actin cytoskeleton exploitation by listeria Flashcards
what is listeria monocytogenes
a bacterium
contaminates foods
gram +ve - thick peptidoglycan
risk factors for infection by listeria *
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
what is it about listeria that makes it dangerous *
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)
how do listeria cross the membrane *
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
mechanism listeria use for phagocytosis *
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
describe E cadherin *
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
why does listeria have to be able to spread through the cytoplasm *
Without motility the bacteria cannot spread through the infected cell efficiently and cannot exit the cell or invade neighbouring cells.
describe the mechanism of motility of the bacterial cells *
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
effect if mutation in ActA *
can form microcolonies in cells, but cant spread from cell to cell so are not virulent
what are the phases in actin polymerisation *
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
how does listeria invade neighbouring cells *
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
what is the enzymatic role of actin
hydrolyses ATP to ADP slowly
what are the 2 ends of actin *
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
what angle is actin branched at *
70 degrees - because the active siute of the protein involved is 70degrees apart
what is the structure of ActA *
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
what is the immune response to listeria *
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
what is the worse consequence of listeria
meningitis (because cross BBB)
can effect the cortex = siezures
what side of the comet tail are actin added to *
the bacterial side
where does the energy for movement of listeria come from *
growth of filament goves propulsion
how can you stop the spreading of listeria
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
where is listeria found *
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
decribe nucleation *
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
describe elongation *
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.
what are the effects of different actin binding proteins *
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).
