Bacteria 9-9-24.pdf

Bacteria Hongwei Yu, PhD Department of Biomedical Sciences [email protected]  Learning Objectives Describe the bacterial structure, Gram stain, cell walls, functions, and how these structures activate macrophages causing cytokine production. Understand stru...

Bacteria Hongwei Yu, PhD Department of Biomedical Sciences [email protected]  Learning Objectives Describe the bacterial structure, Gram stain, cell walls, functions, and how these structures activate macrophages causing cytokine production. Understand structure and function (cell walls, composition, synthesis, mechanism of action of cell wall antibiotics, appendages, virulence factors, ) Know the b-lactam antibiotics resistance mechanisms Know the similarities and differences of cell walls and cell envelopes between Gram negative and positive bacteria Know the bacterial species with unique cell walls  Bacteria defined n Bacteria are unicellular prokaryotes and lack both nuclei and membrane-bound organelles. n Nuclear materials are free inside cells. n No mitochondria, but have electron transport chain in the cytoplasmic membrane (aerobe). n Most but not all bacteria have cell walls. n Bacterial cell walls are unique, e.g., peptidoglycan. n Target for immune system n Target for antibiotics n Differentiation of bacteria “Every bacterium’s wish is to be bacteria”  Bacterial Structure Thick cell wall b-lactamase  Bacterial cell wall: Gram stain n Stain cells with Gram stain (crystal violet) n Treat cells with a mordant dye, Gram’s iodine which forms a complex with crystal violet. n both G+ and G- would appear blue under the microscope n Wash cells with alcohol. n Alcohol has the effect of dehydrating the cell wall esp. peptidoglycan layer. n CVI is effectively trapped inside in gram+ n Counterstain with safranin (pink- red). n G+ cells are blue-purple and G- cells are pink-red  Gram positive cell wall n Key Features: n Thick peptidoglycan overlaying plasma membrane which retains the gram stain. n Lipoteichoic acid (LTA, vertical) n Plasma (Cytoplasmic) membrane  What is peptidoglycan? n Complex macromolecule consisting of glycan cross-linked by peptide chains. n Mesh or network or sacculus, outside cytoplasmic membrane n Only found in bacteria n Glycan chains consist of alternating acetylated sugars n N-acetylmuramic acid (NAM) n N-acetylglucosamine (NAG) n b-1,4 linkages (breakdown by lysozyme) n Peptide chains coupled to NAM only n Penta-glycine bridges present only in gram positives  Peptidoglycan functions (1) Allows diffusion of small molecules to the plasma membrane (2) confers rigidity and shape to bacterial cell (3) interferes with phagocytosis and is mitogenic (4) Site of action some antibiotics and enzymes Penicillin, cephalosporins, vancomycin and lysozyme  Peptidoglycan biological activities § Muramyl dipeptide (MD, NAM-A-G) is a product of peptidoglycan degradation in the host, and is PAMP recognized by intracellular NOD-2 (PRR) and activates production of inflammatory cytokines § adjuvant (stimulates immune response to an antigen) § mitogen (stimulates cell division) § pyrogen (fever-causing) § somnagen (sleep-inducing)  Peptidoglycan Layer Synthesis 1. Before PG cross-linking, PG chains feature NAM moieties connected to 5-amino-acid peptides M (ordered A-G-L-A-A). The last two amino acids G M (dipeptide, D-ala-D-ala) is required for cross- G M M linking, which is similar to structure of a penicillin. G M The 3rd amnio acid must be a di-amino acid such as lysine, or diaminopimelic acids (DAP). M 2. During PG cross-linking, transpeptidase (a type of PBP, Penicillin-Binding Protein) removes the terminal A and links the 2nd A to neighboring L, PG = peptidoglycan thus forming a connected latticework (“—” G = N-acetylglucosamine (NAG) between A and L) M = N-acetylmuramic acid (NAM) A = L-alanine 3. After PG cross-linking, the resultant scaffold G = D-glucosamine provides structural integrity to the bacterial cell L = L-lysine A = D-alanine = Cross-links β-lactams antibiotics are structural analogs of the D-Ala-D-Ala substrate and covalently bind to the transpeptidase active site to block its activity → Cell wall breakdown > cell wall synthesis → autolysis (bactericidal)  Antibacterial therapy target b Lactams  Penicillin (b-lactams) Resistance § Altered penicillin binding proteins (major mechanism in Gram+ bacteria) o Results from genetic mutations, MRSA (PBB2a); Penicillin-resistant S. pneumonia produces altered PBP2x. § Reduced bacterial penetration o Gram negative bacteria: poor penetration, decreased number of porins § b-lactamase enzymes, present in both G+ and G-ve bacteria (but a major mechanism in G-ve bacteria)  β-Lactamases Ribbon structure of β-lactamase n A family of enzymes synthesized in cytoplasm and secreted to periplasm in Gram-negatives. Gram-positives synthesized b- β-lactam drug lactamases in cytoplasm and secreted extracellularly. n Most are on plasmids, sometimes can be on chromosome n “AmpC” β-lactamases may be encoded on chromosome of “SPACE” Gram-negative organisms (Serratia marcescens, Pseudomonas aeruginosa/Proteus spp., Acinetobacter spp., Citrobacter spp., Enterobacter spp.) n Three types of β-lactamases: 1. Penicillinases inactivate penicillins 2. “Extended-spectrum β-lactamases” (ESBLs) inactivate most β-lactams except for carbapenems 3. Carbapenemases (e.g., OXA, KPC, metallo-β-lactamases) inactivate carbapenems Hydrolysis of β-lactam ring inactivates the drug Gram-negative cell envelope Key Features (1) Unique outer membrane which contains LPS (outermost layer) (2) Thin peptidoglycan overlaying plasma membrane (3) Periplasmic space (4) Inner (plasma) membrane  GRAM NEG OUTER MEMBRANE n Outer membrane structure: a lipid bilayer composed of phospholipids and a complex molecule called lipopolysaccharide (LPS). n LPS is in outer leaflet only. n Inner leaflet is phospholipids n LPS is composed of O-antigen + core polysaccharide + lipid A (most toxic part), triggers IL-1, TNF, and IL-6 release by macrophages. Binds to Toll-Like Receptor (TLR4) along with CD14 on macrophages. When large amounts of macrophages activated in this manner, gram- septic shock. n OMPs are antigenic, and porins also present.  LPS COMPONENTS: LIPID A n Lipid A portion is responsible for endotoxic activity. n anchors LPS molecule in the outer membrane n Outer leaflet of outer membrane. n Lipid A is a phosphorylated disaccharide with esterified fatty acids.  LPS COMPONENTS: CORE n The Core Polysaccharide contains 9-12 sugars n An unusual eight carbon sugar 2-keto-3- deoxyoctulosonic acid (KDO) n Core polysaccharide is essential for LPS structure and bacterial viability  LPS COMPONENTS: O ANTIGEN n O antigen is a long linear polysaccharide (50- 100 repeating units) attached to the core n O antigen extends to the exterior of the cell n Basis for serotype of bacterial strains and therefore useful for identification. n Example: EHEC O157:H7  LPS BIOLOGICAL PROPERTIES n Powerful immune stimuli, but induces a weak antibody response (poorly antigenic), no vaccine. n LPS is shed into bloodstream during bacteremia (bacterial lysis or blebs from living cells different from exotoxins) n complement and coagulation cascades are also activated. n LPS activates B cells and induces macrophages and dendritic cells to release IL-1, IL-6 and TNF and other factors via binding to CD14 and TLR4 (PRR) n Heat-stable. Fever, shock (hypotension), DIC.  Other outer membrane structures § Porins are protein complexes that form channels that allow passive diffusion of small (less than 600 MW) charged molecules. § Found only in outer membrane of G- bacteria.  Outer membrane functions (a) serve as permeability barrier to hydrophobic or large molecules (like lysozyme and some antibiotics); (b) maintain structural rigidity of the cell (c) protects peptidoglycan and plasma membrane (d) sieve for small water-soluble molecules (e) presents sites for host cell and phage attachment (f) possesses LPS and other molecules that serve as molecular signals to host organism  Gram negative peptidoglycan DAP: diaminopimelic acid n 1-2 layers, much thinner than G+ n Glycan (M-G-M-G-M-G) polymer is cross-linked via direct covalent bond between terminal D-ala of one tetrapeptide and either lysine or DAP of another tetrapeptide n So, no pentaglycine bridging n Less overall crosslinking, so more porous than G+. n Floats within periplasmic space.  Periplasmic space (gel) q contains a variety of hydrolytic enzymes including proteases, lipases, nucleases, and components of sugar transport systems. q Gram negative pathogens can also have collagenases, hyluronidase and b-lactamases. q Contents of the periplasm can be released by shedding off the outer membrane but cellular delivery mechanisms are unknown. q Plasma or inner membrane is similar to G+ plasma membrane.  Bacterial ribosomes n Site of protein synthesis in bacteria n Two subunits: 50S and 30S n Different from ribosome in eukaryotic cells, allows selective toxicity of antibiotics n Site of action of antibiotics n Tetracyclines: bind to 30S subunit n Aminoglycosides: interferes with 30S protein synthesis n Macrolides inhibit the 50S ribosomal subunit  Plasma (cytoplasmic) membrane n Lipid bilayer similar in structure to euk. membranes n Has permeases that are required for active transport n Has respiratory chain enzymes (involved in ATP generation) and proton pumps n sites for the synthesis for the biosynthesis of peptidoglycan and outer membrane components (like LPS in g- organisms)  Surface Appendages-1 § Pili (fimbriae): § thin, rigid appendages composed of pilins § allow cells to adhere to host cell or other bacterial cells. § Neisseria pili are required for adherence to host epithelia/endothelia. N. Gonorrhea pili mutation: antigenic variation. § Gram negative F pilus is required for conjugation § E. coli (UTI/pyelonephritis, P pili)  Surface Appendages-2 § Flagella are required for movement of cell, toward a nutrient or away from a toxic substance in a process called chemotaxis. § Flagella are primarily composed of one protein called flagellin. § Flagellar antigen is designated as H, useful in strain identification. § E. coli O157:H7  Capsule (glycocalyx): n thick viscous layer to thin slime layers n gives colony a smooth or shiny appearance on agar n confers resistance to phagocytosis n Streptococcus pneumoniae capsule n Polysaccharides in capsule are weakly antigenic, and induces IgM and fails to cause class switching. n usually polysaccharide, but occasionally protein n Bacillus anthracis capsule is poly-D-glutamate polypeptide  Bacterial Spores n Some Gram-positive bacteria can enter a dormant state of growth called spore when nutrients are limited. n Resistant to heat and chemicals. n No metabolic activity n Can survive long periods of starvation n Must autoclave to kill spore by steam at 121 C for 15 min. n Endospores germinate into growing cells when adverse conditions wane n Dipicolinic acid, an abundant compound within the endospore, may stabilize and protect the DNA against damage. n Spore-forming bacteria n Bacillus anthracis (anthrax) and B. cereus (food poisoning) n Clostridium botulinum (botulism), C. difficile (pseudomembrane colitis), C. perfringens (gas gangrene), and C. tetani (tetanus)  Comparison of Gram Negative and Gram Positive Cell Envelopes Gram Negative Gram Positive Outer membrane Present Absent Peptidoglycan Thin Thick Lipopolysaccharide (LPS) Present Absent Endotoxic properties Present (LPS) Some (LTA) Teichoic Acids Absent often present Spore formation None Some strains Exotoxin production Some strains Some strains Capsule Some strains Some strains Lysozyme Resistant Sensitive  Bacteria with unique cell walls n Mycoplasma n No cell wall n Does not gram stain n Cell membrane has sterols for extra stability n Mycobacteria n Cell wall has mycolic acid (long hydrocarbon, waxy) n Does not gram stain well n Special stain (Ziehl-Neelsen) n Chlamydia n Lacks muramic acid