Lecture 2 & 3 010908 011108

Lecture 2 & 3 010908 011108 - MMG 461: Molecular...

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Unformatted text preview: MMG 461: Molecular Pathogenesis Prokaryotic Structure and Function M. H. Mulks Lectures 2 & 3 - January 9 & 11, 2008 Prokaryotic versus eukaryotic Eukaryotic = true nucleus Prokaryotic = primitive nucleus Eukaryotic organisms Animals, including vertebrates, arthropods, reptiles, amphibians, avians, echinoderms, roundworms, etc. Plants, including unicellular green algae up through fruits, vegetables, coniferous and deciduous trees, etc. Fungi, including yeast, molds, mushrooms Protista, or unicellular eukaryotes, including protozoa (amoebas, euglenoids, ciliates, etc.), slime molds, red and brown algae Composite eukaryotic cell Composite prokaryotic cell Eukaryotic cells a true, distinct nucleus, with nucleoli, bounded by a nuclear membrane individual linear chromosomes within the cell nucleus, complexed with histones membrane-bound organelles in the cytoplasm, such as mitochondria, lysosomes, Golgi complexes, and chloroplasts Eukaryotic cells complex internal membranes, smooth and rough endoplasmic reticulum 80S ribosomes, often bound to internal membranes cytoskeleton with microtubules, microfilaments, and cytoplasmic streaming Eukaryotic cells cell division by mitosis, with mitotic microtubular spindle sexual reproduction with rearrangement and division of chromosomes during meiosis Prokaryotic organisms Eubacteria = true bacteria all known bacterial pathogens belong to this group Archaebacteria or Archaea - include methanogens, extreme halophiles, and thermo-acidophiles live in extreme environments and carry out unusual metabolic processes, e.g., production of methane Prokaryotic cells a single circular chromosome that is not contained within a distinct membrane-bound nucleus no nucleus, nucleoli, or nuclear membrane at most very simple internal membranes, so no endoplasmic reticulum Prokaryotic cells no membrane-bound internal organelles, therefore no mitochondria, no Golgi, no chloroplasts, no lysosomes most functions of eukaryotic organelles are handled by cytoplasmic membrane in prokaryotes Prokaryotic cells 70S ribosomes, not complexed to internal membranes no cytoskeleton, no microtubules or microfilaments cell division by binary fission, no mitotic spindle no sexual reproduction or meiosis Comparison of Prokaryotic and Eukaryotic Cells PROPERTIES Groups PROKARYOTES Eubacteria, archaebacteria EUKARYOTES Algae, fungi, protozoa, plants, animals Size of cell 0.1 - 3.0 m in diameter 5 - 100 m in diameter NOTE: resolution of a light microscope is ~0.2 m . Relative sizes of eukaryotic and prokaryotic cells Comparison of Prokaryotic and Eukaryotic Cells PROPERTIES Nucleus PROKARYOTES EUKARYOTES No nuclear membrane True nucleus, with or nucleoli nuclear membrane and nucleoli Single circular chromosome, lacks histones; haploid (note - some bacteria have more than 1 chromosome) Multiple linear chromosomes, complexed with histones; haploid, diploid, or multiploid Chromosome arrangement Comparison of Prokaryotic and Eukaryotic Cells PROPERTIES Cell division PROKARYOTES Binary fission Doubling time in minutes EUKARYOTES Mitosis; microtubular spindle Sexual reproduction Unidirectional transfer Meiosis; reassortment of of DNA fragments only; chromosome complement no meiosis Rare Often in plasmids Common In organelles Introns in genes Extrachromosomal DNA Comparison of Prokaryotic and Eukaryotic Cells PROPERTIES Plasma membrane PROKARYOTES No carbohydrates; lacks sterols EUKARYOTES Sterols and carbohydrates present Complex; endoplasmic reticulum and Golgi Present: mitochondria, Golgi, lysosomes, chloroplasts, etc In mitochondria Internal membranes Simple , limited Membrane-bound organelles Absent Respiratory system Part of plasma membrane Comparison of Prokaryotic and Eukaryotic Cells PROPERTIES Ribosomes Cell walls PROKARYOTES Smaller size (70S) Usually present, rigid; contain peptidoglycan and D-amino acids No cytoskeleton or cytoplasmic streaming EUKARYOTES 80S; 70S in organelles When present, chemically simple, usually polysaccharide Cytoskeleton with microtubules and cytoplasmic streaming Cytoplasm Prokaryotic vs eukaryotic cells: What are some of the big differences? What are some of the big differences? Cell wall & peptidoglycan Lipopolysaccharide (LPS) Protein synthetic machinery Chromosome structure Lack of internal organelles Metabolism? Prokaryotic cell structure Bacterial cells contain, from the inside out cytoplasm, with DNA, ribosomes, etc., but no nucleus and no organelles and no internal membranes cytoplasmic membrane = cell membrane = plasma membrane cell wall some also have surface appendages such as capsules, flagella, pili Prokaryotic cells Bacterial cells contain, from the inside out cytoplasm, with DNA, ribosomes, etc., but no nucleus and no organelles and no internal membranes cytoplasmic membrane = cell membrane = plasma membrane cell wall some also have surface appendages such as capsules, flagella, pili The bacterial cell wall Bacterial cell wall is rigid, gives shape to cell Prevents osmotic rupture of cell Acts as a coarse molecular sieve = barrier against some large molecules but highly permeable to nutrients Unique macromolecule = peptidoglycan Gram stain = a key method of classification of bacteria = based on chemical and structural differences in cell walls Peptidoglycan = murein Polymer found only in prokaryotes, in cell walls Backbone = linear chain of two alternating sugars = glycan chain N-acetylglucosamine (NAG) N-acetylmuramic acid (NAM) Muramic acid is found only in peptidoglycan Glycan chains are cross-linked by peptide side chains Peptidoglycan structure NAM NAc L-alanine O NAG NAc O NAM NAc L-alanine O NAG NAc O NAM NAc L-alanine O NAG NAc O | D-glutamate | DAP ------------ D-alanine | | D-alanine DAP | | D-alanine D-glutamate | L-alanine | D-glutamate | DAP ------------- D-alanine | | D-alanine DAP | | D-alanine D-glutamate | L-alanine | Peptide bond D-glutamate | DAP ------------- D-alanine | | D-alanine DAP | | D-alanine D-glutamate | L-alanine NAc NAM O NAc NAG O NAc NAM O NAc NAG O NAc NAM O NAc NAG O The energy from this bond can be used in a transpeptidation reaction to form another crosslink. Peptidoglycan Attached to each muramic acid residue is a peptide side chain synthesized as a pentapeptide, usually L-alanine D-glutamate Meso-diaminopimelic acid (or lysine) D-alanine D-alanine terminal D-alanine removed during crosslinking contains D-amino acids (rare) and diaminopimelic acid (unique to peptidoglycan) Peptidoglycan precursor structure CM Cytoplasm O COCH3 HN H OH B-P-P - O C C C C C- CH2OH -1,4 linkage GlcNAc Add lactyl ether -> MurNAc Pentapeptide Attach to bactoprenol Add GlcNAc H H O H H O = C C CH3 H L-Ala D-Glu DAP D-Ala D-Ala UDP-NAM UDP NAM - AA3 (L-ala - D-glu - DAP) UDP-NAG L-ala NAG-P UMP UDP NAM - AA3 - D-ala - D-ala D-ala Cycloserine Synthesis of peptidoglycan Cytoplasmic membrane B - P - P - NAM - NAG L-ala D-glu DAP D-ala D-ala Bactoprenol-P and P Bacitracin B-P-P Periplasm or cell surface D-ala D-ala D-ala DAP D-glu D-ala D-ala DAP D-glu D-ala D-ala DAP D-glu Vancomycin Peptide bond Glycosyl bond NAM - NAG - NAM - NAG - NAM - NAG - NAM L-ala L-ala L-ala L-ala D-glu D-glu D-glu DAP D-ala D-ala D-ala D-ala D-glu DAP D-ala D-ala DAP D-glu DAP D-ala D-ala Penicillin DAP D-ala D-ala L-ala L-ala L-ala L-ala NAM - NAG - NAM - NAG - NAM - NAG - NAM Peptidoglycan structure NAM NAc L-alanine O NAG NAc O NAM NAc L-alanine O NAG NAc O NAM NAc L-alanine O NAG NAc O | D-glutamate | DAP ------------ D-alanine | | D-alanine DAP | | D-alanine D-glutamate | L-alanine | D-glutamate | DAP ------------- D-alanine | | D-alanine DAP | | D-alanine D-glutamate | L-alanine | Peptide bond D-glutamate | DAP ------------- D-alanine | | D-alanine DAP | | D-alanine D-glutamate | L-alanine NAc NAM O NAc NAG O NAc NAM O NAc NAG O NAc NAM O NAc NAG O The energy from this bond can be used in a transpeptidation reaction to form another crosslink. Prokaryotic cell structure Cell wall and peptidoglycan are unique Muramic acid DAP (diaminopimelic acid) D-amino acids Gram positive vs gram negative cells Gram stain relates to cell wall structure There are two classes of bacteria based on differences in cell wall structure as detected by Gram stain: 1. Gram positive = retains the crystal violet dye during decolorization = purple 2. Gram negative = loses crystal violet stain during decolorization, must be counterstained to be seen = pink Gram stain procedure Gram positive cell wall Two major components: peptidoglycan and teichoic acid Gram positive cell wall Thick three-dimensional layer of cross-linked peptidoglycan, anchored to the plasma ( = cytoplasmic = cell) membrane by lipoteichoic acid Covalently linked to teichoic acid Teichoic acid Unique to Gram positive cells Polymer of glycerol phosphate or ribitol phosphate to which glycosyl, D-alanyl, and other esters can be attached Teichoic acid is covalently linked to muramic acid Lipoteichoic acid = anchors cell wall to plasma membrane Teichoic acid Together with peptidoglycan, forms an anionic surface matrix that helps regulate surface charge and trafficking of ions, etc, into cell Vectorial synthesis on bactoprenol carrier lipid, similar to peptidoglycan Can be an important surface antigen and adhesin Effectively triggers the alternative complement pathway Teichoic acid structure ________Glycerol phosphate ______ | | O | O | O | | | ( - P - O - CH2 - CH - CH2 - O - P - O - CH2 - CH - CH2 - O - P - ) n | | | | | O O O O O | | NAG D-alanine Gram negative cell wall Thin layer of peptidoglycan within periplasmic space between plasma membrane and outer membrane Peptidoglycan linked by lipoprotein to outer membrane Gram negative cell wall Periplasmic space = region between inner and outer membranes, contains hydrolytic enzymes, etc. Outer membrane with lipopolysaccharide (LPS) Gram negative cell wall Outer membrane phospholipoprotein bilayer membrane inner leaflet = phospholipids outer leaflet contains lipopolysaccharide = LPS lipoprotein links OM to peptidoglycan layer many proteins, including porins, receptor proteins, etc. Major function = selective permeability barrier Lipopolysaccharide = LPS = endotoxin Found only in Gram negative bacteria Toxic to most mammals, triggers a variety of inflammatory responses, can cause endotoxic shock and disseminated intravascular coagulation (DIC) Major virulence factor in Gram negative bacteremia (sepsis) Lipopolysaccharide Lipopolysaccharide molecules have three main components: 1. Lipid A = a phospholipid containing glucosamine 2. Core polysaccharide 3. Repeating oligosaccharide = O antigen Lipopolysaccharide structure P ~~~~~| | ~~~~~| GlcN - KDO - KDO - Hep - Hep - Sugar - Sugar - Sugar - (Sugar - Sugar - Sugar)n ~~~~~| | | | | | | | | KDO P Hep Sugar Sugar Sugar ~~~~~| | | | ~~~~~| GlcN P P ~~~~~| | | | P E E | E |__________| |________________________________________________________| |_______________________| | | | Lipid A Core polysaccharide O antigen P = phosphate group, E = ethanolamine, GlcN = glucosamine, ~~~~ = fatty acid side chains, Hep = heptose KDO = 2-keto-3-deoxy octulonic acid Lipooligosaccharide Some species, such as Neisseria, do not make a repeating oligosaccharide, just the lipid A plus core polysaccharide Often termed lipooligosaccharide or LOS Serologic typing of Gram negative bacteria Traditionally uses K, O, and H antigens on cell surface to determine serotype K = capsular antigens O = repeating oligosaccharide of LPS H = flagellar antigens For example, E. coli O157:H7 is the strain that causes hemolytic-uremic syndrome Lipopolysaccharide Lipid A = a phospholipid containing glucosamine buried in outer membrane primary toxic moiety of endotoxin phosphorylated glucosamine disaccharide to which fatty acids are attached very hydrophobic conserved basic structure in eubacteria Lipopolysaccharide Core polysaccharide Contains KDO residues (2-keto-3-deoxy-octulonic acid) plus 5-8 additional sugar residues, often heptoses Acts as bridge between lipid A and repeating oligosaccharide Structure generally conserved within closely related species or genera of bacteria Lipopolysaccharide Repeating oligosaccharide = O antigen = smooth LPS highly variable within species number of repeats varies even within a single cell very hydrophilic; helps exclude hydrophobic compounds a major surface antigen of Gram negative cells structure can be modulated in response to environmental conditions Lipopolysaccharide structure P ~~~~~| | ~~~~~| GlcN - KDO - KDO - Hep - Hep - Sugar - Sugar - Sugar - (Sugar - Sugar - Sugar)n ~~~~~| | | | | | | | | KDO P Hep Sugar Sugar Sugar ~~~~~| | | | ~~~~~| GlcN P P ~~~~~| | | | P E E | E |__________| |________________________________________________________| |_______________________| | | | Lipid A Core polysaccharide O antigen P = phosphate group, E = ethanolamine, GlcN = glucosamine, ~~~~ = fatty acid side chains, Hep = heptose Lipopolysaccharide = LPS = endotoxin Causes fever and endotoxic shock Present in cell envelopes of all gram negative bacteria and NOT in gram positive bacteria Lipid A = primary toxic entity Major virulence factor in gram negative bacteria Gram negative cell wall Unique structures include lipopolysaccharide in the outer membrane (and LPS components such as KDO), periplasmic space, conserved lipoprotein that anchors peptidoglycan to OM Composite prokaryotic cell External structures Capsules Flagella Pili Capsule Loose, relatively unstructured hydrophilic layer around the cell, outside the outermost layer of the cell wall & membranes Found in many bacteria, not unique to either gram positive or gram negative species Usually polysaccharide, may be protein Usually antigenic Often important virulence factor - impedes phagocytosis & complement-mediated killing Bacterial capsule Functions of capsule Prevention of dessication Adherence Resistance to non-specific host defenses such as phagocytosis and complementmediated lysis,, because inhibits binding of complement component C3b to surface Resistance to specific host immunity Poor antibody response to capsule (e.g., Neisseria meningitidis group B) Antigenic variation Biosynthesis and export of capsule Several mechanisms of biosynthesis have been described, varying with chemical composition of capsule One conserved method involves synthesis of repeat units in cytoplasm and transport across IM attached to bactoprenol (undecaprenolPP); polymerization occurs in periplasm, and polymer is secreted through a pore in the OM Biosynthesis and export of capsule Another conserved method uses highly conserved export gene clusters, which flank variable capsule biosynthetic genes In this system, the capsule polymer is synthesized in the cytoplasm and secreted through an ABC transporter-like pore F E D U C S D C B A T M kps kfi kps Flagella Long helical protein filaments that provide motility for bacteria Complex structure with basal body inserted in cell envelope, hook structure, and flagellin Can be found on both Gram + and Gram- Number and location vary = taxonomic criterion polar, bipolar, polar tuft, peritrichous Flagella - polar tuft http://www.pbrc.hawaii.edu/~kunkel/gallery/ Peritrichous flagella http://www.pbrc.hawaii.edu/~kunkel/gallery/ Flagella Motility and chemotaxis Flagella can also be important surface antigens ("H" antigens in enterics) some bacteria can switch from production of one antigenic type of flagella to another = phase variation = mechanism to avoid elimination by host immune response Pili or fimbriae Hair-like protein projections on the bacterial cell surface Common pili = often are adhesins = allow attachment of bacteria to host cells Sex pili = involved in exchange of genetic material in some species Can inhibit phagocytosis Some can undergo phase and antigenic variation Pili vs Flagella http://www.pbrc.hawaii.edu/~kunkel/gallery/ Escherichia coli - pili http://www.pbrc.hawaii.edu/~kunkel/gallery/ Escherichia coli - common pili and sex pilus http://www.pbrc.hawaii.edu/~kunkel/gallery/ Neisseria gonorrhoeae pili Composite prokaryotic cell ...
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