Lipopolysaccharide, periplasm, flagella

Question 1

What are lipopolysaccharides (LPS) and what are its components?

Answer 1

• large complex molecules containing lipid and carbohydrate units
• called endotoxins when free in host
  made of: Lipid A, core polysaccharide, O side chain

Question 2

What are lipopolysaccharides called when they are free in a host?

Answer 2

endotoxins - can induce septic shock/massive immune response

Question 3

Which parts of an LPS are variable/constant?

Answer 3

o specific = variable

core polysaccharide + lipid A = constant

Question 4

What is the structure of lipid A?

Answer 4

• 2 glucosamine + 1 phosphate each + linked to 2 fatty acids each: one GlcN has fatty acids with side chains
• lipid A integrated into outer membrane
• remaining LPS projects from the cell surface

Question 5

What is the structure of the core polysaccharide?

Answer 5

(also called R-antigen or R-polysaccharide)

• 1-4 molecules of KDO
• unusual sugar residues e.g. glucose, galactose, heptulose (x2) in salmonella
• side chains of Glu-Nac, phosphate and ethanolamine++

Question 6

What is the structure of the O-specific polysaccharide chain?

Answer 6

• highly variable composition (at least 20 diff sugars)
• rough/smooth bacterial variants depends on side chain length
• lipid A + core polysaccharide = straight, O specific = flexible and bent

Question 7

Why is the O-specific polysaccharide significant for hosts?

Answer 7

They are the variable regions responsible for antigenic makeup of bacteria
- key diagnostic tool
- extend outwards from cell so is the first contact a cell makes with a host
- different O-sp polys linked to specific diseases

Question 8

What are the functions of the LPS?

Answer 8

• lipid A stabilises the outer membrane
• core polysaccharide charged due to phosphate groups (-ve) = negative charge on surface
• charged, hydrophilic external layer = less permeable to hydrophobic substances e.g. bile salts, antibiotics
• protects against host defences

Question 9

How does the LPS protect against host defences?

Answer 9

• rough variants more susceptible to phagocytosis
• loss of O-antigen (in E.coli and salmonella) = reduced virulence + less likely to be engulfed in phagocytosis

Question 10

When are endotoxins released and why are they dangerous?

Answer 10

during cell division + lysis of bacterial cells - cause septic shock syndrome + no direct treatment

immunogenic even when the cell it originated from is dead - fragments of dead cell

LPS of some non-pathogens can also be an endotoxin

Question 11

Why might endotoxins be useful?

Answer 11

can act to prime immune system against a pathogen as they are constantly in our bodies in small amounts - prep antibodies + immunological memory

Question 12

Test for endotoxins?

Answer 12

Rabbit Pyrogen test

Limulus amoebocyte lysate (LAL) assay

Question 13

How does the limulus amoebocyte lysate (LAL) test work?

Answer 13

amaebocytes - RBCs of horseshoe crab

contain a clotting factor that is released when bacterial endotoxin is present - natural immune mechanism against infection

Question 14

What are the important properties of endotoxins?

Answer 14

• heat stable
• toxic in nanogram amounts
• triggers release of cytokines in a cascade, activates transcription factors such as interferon-β and tumour necrosis factor (TNF) which kills infected/dmged cells

(can result in: inflammation, fever, vasodilation, thrombosis, acute disseminated intravascular coagulation, depletion of platelets/clotting factors -> internal bleeding/hemorrhage, shock sometimes death)

Question 15

What are porins and where are they located?

Answer 15

• homotrimetric transmembrane protein channels
• permit passage of small molecules up to 600 Da (molecular weight units in size)
• highly conserved structure
• located in the outer membrane -> make it more permeable than the inner membrane
• form water filled channels in OM -> links periplasmic space to the outside -> transfer water soluble molecules
• resistant to protease and detergent degradation: essential for the survival of gram-ve bacteria in harsh environments

Question 16

How do larger molecules cross the outer membrane?

Answer 16

attached to carriers through ACTIVE TRANSPORT SYSTEMS, not through porins !

Question 17

In what ways are porins selective?

Answer 17

• mostly non-selective (some in E.coli are selective for example: maltose and maltodextrin selective porin which is also the selective porin for lambda - bacteriophage)
• <600 Da only
• most porins slightly cation selective

Question 18

What is the structure of a porin?

Answer 18

• 16 stranded antiparallel β
• exceptionally stable
• extra stability from formation of a salt bridge between the N- and C-termini

Question 19

What is the cross section of a porin monomer like?

Answer 19

• hourglass channel with a central constriction
• hydrophobic band of 25Å that sits in membrane
• charges inside the pore define size of solute that can traverse the channel

Question 20

What is the periplasmic space and its features?

Answer 20

• space between the outer and cytoplasmic membrane
• compounds diffuse via porins into periplasm
• 1-70nm in size
• can be up to 40% in cell vol
• gel like consistency due to lots of proteins

(removal of cell walls without lysing the cells allows study of the proteins and enzymes present in this space)

Question 21

Enzyme Activity in Periplasm?

Answer 21

• Nutrient acquisition w/ hydrolytic enzymes
• 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

Question 22

Why are proteins exported into the periplasm?

Answer 22

SEC PATHWAY
- most/all proteins made in the cytoplasm
- needs translocating to the periplasmic space to be folded/modified
- ribosome attached to cytoplasmic side of membrane + pushes proteins through a translocase into periplasm
- each translocase is v specific to the protein so proteins have an N-terminal signal peptide

Question 23

What is the TAT pathway? (twin arginine translocase)

Answer 23

exports fully folded enzymes across cytoplasmic membrane

proteins have twin arginine in N terminal region

Question 24

What are the 3 classes of membrane transporting systems?

Answer 24

simple transport, group translocation, The ABC system

Question 25

Explain group translocation

Answer 25

molecule transported across the membrane and is transformed e.g. phosphorylation during transport

Question 26

Explain the ABC system of transport

Answer 26

protein picks up molecule, attaches to the transporter, active transport to move the molecule into the cytoplasm

Question 27

What are the 3 types of transport events?

Answer 27

uniporter (one molecule), symporter (2 molecules same direction), antiporter (2 molecules different direction)

Question 28

What are the properties of a bacterial flagella motor?

Answer 28

~ 50nm in diameter
~ 20 diff proteins
- rotates at ~ 300 to ~1100rpm (r=revolutions) for E.coli
- complex ring structures anchored into the membrane + cell wall/outer membrane - rings and hook are rigid and attached
- 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 (periplasm to cytoplasm)

Question 29

What are flagella?

Answer 29

• long, thin extracellular helical structures made of protein subunits that aid in motility
• up to 20µm (much longer then the cell)
• connected to a motor that spins them clockwise/anticlockwise -> swim and change direction (twitching motility - run and tumble)

Question 30

What is the structure of the flagellum filament?

Answer 30

• filament made of a single protein called FLAGELLIN (38 - 53 kDa in most species)
• approx 20,000 flagellin subunits per flagellum
• grow constantly to repair shear dmg caused by movement
• n-terminal sequence v homologous in many bacteria
• single protein connecting the shaft to the motor

Question 31

N-terminal

Answer 31

• end of peptide chain with the free amino group (c-terminal = carboxyl end)
• N-terminal sequence = seq of amino acids from the N-terminal end

Question 31

Hook of the flagella

Answer 31

• slightly bent section between L-ring and flagellin shaft/filament - diff subunits to the shaft
• anchored to the whole cell wall structure

1) L-ring in Lipopolysaccharide
2) P-ring in Peptidoglycan
3) S-M ring in and on Membrane
4) C-ring in Cytoplasm

Question 32

How does a flagella grow for repair?

Answer 32

growth occurs at the tip
- subunits made in cytoplasm
- exported in periplasm
- pass up 3nm channel in flagellum
- polymerise at the tip

Question 33

Order of flagella synthesis

Answer 33

1) MS and C rings
2) motor proteins
3) P, L ring
4) Hook
5) cap
6) flow of flagellin through hook, growth of filament

Question 34

How does the motor work?

Answer 34

• proton motive force: protons move across the cytoplasmic membrane through the MOT complex
• one rotation = 1000 protons translocated

Question 35

What is the difference between flagella in Gram-ve and Gram+ve bacteria?

Answer 35

• in gram+ve = no L, P rings (S-M, C, P, L rings present in gram-ve)
• anchor in the membrane layer more complex for gram+ve
• motor proteins surround inner ring and movement of these relative to each other provides the force

Question 36

Flagellum are known as which antigen?

Answer 36

Flagella are known as the H antigen - used when referencing different strains of bacteria e.g. E.coli O157:H7 - best known strain causing food-bourne illness + diarrhoea and kidney failure