Lecture 5: LPS, periplasm, flagella Flashcards
What does LPS stand for?
Lipopolysaccharide
Describe a Lipopolysaccharide
- Large complex molecule containing lipid and carbohydrate
- Contains: lipid A, core polysaccharide, and O side chain
What are 2 examples of bacteria that the LPS is found in?
Salmonella and E.coli
What is the LPS called when it is free in its host cell?
An endotoxin
Is it true that LPS can induce an immune response (i.e septic shock) ?
Yes, and whilst this isn’t a specific function of the LPS, it does induce an immune response when it comes into contact with an innate immune system
do gram negative bacteria have an LPS?
yes
What are the 3 main components of the LPS?
Lipid A
Core polysaccharide
O side chain
Describe the Lipid A component of the LPS
- Made up of 2 glucosamine residues linked to fatty acids and phosphate (occasionally pyrophosphate)
- Integrated into the outer membrane and so can act as an endotoxin and trigger an immune response
- Straight
- Very variable composition
- Stabilises the outer membrane structure
Describe the core polysaccharide component of the LPS
- Also referred to as R-antigen or R-polysaccharide
- Has side chains of NAG, phosphate and ethanolamine
-
Describe the O side chain component of the LPS
- Has a variable region that extends outwards, which is responsible for the antigenic makeup of bacteria. Different O side chains are linked to different diseases.
- O side chain is flexible and bent
- Highly variable composition (at least 20 different sugars)
- Rough/smooth variants depend on the side chain length
Why do bacteria move between rough and smooth variants?
To protect them from the immune system
What are the functions of the LPS
- Stability of the outer membrane (provided by Lipid A)
- Contributes to negative charge on the surface of the bacteria (due to the core polysaccharide being charged)
- Charged, hydrophilic external layer reduces permeability of hydrophobic substances (e.g. preventing entry of bile salts, antibiotics
). - Protects against host defences
-
Is the core polysaccharide negatively charged?
Yes
What’s more susceptible to phagoctytosis, rough variants or smooth variants?
Rough variants
What does the loss of the O antigen in salmonella lead to ?
Reduced virulence
Describe endotoxins
- Produced by many pathogens and are released during cell division or by lysis of bacterial cells, and trigger an immune response
- Can act to prime immune system against a pathogen
- If LPS is in the blood, can cause septic shock syndrome, of which there is no direct treatment
- Immunogenic even in absence of living cells
- Can be tested for using the Rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) assay
What are some important properties of endotoxins?
- Heat stable
- Toxic in small amounts
- Interacts with innate immune system cells
- Has serious infects, including: inflammation, fever, shock, and even death
How can endotoxins interact with innate immune system cells?
Can trigger the release of cytokines in a cascade
Activates transcription factors such as interferon-β and tumour necrosis factor (TNF)
Describe porins
- Make the outer membrane more permeable than the inner membrane
- Links the inter membrane space with the outside
- Protein channels that facilitate the diffusion of small molecules
- Made up of transmembrane proteins
- Highly conserved structure
- Form water-filled channels in the outer membrane
- Most porins are non-specific, but some are selective
- High thermal stability
- Resistant to protease and detergent degradation
- Have an unusual, stable, and closed structure
-
Is it true that compounds diffuse through porins and into the periplasm?
Yes
What is the periplasm?
the space between outer membrane and cytoplasmic membrane that has a lor of proteins
what does the periplasm range in size from?
1 to 70 nm
is it true that the periplasm can be up to 40% of the cell volume?
Yes
why does the periplasmic space have a gel like consistency?
Due to the abundance of proteins
What allows for the study of the proteins and enzymes present in the periplasmic space?
Removal of cell walls without lysing the cells
Describe the enzyme activity in the periplasmic space
- Nutrient acquisition: Hydrolytic enzymes such as alkaline phosphatase
- Energy conservation: e.g. electron transport proteins
- Some peptidoglycan synthesis enzymes are periplasmic
- Periplasmic binding proteins: Deliver specific compounds to ABC transporters in cytoplasmic membrane
- Chemoreceptors: Involved in chemotaxis
How can proteins be exported into the periplasm ?
Through the SEC pathway and through the TAT pathway (twin arginine translocase)
Descrine 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
Describe the TAT pathway
- Exports fully folded enzymes across cytoplasmic membrane
- Proteins have a twin arginine in N terminal region
What 3 ways can transport occur across a cytoplasmic membrane?
- Simple transport
- Group translocation (substance is changed/phosphorylated as it crosses the membrane)
- The ABC system (active transport)
What are the 3 types of transporter events?
- Uniporter
- Antiporter (hydrogen)
- Symporter
Describe flagella
- Long extracellular helical structures made of protein subunits that aid in motility
- Up to 20 µm long (much longer than the cell)
- connected to a motor that spins them clock- or anti-clockwise allowing bacteria to swim
- Complex ring structures anchored into the membrane and cell wall/outer membrane
- The flagella can act as an antigen (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)
-
Describe the motor of a flagella
- 50 nm in diameter
- 20 different proteins
- Rotates at ~300 rpm (E. coli) to 1100 rpm (Vibrio alginolyticus)
Describe the structure of flagellum filament
- made of a single protein called flagellin
- 38 – 53 kDa in most species
- N-terminal sequence very homologous (the same) in many bacteria
- The base is called the hook
- The hook and the shaft have have different protein subunits
- Single protein connecting the shaft to the motor
- The motor moves like a rotary motor
Is it true that the hook of the flagellum is anchored to the whole cell wall structure?
Yes
Where does growth occur in a flagellum?
At the tip of the flagellum
In gram negative bacteria, where is the L ring, P ring, S-M ring, and C ring located?
L ring in Lipopolysaccharide
P ring in Peptidoglycan
S-M ring in and on Membrane
C ring in Cytoplasm
Describe how the flagella is synthesised
- Subunits made in cytoplasm and are exported to periplasm
- Pass up 3 nm channel in flagellum, polymerise at the tip
-
Approximately how many flagellin subunits are there per flagellum?
20,000
Why is the flagellum constantly growing?
to repair shear damage caused by movement
Describe the order of steps of flagellum synthesis
1) MS and C rings in cytoplasmic membrane
2) Motor proteins
3) P and L ring, hook and cap
4) Flow of flagellin through hook, growth of filament
What type of motion is the motor?
A rotary motion
What powere the motor?
- Protons move across the cytoplasmic membrane through the mot complex
- This provides the energy for the motor to spin
Does mutational analysis suggest that the motor depends on an Asp residue in Mot B? And describe this
Yes
- Hypothesis: Changes in charge lead to conformational change of MotA, causing movement relative to MotB
- Second conformational change upon loss of charge may provide another step of the motor
Are the flagella in gram positives different to the ones in gram negatives?
Yes
How are the flagella in gram positives different to the flagella in gram negatives?
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 H antigen?
Flagellum
Is it true that Escherichia and Salmonella serotypes are defined in part by flagella structure?
Yes
What is E. coli O157:H7 best known for?
Strain causing food-borne illness including hemorrhagic diarrhoea and kidney failure
What is E. coli O104:H4 known for?
causing the 2011 Germany outbreak
Why isn’t the H antigen of much diagnostic value?
Because there’s many fewer H serotypes than O serotypes (LPS O polysaccharide), better to be used in conjuction with O serotypes.
