Lecture 10 - Listeria Monocytogenes Flashcards
Listeria monocytogenes is a gram-______, ___-shaped bacterium.
It is an _____ pathogen and its life cycle reflects ______ to survival in host cell.
It is ______ in the environment (soil, water, food products, humans, and animals).
It causes the food-borne disease called ______.
It mainly affects _______ individuals (elderly, pregnant women, and newborns).
positive; rod
intracellular; adaptation
ubiquitous
listeriosis
immunocompromised
What can listeriosis manifest as?
Why is there so many diverse clinical manifestations?
It can manifest as gastroenteritis, meningitis, encephalitis, mother-to-fetus infection and septicaemia.
It is able cross 3 barriers in human host!
What are the 3 barriers L. Monocytogenes can cross in humans?
After ingestion of contamination food, it can traverse INTESTINAL BARRIER where it can then enter the bloodstream and spread to target tissues (liver/spleen).
In some cases (immunocompromised host), it may cross the BLOOD-BRAIN barrier or FETOPLACENTAL barrier and potentially cause fatal meningitis, sepsis, premature birth or abortion.
The survival & replication of several intracellular pathogens requires that they establish a niche either in the host cytosol or within membrane-enclosed compartments.
Endosomes can fuse with lysosomes so these pathogens have evolved strategies to block their encounter with the lysosomal env.
What are the three strategies? Which one is used by L. monocytogenes?
1) Some bacterial pathogens escape from endosomal compartment before fusion with lysosomes.
2) Some bacterial pathogens remain in endosomes, but modify the endosomes in a manner that blocks lysosomal fusion.
3) Some bacterial pathogens are able to adapt to the harsh lysosomal conditions.
L. monocytogenes uses 1!
Match the steps of L. monocytogenes infection in human host.
1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5
A) ESCAPE: Vacuolar escape is mediated by 3 virulence factors:
Phospholipase A (PlcA), Phospholipase B (PlcB) and Listeriolysin O (LLO).
B) GENOMIC EFFECTS: Listeria proteins can lead to changes in histone modifications within the host genome, leading to altered chromatin packing & changes in gene expression. Infection can also lead to DNA damage
C) CELLULAR EFFECTS: LLO can also lead to desumoylation of target proteins (alters signalling), mitochondrial fission, ER stress, lysosomal permeabilization, pore formation & disruption of ion balance.
D) ENTRY: Entry into non-phagocytic cells (i.e. epithelial cells, goblet cells) occurs via receptor-mediated endocytosis. Entry into phagocytes (i.e. macrophages) occurs via phagocytosis.
E) ACTIN-BASED MOTILITY: Following vacuolar escape, Listeria polymerizes actin and can spread from cell to cell via actinbased motility
1) ENTRY: Entry into non-phagocytic cells (i.e. epithelial cells, goblet cells) occurs via receptor-mediated endocytosis. Entry into phagocytes (i.e. macrophages) occurs via phagocytosis.
2) ESCAPE: Vacuolar escape is mediated by 3 virulence factors:
Phospholipase A (PlcA), Phospholipase B (PlcB) and Listeriolysin O (LLO).
3) ACTIN-BASED MOTILITY: Following vacuolar escape, Listeria polymerizes actin and can spread from cell to cell via actinbased motility
4) CELLULAR EFFECTS: LLO can also lead to desumoylation of target proteins (alters signalling), mitochondrial fission, ER stress, lysosomal permeabilization, pore formation & disruption of ion balance.
5) GENOMIC EFFECTS: Listeria proteins can lead to changes in
histone modifications within the host genome, leading to altered chromatin packing & changes in gene expression.
Infection can also lead to DNA damage.
Fill in the blanks regarding the entry into host cells:
1) L. monocytogenes may be internalized by ________ or ________ cells.
2) Phagocytic cells (i.e. macrophages) internalize Listeria via ______.
3) Non-phagocytic cells (i.e. epithelial cells, goblet cells) internalize Listeria via ____-_____ _____ that requires two bacterial proteins called _______ and _______.
4) InlA binds to host cell adhesion molecule ‘_______’
5) InlB binds host growth factor receptor ‘___’
1) phagocytic; non-phagocytic
2) phagocytosis
3) receptor-mediated endocytosis; Internalin A (InlA); Internalin B (InlB).
4)E-cadherin
5) Met
*different cell types express E-cadherin or Met
*receptor mediated endocytosis can promote uptake of host receptors to regulate cell signalling - Listeria hijacks this process for its own uptake
What happens when InIA interacts with E-cadherin or when InIB interacts with Met?
Interaction leads to E-cadherin or met clustering, phosphorylation and ubiquitination, causing drastic changes in the actin cytoskeleton to allow for bacterial uptake.
1) What is Listeriolysin O (LLO)?
2) How many domains does it consist of?
3) Which family is it part of?
4) What is a unique feature of Listeria with LLO?
5) How does it differ from other pore-forming toxins?
1) Primary virulence factor facilitating escape from the phagosome and enables cell to cell spread.
2) 4 main domains
3) Member of the pore-forming cholesterol-dependent cytolysin family secreted by several gram positive bacteria.
4) A unique feature of Listeria is that it can secrete LLO when it is INSIDE the host cell.
5) Unlike other types of pore-forming toxins, LLO can form pores at low pH (<6).
How does the LLO cytoplasm form pores in host membranes?
LLO activity is OPTIMAL AT ACIDIC pH (within phagosome). At acidic pH, the soluble LLO monomer interacts with endosomal host membrane (cholesterol-dependent).
Upon membrane contact, structural changes occur in LLO. Structural changes in each monomer exposes residues that can form HYDROGEN BONDS with other monomers, promoting the formation of the PRE-PORE OLIGOMER.
Following oligomerization, regions from D3 (domain 3) of each monomer extend to form TRANSMEMBRANE b-hairpins that can ultimately PUNCTURE THE MEMBRANE.
How is LLO controlled by acidity? What happens during neutral pH?
At neutral pH (i.e. in host cytoplasm), domain 3 (D3) of the monomer UNFOLS PREMATURELY which leads to the INACTIVATION pore-forming abilities of LLO!
(T/F) The only function of LLO is to generate pores within endosomal membranes.
False!
LLO seems to have newly discovered roles at host cell plasma membrane BEFORE ENTRY and within the host cell AFTER VACUOLE ESCAPE.
Once Listeria has been internalized into the host cell and succeeds in escaping from the phagosome. it then ______ & ______ actin tails, allowing bacterium to migrate to neighbouring host cell.
nucleates; polymerizes actin
*Listeria hijacks the host actin cytoskeletom
*F actin = filamentous actin
Fill in the blanks regarding the model for actin filament nucleation by the Arp2/3 complex.
1) In the absence of an ______ factor, Arp2/3 reside in a conformation that _______ actin nucleation.
2) When an activating factor binds Arp2/3, it establishes a new confirmation that ______ the end of an actin filament.
3) _____ _____ then assemble from the Arp2/3 complex.
4) Arp2/3 nucleates actin filaments most efficiently when it is bound to a __-_______ ______ (extends at a __˚ angle)
activating; prevents
mimics (this attracts actin monomers)
actin subunits
pre-existing filaments; 70˚
1) What is ActA?
2) What is its mechanism of action?
3) What is cofilin?
4) Where is actin polymerization focused at of the bacterium? Why?
1) ActA resembles the “activating factor”; it is a Listeria cell-surface protein that polymerizes host actin.
2) ActA binds and activates the Arp2/3 complex to nucleate filament assembly along the sides of existing filaments. Filaments grow at their plus end until capped by capping protein.
3) Actin is recycled through the action of COFILIN, which enhances depolymerization at the minus end of the filaments.
4) Actin polymerization is focused at the rear surface of the bacterium which ultimately propels it forward (uni-directional movement)
What are the four roles of extracellular LLO and the cellular effects they cause?
1) Formation of LLO pores in HOST PLASMA membrane shown to cause Ca2+ influx, which leads to MITOCHONDRIAL FRAGMENTATION.
2) Formation of LLO pores in host plasma membrane shown to cause K+ efflux, which leads to INFLAMMASOME ACTIVATION and HISTONE MODIFICATION (gene expression alteration).
3) LLO promotes degradation of the E2 component of the SUMOylation pathway, resulting in disruption of a range of cellular processes (transcription, genome integrity, stress response)
4) LLO can induce autophagy
