Lecture 5. Lipopolysaccharide, Periplasm And Flagella Flashcards
What is lipopolysaccharide (LPS)?
Large complex molecules containing lipid and
carbohydrate (lipid A, core polysaccharide, O side chain)
What is the best described form of LPS?
Salmonella
E. coli LPS is nearly identical and also well studied
What is LPS known as when free in host?
Endotoxin
What makes up lipid A?
Two glucosamine residues linked to fatty acids and
phosphate (occasionally pyrophosphate)
What makes up the core polysaccharide?
In Salmonella, 10 (mostly) unusual sugar residues: glucose; galactose; heptulose (x2); 2-keto-3-deoxyoctonate (x3)
Side chains of NAG, phosphate and ethanolamine
What is the O side chain?
Variable region, responsible for antigenic make-up of bacteria
Lipid A and core polysaccharide are straight and O side chain flexible and bent
Highly variable composition
Rough (R)/smooth (S) variants depend on chain length
What are the functions of LPS?
Lipid A stabilises outer membrane structure
Core polysaccharide charged
Charged, hydrophilic external layer reduces
permeability of hydrophobic substances
Protects against host defences
What are endotoxins?
Released during cell division or by lysis of bacterial cells
Can act to prime immune system against a pathogen
If in the blood LPS can cause septic shock syndrome
What is the limulus amaebocyte lysate (LAL) test?
An approved procedure for assaying for LPS
Amaebocytes are the blood cells of Limulus polymephus
Contains a clotting factor that is released if coming into contact with bacterial endotoxin
Natural immune mechanism that contains bacterial infection
What are important properties of endotoxins?
Heat stable
Toxic in nanogram amounts
Interacts with innate immune system cells
Can result in inflammation, fever, vasodilation, thrombosis, acute disseminated intravascular coagulation, depletion of platelets/clotting factors leading to internal bleeding (haemorrhage), shock, sometimes death
Why is the outer membrane more permeable than the inner?
Due to presence of porins
Porins are protein channels that permit passage of small molecules up to around 600 Da
What are porins?
Homotrimeric, transmembrane proteins
Highly conserved structure
Form water filled channels in OM
Are most porins selective or non-selective?
Most are non-specific channels that allow passage of small (<600 Da) hydrophilic molecules (Most porins are slightly cation selective)
Some porins are selective
What is common across all porins?
Unusual structure consisting of a 16-stranded antiparallel β-barrel
Exceptionally stable
What can be found in a cross-section of a porin monomer?
Hourglass shape with a central constriction
Hydrophobic band of 25 Å that sits in membrane
Charges inside pore define size of solute that can traverse the channel
What do compounds diffuse through porins into?
The periplasm
What is the periplasm?
The space between outer membrane and cytoplasmic membrane (Ranges in size from 1 to 70 nm and can be up to 40% of cell volume)
Gel like consistency due to abundant proteins
Removal of cell walls without lysing the cells allows study of the proteins and enzymes present in this space
What enzyme activity occurs in the periplasm?
Nutrient acquisition
Energy conservation
Some peptidoglycan synthesis enzymes are periplasmic
Periplasmic binding proteins
Chemoreceptors
What is the Sec pathway?
Exports nascent polypeptide through cytoplasmic
membrane using a translocase
Folding of protein occurs after translocation
Proteins have an N-terminal signal peptide
What is the TAT pathway (twin arginine translocase)?
Exports fully folded enzymes across cytoplasmic membrane
Proteins have a twin arginine in N terminal region
What are flagella?
Long thin extracellular helical structures made of protein subunits that aid in motility
What is the structure of the flagellum?
Complex ring structures anchored into the membrane and cell wall/outer membrane
Flagellum shaft is easily removed by vigorous shaking
Different antigenic properties of shaft, tip of shaft and hook
The motor is driven due to transfer of protons through the ring structure (from periplasm to cytoplasm)
What is the structure of the flagellum filament?
Filament made of a single protein called flagellin
Base is different to the shaft and is known as the hook
Single protein connecting the shaft to the motor
Moves like a rotary motor
Where does growth in flagella take place?
At the tip
Process of flagella synthesis
- MS and C rings in cytoplasmic membrane
- Motor proteins
- P and L ring, hook and cap
- Flow of flagellin through hook, growth of filament
How does the motor in flagella work?
Rotary motion provided by the basal structures
Powered by proton motive force
Mutational analysis suggests dependence on Asp residue in MotB
How are flagella different in Gram +ve than Gram -ve bacteria?
In Gram +ve, no L and P rings
Anchor in the membrane layer is more complex
Mot proteins surround inner ring and movement of these relative to each other provides the force
What is the flagellum also known as?
H antigen
