial spores. Most are saprophytic – Living on dead or decomposing matter. ThinkOutLoudApparel  Fungi versus Bacteria Property Fungi Bacteria Cytoplasmic Membrane Sterols present Sterols absent Cell Wall Glucans Peptidoglycan Mannans Muramic acid Chitin Cross-linking peptides Glucosamine Metabolism Heterotrophic Obligate and facultative -Aerobic or facultative aerobes and anaerobes. anaerobes. -No autotrophs or obligate anaerobes.  Fungal Infections Inoculum size or repeated Superficial mycoses exposure are critical – Outermost layers of skin factors to establish and hair. infection. Cutaneous mycoses Only Candida species are – Epidermis and/or hair and considered normal flora. nails. Mycotic diseases are Subcutaneous mycoses usually not contagious – Dermis and subcutaneous but are typically acquired tissues. from the environment. Systemic mycoses – Normally originate in the lungs and spread to other organ systems.  Anti-Fungal Immunity High resistance to fungal infections. – Epithelial barrier. – Cell sloughing, ciliated layers, pH, etc. – Normal microbiology flora. Innate immune mechanisms. Cell-mediated immunity. – Especially T cells. Nature Reviews Immunology 11, 275-288 (April 2011)  Parasitology Parasites are generally divided into three categories: – Protozoans Single-celled, nucleated eukaryotes. – Helminths Metazoan worm-like organisms – Arthropods (not covered in this course) Ticks or insects http://parasite.org.au/para- site/introduction/introduction.html  Helminths Metazoan multicellular organisms. Size from 1 cm to 10 m in length. Covered by a cuticle or tegument to protect from digestion and environmental stress. Can be different sexes or hermaphroditic. Three Classes: Nematodes – Roundworms Cestodes – Tapeworms Trematodes – Flukes https://www.autism.com/statement_helminths  Helminth Life Cycle Life cycle includes several stages. – Egg – Larva Can be multiple stages. Sometimes a cyst. – Adult Transmission can be from several stages. – Ingestion of eggs or larvae. – Skin penetration by larvae. – Inoculation from insect bite with larvae. By SuSanA Secretariat - https://www.flickr.com/photos/gtzecosan/15834812242/in/set-72157648845463939, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=36876123  Helminth Transmission Infection Course Factors that favor Many are asymptomatic. completion of the life-cycle – Especially if the number of and transmission. parasites is small. – Warm climate Often chronic in nature. – Poor sanitation – Can last many years. – Lack of clean water – Some worms have a long life- – Poor housing span.  Helminth Host Response Tissue destruction Hypersensitivity reactions. Often presents with eosinophilia. – Th2 response. – Response to tissue migration. – Located in tissue or blood. – Correlates with the parasite burden. – May be absent is parasites are well contained. – Absent if confined to the lumen. Ascaris and tapeworms. – Not in response to protozoa infection.  Parasite Immune Evasion Antigenic variation of surface protein expression. – Recombination or differential gene expression. – Trypansosomes, malaria, giardia. Camouflage-binding host proteins on their surface. – Schistosomes. Cleavage of antibodies or complement. – Amoebae, leishmania. Immune suppression or redirection. – Malaria, leishmania, schistosomes.