W2 Microbial Structure (Reid) (1).pdf

Microbial Structure Dr. Sean D. Reid [email protected] Learning Objectives and References 1. Describe the general organization of a bacterial cell. 1.1. Identify major bacterial structures in a cartoon or electron micrograph. 1.2. Describe the composition, structure, and function of the following b...

Microbial Structure Dr. Sean D. Reid [email protected] Learning Objectives and References 1. Describe the general organization of a bacterial cell. 1.1. Identify major bacterial structures in a cartoon or electron micrograph. 1.2. Describe the composition, structure, and function of the following bacterial cell envelope components: plasma membrane, cell wall, outer membrane, periplasm, capsule, slime, pili/fimbriae, and flagella. 1.2.1 Differentiate between basic cellular functions and roles in pathogenesis. 1.3. Differentiate host mimicry, antigenic variation, and phase variation. 1.4. List structures highly conserved among genera. 1.5. List structures often useful for differentiating bacterial strains based on serotyping. 2. Differentiate 6 groups of bacteria based on cell envelope structure: Gram-positive, acid-fast, mollicute, Gram-negative, spirochete, Chlamydiaceae. 2.1 Categorize medically-relevant genera into each of the 6 bacterial groups in LO 2. 3. Compare and contrast the Gram and Ziehl-Neelsen acid-fast stains in terms of technique, basis, and expected results for each of the 6 bacterial groups in LO 2. 4. Outline the biosynthesis of peptidoglycan. 4.1. Describe the general role of PBPs in the bacterial cell. 4.2. Explain how transglycosylase and transpeptidase PBPs create the 3D cell wall structure. 4.3. Explain the importance of bactoprenol (=undecaprenol). References: Murra y 7th ed. Ch.12 Sherri s 5th ed. pp. 61-64, 347-357, a lso chapters on va rious organisms Sherri s 6th ed. Ch 21, cha pters on va rious organisms http://textbookofbacteriology.net ba sic mi cro texts, revi ew a rticles You Will Probably Diagnose and Treat MANY Infections! Madrigal, 2011 Bacterial Structure: General Organization Outer membrane Some structures conserved. Others quite variable. o May be used for differentiating strains or species (serotyping). Some absent in some genera or species. Image modified from Mariana Ruiz Villarreal Plasma Membrane Outer membrane Plasma Membrane: Structure Proteins within a phospholipid bilayer No sterols (except mycoplasmas) Contains cardiolipin o Not found in eukaryotic plasma membranes. o Where (other than bacteria) do you find it? Barrier against aqueous ions. Plasma Membrane: Function o Allows gradients, control over osmotic balance. Most of the functions come from the proteins within the membrane. o Metabolite and nutrient transport o Biosynthesis of lipids, polysaccharides, peptidoglycan Peptidoglycan = Murein ≈ Cell Wall Outer membrane PG layer differs between different types and species of bacteria. Thickness Shape Chemical structure PG PM PG Adapted from Sherris, 5th ed. Peptidoglycan: Structure www.sp.uconn.edu www.ccbcmd.edu Alternating GlcNAc (NAG) and MurNAc (NAM) Peptide tail on NAM covalently linked to tails of other strands. Forms a 3D multilayered net—porous. Peptide part can vary a bit, MurNAc can be ‘decorated,’ but in general highly conserved. Peptidoglycan: Function Essential to most bacteria. o Exceptions: mollicutes, Chlamydiaceae, some other intracell. Protects against osmotic lysis. Provides shape. o Why do bacteria have different shapes? Protects against some chemicals and large molecules. o Highly polar—some chemicals cannot pass. o Pores block passage of large molecules. Anchor site of some proteins and polysaccharides to cell Peptidoglycan: Biological Effects A PAMP (Pathogen Associated Molecular Pattern) o Unique to bacteria, present/similar in most bacteria o Induction of IL-1, IL-6, GCSF, TNF Cytotoxin—specific type of PG fragment in some organisms o e.g., Bordetella pertussis—tracheal cytotoxin, targets ciliated respiratory epithelial cells Teichoic and Lipoteichoic Acids In Gram-positives only Polymers of negativelycharged sugars o Anchored to different parts of the cell envelope o Lipo- have lipid anchor General role in regulation of autolysis Adhesins PAMPs Other roles too! Sherris, 4th ed. Outer Membrane Outer membrane Outer Membrane: Structure In Gram-negatives and Chlamydiaceae only Asymmetric o Lipopolysaccharide (LPS) outer leaflet o Phospholipid inner leaflet Contains many proteins, including Outer membrane porins (OMPs) Raetz and Whitfield, 2002 Outer Membrane: Function Selective barrier Protection against lysis Impermeable to large and/or aqueous molecules o Lysozyme o Cationic peptides o Bile salts Porins/transporters to let things through Host mimicry and antigenic variation via sugars (O-antigen) and proteins Hiding from the Host Immune System HOST MIMICRY ANTIGENIC VARIATION Microbial surface structure is antigenically similar to a host structure. If structure is abundant, host has difficulty mounting an effective immune response against the pathogen. Genetic mechanisms cause changes in certain microbial surface antigens. Acquired response no longer recognizes pathogen. (Phase variation: structure present/absent) Figueiredo Periplasm: Structure Viscous solution of proteins and solutes o Between plasma membrane and PG/outer membrane Protection against osmotic lysis via solutes Biosynthesis Periplasm: Function o Peptidoglycan, fimbriae Nutrient binding Macromolecule degradation Detoxification Chemotaxis receptors No single major function Capsules and Slime Outer membrane Capsules and Slime: Structure Long chains of sugars, sugars/amino acids, or amino acids “Glycocalyx” Capsule o Organized, tightly cellassociated o Anchored via lipid in membrane or covalently onto PG Slime o Disorganized, loosely-associated (no anchor) Some pathogens make HUGE amounts of capsule; looks like slime. Capsule: Function Antiphagocytic—major role o Negatively charged for many pathogens o Reduction of complement-mediated opsonization May permit host mimicry. One species may express many different structures; strainspecific. o ‘K’ antigen in some serotyping schemes, numbered in others, letters in others e.g., K1, K5 capsule types in E. coli related to invasive disease Vaccine target (e.g., pneumococcus, meningococcus) Can have other roles (adhesion, protection from desiccation). Slime: Function Adhesion to surfaces or cells o Biofilm formation Pili/Fimbriae Outer membrane Pili/Fimbriae: Structure and Function Short protein strands, all over cell surface Functions o Adhesion to host cell receptors (e.g., P-pili in uropathogenic E. coli) o Twitching motility (only a few species, e.g., Neisseria; Type IV pili) o Conjugation (specifically Fpilus) o Occasionally antiphagocytic o Often undergo antigenic variation. http://2010.igem.org/Team:Michigan/Project Charles C. Brinton, Jr. Flagella Outer membrane Flagellum: Structure and Function Helical protein filament Anchored in membrane(s). Made of flagellin subunits. o Called the ‘H’ antigen in serotyping. o Also a PAMP CDC A variety of arrangements Usually on rod/spiralshaped bacteria Major function MOTILITY commons.wikimedia.org Gram-Positive Thick layer of PG, no outer membrane Contains teichoic acids and proteins. Strong cell wall helps resist deformation upon desiccation. o Often see G+ in drier environments (soil, skin). Prescott’s Principles of Microbiology Gram-Positive Genera Staphylococcus, Streptococcus, Enterococcus Listeria Bacillus, Clostridium Actinomyces Mycobacterium, Nocardia, Corynebacterium There are more!!! Gram-Negative Genera Thin layer of PG Outer membrane with LPS and porins Many genera… Escherichia, Klebsiella, Citrobacter, Enterobacter, Serratia, Salmonella, Shigella, Yersinia, Proteus, Vibrio, Campylobacter, Helicobacter, Bacteroides, Pseudomonas, Burkholderia, Stenotrophomonas, Neisseria, Haemophilus, Moraxella, Acinetobacter, Bordetella, Brucella, Pasteurella, Legionella, Francisella, Kingella, Actinobacillus… G+ vs G- Cell Envelope Structures (e.g., Staphylococcus) (e.g., Pseudomonas) Gram Stain G+ Crystal violet Colors G+ and G- Iodine Complexes form. Alcohol Decolorizer Safranin Lipids washed away; peptidoglycan remains. Counterstain is visible in decolorized cells. G- Gram Stains © R. Buxton C. jejuni B. anthracis Acid-Fast G+ but more complex o Mycolic acids (waxy) C60-C90 o Arabinogalactan o Lipoarabinomannan Mycolic acids o Resist chemicals, including those found in phagolysosome and some antibiotics Mycobacterium, Nocardia (partial), Corynebacterium (very weak acid-fast, will usually stain G+) Esko, 2008 Acid-Fast Staining Don’t Gram stain well Very resistant cell wall permits specific staining techniques with very strong decolorization Acid-Fast stains o Ziehl-Neelsen stain o Kinyoun stain o Auramine stain CDC Ziehl-Neelsen Staining Procedure Acid-fast Heated Colors Aniline dye EVERYTHING. (Carbolfuchsin) Acidified Alcohol Decolorizer Washes away lipids and damages peptidoglycan. Methylene Blue Counterstain is visible in decolorized cells. Other Mollicutes Soft, pleomorphic structure-NO CELL WALL Extracellular bacteria Membrane contains sterols, acquired from host. o Give some rigidity. Very small (0.2-0.3 µm) bioweb.uwlax.edu o Can pass through most filters. o Too small to see by light microscopy. Gram stain not useful. Genera of importance are Mycoplasma, Ureaplasma. H. Lunsdorf Spirochetes Kaiser, 2009 Gram-negative Rosa et al., 2005 Flagella between PG and OM = endoflagella Turning of flagella causes flexible body to twist, propelling organism. VERY thin, so not visible by light microscopy o Gram stain not useful. o Use dark field microscopy, fluorescence microscopy. Treponema, Borrelia, Leptospira Chlamydiaceae Have an outer membrane with LPS but no PG o Instead, have Cys-rich envelope proteins. Unrelated to G- bacteria Usually unreactive in Gram stain (intracellular, cell wall structure). Genera of importance: Chlamydia, Chlamydophila Protein layer PBPs Penicillin-binding proteins Carry out various jobs in the PG biosynthetic process. o Transglycosylase o DD-transpeptidase = transpeptidase o DD-carboxypeptidase—removes last D-ala from peptide tails that have not been crosslinked. o DD-endopeptidase—cleaves PG crosslinks, to allow cell growth and separation; one of several type of autolysins. Enzymes that degrade some beta-lactam antibiotics, called beta-lactamases, are also classified as PBPs. Bacteria have many, often overlapping in function (important because PG essential). PG Biosynthesis:Phase II: Bridge Peptide (if present) If a bridge peptide is present, it is added in phase II. o S. aureus has a pentaglycine bridge; other species can have others. o Many bacteria do not have a bridge. PG Biosynthesis: Phase I: Monomer Production PG is built from the polymerization of PG monomers, which are comprised of NAG-NAM-peptide tail. Monomer production occurs in the cytoplasm. The monomer is completed on bactoprenol-P o a.k.a undecaprenol-P, C55-P o This scaffold is used for production of many extracellular polysaccharides. PG Biosynthesis: Phase III: Translocation and Transglycosylation The completed monomer is translocated across the PM. Transglycosylases incorporate the new monomer into the PG macromolecule. The bactoprenol-PP is recycled. o Very expensive molecule PG Biosynthesis: Phase IV: Transpeptidation Transpeptidases cross-link strands through peptide tails/bridges. The final D-ala is cleaved by the transpeptidase to provide energy to the reaction. Cross-linking is not complete; some tails remain unlinked. Peptidoglycan Synthesis as a Drug Target Major target o Unique to bacteria o Essential for most bacteria -lactams (penicillins, cephalosporins, etc.) o Inhibit transpeptidases. o New PG is weak, leads to cell lysis as autolysins continue to function. Peptidoglycan Synthesis as a Drug Target Glycopeptides (vancomycin) o Bind the stem peptide D-ala-D-ala. o Prevent transpeptidation and transglycosylation. Peptidoglycan Synthesis as a Drug Target Bacitracin (in Polysporin…) o Prevents recycling of bactoprenol-PP. o Affects synthesis of several cell-surface polymers. Peptidoglycan Synthesis as a Drug Target Fosfomycin (phosphomycin) o Inhibits MurA. o Cycloserine (antimycobacterial) o Inhibits alanine racemase and D-ala-D-ala synthetase. Peptidoglycan Synthesis as a Drug Target Tunicamycin o Nucleoside analogue o Prevents transfer of NAM-P to Und-P. Peptidoglycan as a Drug Target Also target for innate antibacterial lysozyme o Cleaves bond between NAG and NAM. o In mucus, tears, saliva O-acetylation of PG inhibits lysozyme. o Staphylococcus aureus Outer membrane impedes access of lysozyme. Plasma Membrane as a Drug Target Unique composition Disruption, usually by pore formation o Destruction of chemical gradients o Loss of nutrients and ions Targeted by o Cationic polypeptides (e.g., polymyxin B) o Newer glycopeptides o Lipopeptide antibiotics (e.g., daptomycin) Summary The cell envelope: o Mediates many interactions with the host—how does each structure function and what kind of bacterium are they found in? This helps in understanding the diagnosis and pathogenesis of individual organisms o Provides a basis for broadly differentiating bacteria, which we use to help diagnose infectious diseases. o Presents many targets useful in battling infections—what are they, why are they important to the bacterium and how can we mess them up?