lecture 5

Question 1

define lipopolysaccharide LPS

Answer 1

it’s a large complex molecule that contains lipid and carbohydrate (lipid a, core polysaccharide, o side chain)

it’s found on the surface of gram -ve cells

it is also called endotoxin when it is free in the host (can induce massive immune response when it interacts with the innate immune system)

Question 2

basic structure of LPS

Answer 2

lipid A
core polysaccharide
o-specific polysaccharide
can have extra glucose as side chains

LPS is anchored into the outer membrane via the lipid

Question 3

component 1 lipid A

Answer 3

2 glucosamine residues linked to fatty acids and phosphate
integrated into the OM
induces the largest immune response when it’s present as an endotoxin

( So the presence of the lipid is what causes the issue if you get LPS circulating in your body. We have a lot of LPS in our body fairly regularly because we have so many bacteria associated with our gut and ur skin.)

Question 4

component 2 core polysaccharide

or R-antigen, or R-polysaccharide

Answer 4

sugar residues (glucose, galactose, heptulose, 2-keto-3-deoxyoctonate
has side chains of NAG, phosphate and ethanolamine

Question 5

component 3 O side chain

Answer 5

Responsible of antigenic characteristic - spotted by the immune system.
It’s the part that our immune system makes antibodies to.
Different diseases have different O serotypes.

O side chain is very variable, flexible, and can bend or twist.
It has a lot of modifications

Question 6

difference between o side chain of smooth and rough colonies

Answer 6

Smooth colonies have shorter non antigenic o side chains than the rough colonies

Question 7

function of LPS

Answer 7

lipid A stabilises the OM structure
core polysaccharide gives the -ve charge
protects against host defences
o-antigen is very variable so is a key diagnostic tool to determine what type of cell has caused the infection

Question 8

define endotoxins

Answer 8

LPS is only called endotoxin when it is broken off of the bacteria and is now in our bloodstream
Many pathogens produce endotoxins - as their cells are attacked or destroyed by the immune system, bits of their membrane can end up being produced.

Question 9

levels of LPS when we have an active infection

Answer 9

If we have an active infection, our levels of LPS rise.
LPS acts as a prime immune system - allows the immune system to get ready for the presence of a certain type of bacteria
If the level of LPS gets too high, it will cause a septic shock because we already destroyed the bacteria that was producing it.

Question 10

test for endotoxins

Answer 10

Rabbit pyrogen test - injecting rabbits with a sample from the patients and seeing if they produce an immune response of inflammation
Now use cells that allows us to test it in culture.

Question 11

properties of endotoxins

Answer 11

heat stable
toxic in nanogram amounts
interacts with innate immune system cells (triggers release of cytokines, activates transcription factors that are tehre to destroy infections)
can result in inflammation, fever, vasodilation, and blood clotting which is what causes the septic shock

Question 12

define the LAL test (Limulus Amaebocyte Lysate)`

Answer 12

it’s an approved procedure for assaying for LPS
amaebocytes are the blood cells of limulus polymephus. they contain a clotting factor that is activated when in contact with bacterial endotoxin

Question 13

define porins and their role

Answer 13

Outer membrane is not a fundamental barrier to the outside env in gram -ve cells.
They have designed structures called porins that allow substances from the outside to enter the cell.

Question 14

define the outer membrane’s structure

Answer 14

OM more permeable than the IM due to porins
porins are protein channels that will allow small molecules through (if the pathogen is small enough, it will pass through as well) <600 Da

Larger molecules have to be carried through by active transport systems

Question 15

porin superfamily

Answer 15

homotrimeric (made up of 3 identical subunits), transmembrane proteins
highly conserved structure (similar aa that bind and transfer molecules across the membrane)

Question 16

porin selectivity

Answer 16

Most porins are non-selective and allow all molecules that are small enough, but they still have a cation selectivity
Some porins are very selective and act as receptors for bacteriophages

Question 17

porin characteristics

Answer 17

high thermal stability

resistant to protease and detergent degradation (essential for the survival of gram -ve bacteria in harsh env)

Question 18

porins have common beta strand structure

Answer 18

very unusual structure but it is still very common across all porins (consists of a 16-stranded antiparallel B-barrel)
Each B barrel strip are the bits that go through the membrane
Very stable because it joins its N terminus to its C terminus via a salt bridge and forms a complete circle to stick itself together
Very stable because it forms a circular protein molecule and holds together in the membrane

Question 19

charged structure of porins

Answer 19

hourglass shape with a central constriction
hydrophobic band sits in the membrane
charges inside the pore define size of solute that can traverse the channel

Question 20

periplasmic space

Answer 20

between the outer and inner membranes.
Gel like consistency because it is packed with proteins
can be 40% of cell volume

Question 21

enzyme activity in periplasm (5)

Answer 21

• nutrient acquisition (break down large compounds for easier acquisition)
• energy conservation (uses energy from e- transport chain)
• peptidoglycan synthesis enzymes (synthetic enzymes in the periplasm)
• periplasmic binding proteins (binding proteins linked to ABC transporter system and deliver compounds to it)
• chemoreceptors (involved in chemotaxis)

Question 22

protein export into periplasm through 2 pathways

Answer 22

• sec pathway
  exports polypeptides using translocase, through the cytoplasmic membrane before they fold.
  Proteins have a very specific N-terminal signal peptide that direct them into this sec pathway for transportation
• tat pathway
  It exports fully folded proteins across the plasma membrane
  Proteins have a different signal molecule on their N-terminal region, they have a twin arginine which directs them to the tat pathway.

Question 23

3 classes of transporting systems across cytoplasmic membrane

Answer 23

• simple transport
  allows a molecule to pass through in both directions. Usually the molecule passes through with a H+ ion. The hydrogen is passing down a conc gradient which supplies the energy to move another molecule through
• Group translocation
  glucose enters the periplasm, moves through the transport system, is instantly phosphorylated, becomes different to the glucose molecules outside the cell to maintain a gradient
• ABC system
  a binding protein brings a compound to the transporter. The transporter translocates it across a membrane by using ATP. these are energised transporting systems

Question 24

Examples of transporter systems (3)

Answer 24

Uniporter - moves one molecule through
Antiporter - moves one molecule in and one out
Symporter - moves two molecules out

Question 25

what are flagella?

Answer 25

long, thin, extracellular, helical structures made of proteins
20 micrometers long
flagella are connected to a motor
motor is a nanotechnological marvel)

Question 26

structure of the flagellum

Answer 26

complex ring structures anchored into the membrane and cell wall/OM
rings (embedded into the membrane) and hook (attached to flagella shaft) are rigid and attached
different antigenic properties of shaft, tip of shaft, hook
motor is driven by transfer of protons through the ring structure

Question 27

structure of flagellum filament

Answer 27

filament is made of flagellin proteins (38-53 kDa)
N-terminal sequence is almost identical in all bacteria because it’s a protein that is transported into the periplasm, out of the periplasm, and into the outside world.
There is a single protein connecting the shaft to the motor, then the rest of the proteins are about the ring structure and the motor itself.
The motor proteins on the outside spin the inside of the motor

Question 28

structure and synthesis of flagella in gram negative bacteria

Answer 28

Only gram negative bacteria have the complex structure where it has:

• L ring inside the lipopolysaccharide of the outer membrane
• P ring in the peptidoglycan in the periplasm
• SM ring sitting on the surface of the cytoplasmic membrane (in and on the membrane)
• C ring on the cytoplasmic membrane

The flagella grows at the tip so it transports proteins up the flagella and makes it longer from the tip rather than growing from the base
So the 3nm channel allows the flagellum to pass up the flagella and polymerise at the tip

Question 29

flagella structure in order

Answer 29

MS and C rings (in cytoplasmic membrane)
motor proteins
P and L ring
hook and cap
flow of flagellin through the hook, growth of filament

Question 30

how does the motor work?

Answer 30

rotary motion provided by the basal structures. it is powered by the pmf (proton motive force) and one rotation needs the translocation of 1000 protons

MotB protein appears to be the main driver of the movement of the motor, this suggests that it is dependent on a single aspartate residue within that MotB protein

Change in charges leads to a conformational change of MotA which causes movement relative to MotB → so the two proteins move across each other

A second conformational change allows them to separate again, so the 2 proteins move and separate repeatedly in a ring structure which causes a smooth circular motion

Question 31

flagella difference in gram +ve and gram -ve cells

Answer 31

gram +ve
does not have 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

Question 32

flagellum antigenic structure

Answer 32

Flagella is also an antigenic structure and known as the H antigen
E coli and salmonella are both defined by their:
O antigen which is on the LPS
H antigen which is on the flagella

there are fewer H serotypes than O serotypes and therefore of limited diagnostic value