actin cytoskeleton exploitation by listeria

Question 1

what is listeria monocytogenes

Answer 1

a bacterium

contaminates foods

gram +ve - thick peptidoglycan

Question 2

risk factors for infection by listeria *

Answer 2

in healthy people immune system is cleared quickly - symtomless

in pregnant women infection can led to problem for fetus and miscarriage

causes pathology in immunocompressed people - elderly, HIV, organ transplant, very young

Question 3

what is it about listeria that makes it dangerous *

Answer 3

can go through the placenta and iovade the fetal organs

can cross BBB and gut barrier

this ability to cross membranes is unique - most bacteria causing food poisening remain controlled in gut (although toxins can go elsewhere)

Question 4

how do listeria cross the membrane *

Answer 4

in the placenta the lining of the blood space is the syncytioblast - there are no clefts between cells, so only way bacterium can cross to fetal sie is through cytoplasm

this is not done by damaging the mucosa in the GI either

it enters by exploiting the structures leading to internalisation - it is phagocytosed by cells that are not phagocytes (ie enterocytes) - therefore engulfed

it uses the proteins internalin A and B

Question 5

mechanism listeria use for phagocytosis *

Answer 5

the receptor for internalin A is E-cadherin (explains the trophism for epithelial cells)

bacteria enters in a vesicle and is taken to the lysosome

forms the phagolysosome

in phagolysosome there is a drop in pH because H+ ions are pumped in that should destroy the pathogen

however this activates a listeria protein that is a toxin (lysin) - allows bacteria to form a pore in membrane of phagolysosome so it can enter the cytoplasm and be free in the cells

Question 6

describe E cadherin *

Answer 6

E cadherin are transmembrane proteins that maintain the integrity of the epi layer - they attach cells together and attach to cytoskeleton

they recieve a signal from the outside of the cell and initiate a downstream cascade to make the response inside the cell

Question 7

why does listeria have to be able to spread through the cytoplasm *

Answer 7

Without motility the bacteria cannot spread through the infected cell efficiently and cannot exit the cell or invade neighbouring cells.

Question 8

describe the mechanism of motility of the bacterial cells *

Answer 8

dependant on the bacterial protein ActA - produced at 1 pole of bacteria

ActA can bind to actin in the host cell cytoplasm to allow limited motility

for max motility ActA binds to cell protein VASP, at teh proline rich region, which is an adaptor protein - this recruits arp complexes which act as nuclei for new actin filaments - increasing rate of comet tail production

VASP binds to profilin

profilin binds to cellular actin and promotes polymerisation by exchanging ADP in favour of ATP - therefore ensures a good supply of ATP-containing G-actin

ATP actin monomers are incorporated into the filaments (new actin filaments are constantly being produced- they propel listeria from 1 side of cell to other

the bundle of actin filaments is called the comet tail

cross linking of the actin keeps the bundle together and gives it directionality

Question 9

effect if mutation in ActA *

Answer 9

can form microcolonies in cells, but cant spread from cell to cell so are not virulent

Question 10

what are the phases in actin polymerisation *

Answer 10

nucleation - need 3 monomers to come together to form stable trimer

Arp2/3 stabalises the trimer - prevent it from depolymerising

elongation - more monomers are added to form F-actin

Question 11

how does listeria invade neighbouring cells *

Answer 11

when it gets to the periphery of the cell it pushes membrane and forms filopodia - move into other cell

it is then encapsulated in membrane from old and new cells - can get out using the protein used in the lysosome to make pores in the membrane

Question 12

what is the enzymatic role of actin

Answer 12

hydrolyses ATP to ADP slowly

Question 13

what are the 2 ends of actin *

Answer 13

Actin has fast growing and slow growing end

fast growing is the leading end (new monomers get incorporated), slow is where the monomers are released

filament keep growing until reachws equilibrium

if want to keep growing the monomors from slow growing are recycled and go to the leading/fast end

Question 14

what angle is actin branched at *

Answer 14

70 degrees - because the active siute of the protein involved is 70degrees apart

Question 15

what is the structure of ActA *

Answer 15

it contains 4 proline-rich repeats flanked by acidic amino acids (aspartate, glutamate) which are homologous to those seen in zyxin, an endogenous protein in host cells

zyxin is found in focal adhesions - where associated with actin stress fibres - it binds to VASP (vasodilator stimulated phosphoprotein) - which binds prolifin and Arp, it is involved in actin rearrangement

ActA also contains region similar to actin-binding region of vinculin that maintains membrane attachment to actin

also have signal peptide - tells ribosome that protein needs to go to the cell surface

has anchor protein that docks the protein onto the cell wall

Question 16

what is the immune response to listeria *

Answer 16

inside the cell so Ab and complement wont be effective

only way to kill listeria is a T cell CD8+ cytotoxic T cell response – if CD8 response is compromised – pose threat - therefore this is why immunocomprimised people are at risk

after cross GI bacteria get into blood and lymphatic system to spleen and liver – in liver 1st line of defence: kupffer cells that are a type of macrophages – normally this is where infection stops because the kupffer cells clear infection – in immunosuppressed no clearance = more proliferation – spreading to brain

Question 17

what is the worse consequence of listeria

Answer 17

meningitis (because cross BBB)

can effect the cortex = siezures

Question 18

what side of the comet tail are actin added to *

Answer 18

the bacterial side

Question 19

where does the energy for movement of listeria come from *

Answer 19

growth of filament goves propulsion

Question 20

how can you stop the spreading of listeria

Answer 20

use a TK inhibitor eg fungal metabolite wortmannin - inhibits TK and so blcoks lifecycle of bacteria = stop spreading

cytochalasin D (fungal metabolite) – able to inhibit the rearrangement of the cytoskeleton - act as capping protein at fast growing end – stop monomers being incorporated and elongation, cant use to treat because it is toxic because would stop cytoskeleton in all cells

AB – they need to be able to get across the cell membrane quickly, because if doesn’t reach the concentration inside cell necessary – wont kill bacteria

Question 21

where is listeria found *

Answer 21

common in soil – small number in uncooked fruit/veg destroyed in cooked or pasteurised but continues dividing in the fridge and can build up to infective concentrations

Question 22

decribe nucleation *

Answer 22

at least 3 G-actin (each binding ADP/ATP) come together in right orientation to form a filament nucleus for further elongation

this is slow unless nucleus is stabalised eg by capping protein or Arp complex

Question 23

describe elongation *

Answer 23

actin monomers added to preexisting filaments at either end, equilibrium differs for the 2 filament ends and accoding to whether actin binds ATP (favours addition) or ADP (favours loss).

ATP hydrolyses to ADP within the filament.

Question 24

what are the effects of different actin binding proteins *

Answer 24

proteins binding free monomer (e.g. β-thymosin) decease polymerisation.

Some (e.g. profilin) enhance the normal rate of replacing ADP with ATP on monomers and favour polymerisation

Capping proteins bind to one or other filament end and prevent polymerisation there.

Severing proteins cause filaments to split, exposing 2 new ends.

Crosslinking proteins bind filaments together, either at right angles (e.g. filamin) or in parallel (e.g. α-actinin).

Question 25

how does the formation of actin filaments push the listeria across cells *

Answer 25

once made, the actin filaments in the comet tail are stationary (because of the crosslinking woth each other, cellular filaments, and a-actin and other cross-linking proteins)

therefore as new filaments are created the bacteria is pushed forward

(the filaments at the distal end of tail depolymerise)

Question 26

how is listeria sopread through the body *

Answer 26

doesnt need to travel through pre-existing wound

is phagocytosed (dont know if through M cells or intestinal epithelial cells)

then end up in macrophages and blood monocytes which carry bacteria to other tissue cells around body

main site of bacterial division is liver

can also invade the syncytiotrophoblast lining the maternal blood spaces in the placenta and infect the fetus

Question 27

what are the epidemiological consequences of where listeria is found *

Answer 27

it is the basis for strict hygiene rules in commercial food production - utensils for uncooked fruit and veg must be kept separate from those for other foods

at risk gps should avoid products made from unpasteurised milk eg soft cheeses and refrigerated foods particularly likely to have listeria eg pates

not to eat food past the use by date