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Gram-negative vs. Gram-positive
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1. PG cross-linking
2. Thickness of PG
Gram-negative = 2 to 3 layers
Gram-positive = as many as 40 layers
The Gram-stain is based on the difference in cell wall thickness which determines the ability of the bacteria to retain a crystal violet-iodine complex.
3. Outer membrane?
Gram-negative = yes
Gram-positive = no
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Summary of Cell Structures
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Bacterial Shapes
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Length: smallest bacteria = 0.5-1 μm; largest = up to a few μm
Basic Structures: rod, coccus (round), and spiral
Shapes: all variations of the basic structure exist (rods: short, long, curved, bent, pleomorphic; ends can be pointed, round, or flat -- cocci: regular, irregular)
Associations: tandem, chains, clusters (regular, irregular)
Cocci:
Rods:
Spiral:
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Dental Plaque = Biofilm
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Glycocalyx (capsule, slime)
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external coating secreted onto the outside of the cell
two types: capsule - closely associated with the cell; slime - secretions loosely adhering to the cell surface, w/o clearly definded border
structure: principal constituent in water; contains polysaccharides and sometimes glycoproteins; structure varies in thickness and can be rigid or flexible; usually connected to cell wall by ionic bonding
functions: protection, attachment (S. mutans produces slime layer in the presence of sucrose), virulence factor (evasion of host defense system--antiphagocutic), antigenic (used in serological classification--K-antigen)
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Pili (Fibrillae)
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longer than fimbriae, thin and flexible, only few per cell
function: receptors for certain bacteriophages, attachment of some pathogens to their host, conjugation
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Bacterial Structures and Virulence
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Bacterial cell walls and surface structures contribute to different stages of infection:
Adhesion to biotic and abiotic surfaces (e.g. LPS, fimbriae, outer membrane proteins, glycocalix)
Evasion of host defenses (e.g. capsule, flagella, teichoic acids)
Invasion of tissues (as a consequence of adhesion and evasion of host defenses)
Stimulation of proinflammatory activities → often resulting in tissue damage (e.g. PAMPs; Lipid A)
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External Bacterial Structures
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These structures may or may not be present depending on the species:
Flagella
Fimbriae, Pili
Glycocalyx (capsule, slime)
These structures can:
contributed to virulence properties
decrease susceptibility to host defense system
trigger the innate immune response
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Cell Wall of Gram-positive Bacteria
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thick (20-80 nm) and relatively homogenous
PG comprises 40-80% of total cell weight and is extensively cross-linked in three dimensions
components other than PG: teichoic acids or teichuronic acids, polysaccharides, proteins
Teichoic acids and surface proteins are used in the identification and serological classification of Gram-positive bacteria.
Potential roles of surface proteins:
can act as adhesins (lipoproteins of oral streptococci)
can be antiphagocytic (M protein of group A streptococci)
antigenic variation to evade host immune system
mimicking of host antigens
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Bacterial Cell Structures
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Surface Proteins
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Teichoic acids and surface proteins are used in the identification and serological classification of Gram-positive bacteria
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Fimbriae
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Short, rigid protein rods; not involved in motility; more numerous than flagella
Originate in CM and extend from cell surface
function: attachment to surfaces and adherence to other cells (bacterial and animal) -- binding to specific sugar residues on glucoproteins and other surface components of target cell; associated with ability to cause disease
are found in Gram-positive and Gram-negative oral bacteria
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Teichoic Acids
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Strengthen and stabilize then PG layer
highly charged (neg. charge)
long acidic plymers of alditols (ribitol or glycerol) linked through phosphodiester bridges
free hydroxyl groups (R) are substituted with amino acids, amino sugars, or mono-, di-, and trisaccharides
Two types of teichoic acids:
Wall teichoic acid - ribitol teichoic acids linked to NAM in PG
Lipo(or membrane)teichoic acid - glycerol teichoic acid linked to a glycolipid in CM; is present in all Gram-positive bacteria; R = often D-Ala
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Prokaryotes vs. Eukaryotes
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Antibiotic Targets
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Cell wall (e.g. penicillin - inhibits cross-linking of PG)
Cell membrane (e.g. polymyxin B - replaces cations on lipids → disrupts CM)
DNA (e.g. quinolones - block DNA gyrase → block DNA replication)
Ribosomes (e.g. gentamycin - 30S; clindamycin - 50S)
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Teichuronic Acids
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highly charged polymers with glucuronic acid as a backbone
contains no phosphorus
Production depends upon the availability of phosphate; phosphate available = teichoic acid; NO phosphate = teichuronic acid
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Acid-fact Bacteria (Mycobacterium)
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Cell wall contains equal amounts of PG and arabinogalactans complexed with lipids.
50% of these lipids are mycolic acids (C32-C90 fatty acids) which are attached to arabinogalactans and form a water-insoluble complex (= wax D).
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Cell Envelope (Cell Wall)
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Located outside of CM
Functions:
shape
rigidity (=couters osmotic pressure; water wants to flow into the cell because of high concentrations of solutes inside the cell)
platform for surface appendages
Clinically:
contributes to the ability of bacteria to cause disease
site of action of several antibiotics
can trigger innate immune response
Composition:
Common component is peptidoglycan (PG) = murein
PG consists of a polysaccharide backbone with peptide cross-links
Polysaccharide chain: alternating residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
Peptide: tetrapeptide synthesized as pentapeptide; linked to polysaccharide chain at carboxyl group of NAM; alternating L- and D-amino acids; peptide chains are almost identical from organism to organism (exceptions where noted below)
Unusual Properties:
contains D-amino acids
contains diaminopimelic acid
contains muramic acid
peptide chain is synthesised by a series of enzymes and NOT by ribosomes
Autolysins:
group of enzymes that cleave PG in specific places
necessary for cell wall synthesis
Amidase = cleaves between NAM of polysaccharide chain and L-Ala of peptide chain
Endopeptidase = cleaves cross-link between two peptide chains
Lysozyme = cleave between NAM and NAG of the polysaccharide chain
Bacteria without cell wall:
Mycoplasma: group of bacteria which lack cell wall naturally; found in environment with high osmotic strength (e.g. moist mucosal surfaces as in lung, genital tract, etc.)
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Pathogen-Associated Molecular Patterns (PAMPs)
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Some bacterial molecular sequences that are recognized by toll-like receptors and other pattern recognition receptors and initiate an immune respose:
lipopolysaccharides
peptidoglycan
lipoteichoic acids
mannose-rich glycans (glycoproteins)
flagellin
pilin
bacterial DNA (due to methylation)
phosphorylcholin and other lipids (bacterial membrane)
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Flagella (internal)
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AKA axial filaments, endoflagella
found in spirochetes
internal structure
2 to >100 filaments extend from both ends of the cell between the OM and CM (located in the periplasm)
Hypothesis: endoflagella rotate in the periplasmic space; this could cause the corkscrew-shaped outer membrane of the spirochete to rotate and propel the bacterium through the surrounding fluid
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Cytoplasmic Constituents
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1. Water: ~70%
2. Macromolecues: ~26% (~90% of dry wt)
Proteins (~50-60% of dry wt, up to 100 different enzymes, ribosomes--up to 25% of cell volume → target for may antibiotics)
Nucleic Acids (DNA: 3% of dry wt, generally one chromosome + plasmids and phages, bacterial genomes contain high % of unmethylated CG dinucleotide sequences; RNA: 10-20% of dry wt, mRNA [mono- or polycistronic], tRNA [carries charged amino acids to growing peptide chains], rRNA [part of 30S and 50S ribosomal units])
Storage granules (stored energy; used under metabolic stress; kinds: glycogen [polymers of glucose, good energy source, most common potential cariogenic trait], poly-beta-hydroxybutyric acid [lipophilic, very high energy], sulfur granules [common in some photosynthetic bacteria], and polyhexametaphosphate [AKA volutin granules, used to store phosphate which is often a limiting nutrient, are metachromatic])
Endospores (small, survival structure produced in response to nutrition deprivation, lack of water, or severe temperature changes; resting structures [metabolically inactive]; resistant to heat, to desiccation, to disinfectants, to UV [major source of problems in infection control, basis for resistance: impermeable surface layer, low water concentration]; spores have high concentrations of calcium most of which is complexed with dipicolinic acid; 3 parts: Core--dehydrated cytoplasm containing DNA, ribosomes, enzymes, etc. everything that is needed to function once returned to the vegetative state, Cortex--modified cell wall/peptidoglycan layer that is not as cross-linked an in a vegetative cell, Coat--several protein layers outside of the cortex that are impermeable to most chemicals [the coat is responsible for the resistance to chemicals]; sporulation process takes 4-8 hrs; germination takes just minutes [can be triggered by heat in the presence of moisture, swelling of endospore, metabolic activity starts, loss of resistance to environmental stress, disintegration of exterior parts of the envelope, development into a fully functional vegetative cell])
3. Small molecules & ions: ~4% (~10% of dry wt)
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Cross-linking of PG Layer
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Cross-linking usually occurs between the third amino acid of one peptide chain and the forth amino acid of the other peptide chain.
Gram-negative bacteria usually have a direct cross-link while gram-positive bacteria often have peptide bridges between cross-linked peptide chains (most commonly peptide bridge consists of 5 gly).
In gram-negative bacteria, only about 50% of the potential cross-links are formed; the remainder are free tetra- or pentapeptides or no peptide at all.
In gram-positive bacteria, all peptide chains are linked, often with additional peptide bridge joining distant glycan chains (PG of gram-positive bacteria is much stronger because it takes advantage of all peptide links).
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Flagella (external)
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on spirilla and most bacilli
function: locomotion (cells move by rotating flagella like a propeller, driving force is proton gradient between cytoplasm and periplasm or outside environment)
Location: 1. Polar flagellation - flagella attached at one (monopolar) or both (bipolar) ends; 2. Peritrichous flagellation - completely surrounded by flagella
Composistion: protein subunits of flagellin form a tube-like structure; hook and basal body are needed for anchorage and rotation
insturment of chemotaxis
enables some pathogenic bacteria to penetrate host defenses
antigenic - serological determinant (H-antigen)
can be object of phase variation: ability of some bacteria to alternate synthesis of different flagella → evasion of host defenses
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Cell Wall of Gram-negative Bacteria
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Periplasm = space between CM and OM
may occupy as much as 30% of cell volume
contains many specialized proteins which ar often involved in nutrient acquisition (binding proteins = bind nutrients [sugars, aa, vitamins] that enter through porins; enzymes = cleave organic molecules to provide nutrients [e.g. phosphatases, proteases, endonucleases])
Peptidoglycan: 2 to 3 layers; cross-linked in two dimensions only; attached to outer membrane (OM)
Murein lipoproteins: small (7.5 kDa); function: anchors OM to PG and maintains structure and organization of OM; protein portion is covalently attached to ~10% of the peptide chains in PG; lipid portion consists of three fatty acids which are embedded in OM
Outer Membrane (OM):
Composition: phospholipids (35%; on the inner side of OM next to PG; composition similar to CM), protiens (15%; porins - create pores, allow small hydrophilic molecules to pass freely; receptors - for nutrients such as vitamins, siderophores [small molecules that bacteria release to capture iron and bring it back to the cell], and phosphate), and lipopolysaccharides (LPS; 50%)
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Mesosomes (Intracytoplasmic Membranes)
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Purpose: increase surface area of CM
Invaginations of the CM
Provide increased membrane surface for enzymes of electron transport
Might be involved with segregation of new replicated chromosomes
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Lipopolysaccharides (LPS)
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Complex unique glycolipid which is composed of three covalently linked regions.
Lipid A: disaccharide linked to hydroxy fatty acids; fatty acid composition differes in bacterial genera but is consistent within species
Core Oligosaccharide (AKA Core Polysaccharide): attached to lipid A; contains various sugars including two unusual ones - heptose and KDO (2-keto-3-deoxyoctolusonic acid)
O-specific Side Chain: attached to sugar core; sticks out from bacterial surface; has variable number (0-50) of repeated oligosaccharide units of 3 to 5 sugars; major serological determinant of Gram-negative bacteria (O-antigen); varies within species and even between strains of a species
Biological activites of LPS:
when cell-bound: strong negative charge assists in evasion of complement system phagocytosis
when released (cell lysis): acts as endotoxin; toxicity resides in lipid A and is enhanced by polysaccharide; active only in sizeable amounts; toxin activates complement and stimulates release of cytokines - overstimulation of defense system results in dropping blood pressure (→ vascular collapse) and fever and septic shock
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Cytoplasmic Membrane
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Functions:
Permeability barrier, prevents cell contents from leaking out (very impermeable to polar and charged molecules--most biomolecules, also small ions such as K+, H+; only water, lipid-soluble materials and gasses pass freely)
Segregation of chromosomal and plasmid DNA
Import and Export (transport proteins--bind specific substrates and transport then to cytoplasm; secretory proteins--involved in secretion of periplasmic and extracellular proteins; this system can play a role in resistance to antibiotics!)
Energy Metabolism (proteins involved in energy metabolis - generation of electrochemical gradient of ions; ATPase--formaiton of ATP by use of proton motive force)
Composistion:
50-70% protein (for: transport, energy metabolis, cell wall synthesis, sensor for two component regulatory system)
30-50% phospholipid (form bilayer in which proteins are embedded; no sterols--except for those parasitic bacteria that use parts of the host cell's membrane; but: hopanoids)
small amounts of carbohydrates
Can be a target for antibiotics (mechanism unknown)
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