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91 Cards in this Set
- Front
- Back
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What are the 2 points of view about phagocytosis?
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1) Actin dependent: endocytic process used by cells to engulf and destroy foreign/apoptotic bodies
2) Common mechanism by which intracellular pathogens invade host cells and thus avoid direct destruction by serum components or by cytotoxic cells (induce their own internalization) |
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What's the difference btw phagocytosis and endocytosis?
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Size of body engulfed
Phago: > 0.5uM Endo: particels <0.5uM |
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What is phagocytosis important for?
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1) Nutrition: need to eat
2) Embryogenesis: for multi cell organisms, used for modificiation of embryo 3) Tissue remodelling: formation of limb, repair wounds 4) Immunity: link btw innate and adaptive |
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What's important about the mb size during phagocytosis?
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Rapidly grows during phagocytosis because can ingest volume of particles greater than the amoeba
->all the particles end up in a vacuole surrounded by mb |
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What are considered professional phagocytes?
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Monocytes/macs
Neutrophils |
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What are considered non-professional phagocytes?
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Epithelial cells
Fibroblasts |
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What are professional phagocytes responsible for?
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Sentinels of the immune system
Destroy senescent, apoptotic or defective cells, pollutant particles and potentially pathogenic microorganisms |
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What are non-professional phagocytes responsible for?
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Low phagocytic competence
"Induced" phagocytosis ->This is what happens to salmonella in the gut |
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Describe the role of phagocytosis in immunity
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Microbes rec'z pathogens through TLRs
Phagocytic receptors rec'z ligands and send signals Phagocytosis of bact into vacuole *All important events for immune response takes place in the vacuole Degrade microbe Present peptides ->Ag that is internalized is presented more efficiently than soluble Ag |
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What kind of signals are involved in phagocytosis?
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Inhibitory and activating signals
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Whatr are the 2 important signals in phagocytosis?
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Rho GTPase: early phase of phagocytosis, actin remodellin
PI3 kinase: generation of phosphoinnositides, can be targetted by intracellular path (salmonella/legionella) |
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Describe phagolysosome biogenesis
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1) Ingestion
make a phagocytic cup, made of polymerized actin, which will grow and create a bud mb seals when pseudopods touch Neutral ph (7-7.5) 2) Phagosome Mainly made of plasma mb When actin polymerized, allows interaction of phagosome with endosomal system Recruitment of H+ pump and hydrolases Acidification of phagosome 3) Phagolysosome: At acidic pH, almost everything destroyed Filled with hydrolases Very microbicidal |
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Which one of these stages are usually inhibited by pathogens?
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Maturation btw phagosome and phagolysosome
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What is focal exocytosis?
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Process where endosome comes to mb and when it touches, phagocytic cup fuses with the microbe
-> mb exocytosis |
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Where does the extra mb come from?
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Late endosome/lysosome
Recycling endosomes ER |
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What are the marker's ass't with the early phagosome?
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EEA-1
rab5 Low lvls of V-ATPase |
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What are the markers of the late endosome?
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Rab7
LAMP-1 Higher lvls of V-ATPase Becomes more acidic |
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What happens to the size of the phagosome?
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Doesn't change
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What is the kiss and run model?
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Explains why the phagosome size never changes
1) Endosome and phagosome move along the microtubule 2) pore formation Exchange of material Fission event 3) Go back on microtubule Keeps happening until it uptakes something that makes it leave Interact with early, then late phagosome, than phagolysosome |
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What is the default phagosome?
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Rapid maturation into phagolysosome
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What does the V-ATPase do?
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pump H+ from cytoplasm to lumen
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How do intracellular professional bact pathogens modulate phagosome maturation?
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Block phagocytosis at a specific (early) step
->Characterized by Rab5 presence Block activity of PI3K and phosphoinosites |
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What does legionella pneumophila do?
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Divert traffic btw golgi and ER
Vacuoles they replicate in are very similar to ER env't |
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What does listeria monocytogenes do?
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Escape out of cell
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What does C. burnnitti do?
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Creates an autophagosome
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What are some host microbicidal factors? How are the fought by pathogens?
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V-ATPase: blocked or excluded by many intracell path
Nitric oxide: some microbes secrete things to get rid of NO NADPH oxidase: inhibited and targetted |
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What are microbial defensive mechs?
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Blocking NADPH oxidase and/or V-ATPase
Cleave AMPs at surface of mucrobes |
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What do phagosomes mediate the formation of?
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peptide:class II MHC complexes
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Are phagosomes fully competent Ag presenting organelles?
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Yes
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What can microbe sdo to the phagosome composition and Ag processing?
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Can alter phagosome composition
Can reduce Ag processing |
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Where does Salmonella stop phagocytosis?
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Early stage (around early endosome)
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Describe Salmonella (G+/-, shape, motility)
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G-
Rod Motile |
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What causes typhoid fever?
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S. typhi
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What causes gasroenteritis?
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S. typhimurium
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Describe the 2 pathogenicity islands of Salmonella?
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SP-I: Best characterized of the 2,40 kb, 29 genes
SP-2: 40 kb, 40 genes |
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What do both salmonella pathogenicity islands encode?
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Type 3 secretion systems
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What is the T3SS apparatus?
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Molecular syringe
Other ptns in the bacteria are effectors Includes an ATPase |
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What do most of the effectos of SPI-I code for?
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Entry of salmonella into non-phagocytic cells
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What moecule is important for phagocytosis?
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Actin important for phagocytosis by profesionnal/non phagocytic cells
Injection of effectors affect host cell stage |
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What do the SPI-2 effectors code for?
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Replication in host
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What is a strategy used by many microorganisms when it comes to phagocytosis?
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Hijacking of host cell's cytoskeleton
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Why do microbes hijack the cytoskeleton of the host?
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* Induce internalization by non-phagocytic cells (Salmonella)
*Evade microbicidal mechanisms (Salonella) -To avoid internalization -Motility (listeria) |
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What is a common target to take over actin polymerization?
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Rho family GTPases
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Describe the Rho family
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Member of Ras
17 members Regulates rearrangements of the actin cytoskeleton Important role in phagocytosis: Rac, Cdc42, RhoA |
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What can microorgs do to modulate the activity of Rho-family GTPases?
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Produce effectors
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How does Salmonella modulate activity of the Rho family?
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Injects molecules that are GEFs/GAPs
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What happens when Rho is attached to GTP?
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Active and can be replaced by effectors
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Which SPI encodes direct inhibition of GTPase?
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SPI-1
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What are the key salmonella effector ptns secreted through the SPI-1 T3SS?
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SopE (on): GEF, directly activates both Rac1 and Cdc42
SptP (off): GAP, specfically simulates GTPase activity both Cdc42 and Rac1 SipA: F-actin binding ptn, reduceds [G-actin] required for polymerization, stabilizes actin SipC: part of TTSS delivery pore, nucleates and bundle s actin SopB (SigD): Inositol phosphate polyphosphatase |
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Why does Salmonella modulate the actin cytoskeleton?
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Invade host cells
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How does Salmonella modulate this activity?
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Salmonella makes syringe (T3SS)
Injects effectors Interfers with GTPase (SopE) Take over GEFs and GAPs |
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What effects does SopE have on Cdc42 or Rac1?
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activating
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What effect does Sp1P have on rac1 and cdc42?
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Inhibitory (its a GAP)
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What does salmonella do to epithelial cells?
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Mb ruffling
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What does activation of GEF that induces Rac (and then RAF) cause?
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Actin polymerization
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What happens if there is too muchmb ruffling?
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Cell dies
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Why is mb ruffling important?
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Cell will be able to better engulf the bacteria
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How does Salmonella induce host cell invasion?
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Induction of mb ruffling
Internaliztion in a vacuole: inject effectors to change composition of pahgosome so it can't fuse with the lysosome |
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What ar the stages of temporal modulation of host cell's cytoskeleton?
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1) Delivery of effectors
2) Activation of Rac1/Cdc42 through SopE 3) Initiation of actincytoskeleton reorganization 4) Localized ruffling process 5) Shut-off Rac1 /Cdc42 activation: sptP inactivates GTPases so cell wnt be killed, host cell can't keep rufling for hours |
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Describe Salmonella entry into host
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Depends on coordinated action of several bacterial effector ptns
Effector ptns exert their activity in a temporally coordinated manner Highly regulated process: effector ptns themselves may have regulatory domains that control their activity |
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What are the 3-phosphoinositides?
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Mb phospholipids
Ex: PI3P, PI4P, PI3,5P |
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What are phosphoinositides important for?
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Phagosome maturations
Phosphoinositides are sites for recognition and recruitment |
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What kind of changes do phosphoinositides cause?
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Transient and localized modifications on the mb lipid composition
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What causes deP of phosphoinositides?
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specific PIP phosphatases
SHIP, PTEN |
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What are the specific domains that recognize the specific ptns?
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PH:lipid: PI(3,4,5)P3, ptns: AKT, PLCdelta (>100)
FYVE: PI(3)P, doma: EEA1 Hrs (v/important in phagocytosis) PC: PI(3)P, dom: p47 phox, p40phox, nexins Kinases makes a specific kind of phophoinositide which is rec'z by dif ptns domains and which then serves to recruits dif ptns (effectors) |
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What are phox ptns important for?
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NADPH oxidase
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Describe phosphinositide metabolism during phagocytosis
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Initially: high level of PIP2
Low levels PIP3, DAG and PA During cup formation: increase in DAG, PA and PIP3 (make PIP3 from P of PIP2) Dec in PIP2 During Phagosome sealing; leveling of or decreasing in all Phagosome internalization: Dec in everything Very transient changes that happen during phagocytic |
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What is important for pseudopod formation?
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PIP2
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What is PIP3 important for?
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formation of vacuole
Later steps of phagocytosis |
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What is PI4P mainly associated with?
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Golgi
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What is there high levels of during phagocytosis?
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PIP3
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Which phosphoinositide does Salmonella most affect?
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PIP3
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What did the GFP-FYVE experiment show?
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Synthesis of PIP3 is very localized (only at the mb)
Transent, only lasts a few minutes When PI is there, it can recruit things to the mb, such as effectors or things required for phagosome maturation |
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Describe the subversion of phosphoinositide metabolism by microorgansms
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Prevent phagocytosis
Interfere with maturation of process of phagolysosome Ex: mycobact work on PIP3 to stop its metab Salmonella blocks PIP3 <-> PI5P |
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What is SopB required for?
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Maintain high levels of PI3P n the mbs of SCVs (mb vacuoles)
SopB required in vacuole to block its maturation |
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What does SopB do?
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DeP Pi(3,5)P2 and PI(3,4,5)P3
Prevents transition of PI3P <-> PI(3,5)P SopB arrests the progression of SCV down the vesicular trafficking path leading to lysosomes ->Get enlargement of SCV |
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What happens if the Salmonella is SopB deficient?
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Can't cause PIP3 accumulation
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Waht does SopB prevent?
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SCV-lysosome fusion
-> without sopB, can't block maturation process of phagocygtic vacuole .: bacteria will die |
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How does M tb manipulate phagosomal PI3P lvls to remodel their habitat?
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Prevents reproduction of PIP3 (earlier step)
Inhivits glycolipid Inhibit specific PIP kinase Prevent generation of PIP3 Also makes enz to deP PIP3-> PI STOP maturation 1 step earlier on maturation prcess because prevents formation of PI3P |
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How does Salmonella enterica manipulate phagosomal PI3P lvls to remodel their habitat?
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Prevents disappearance of PIP3
High lvl PIP3 on vacuole mb Affects phagosome maturation |
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What is required for intracellular survival of salmonella?
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SPI-2 TTSS
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Describe the effectors of SPI-2 secretion system
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SpiC: inhibits phagosome-lysosome fusion
No homologues in the sequence databases and motifs that predict fcts SifA: formation of Salmonella induced filaments SseD: exclusion of NADPH oxidase from the phagosome |
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Describe the Salmonella containing vacuole (SCV)
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The SC ends up surrounded by actin mesh
This is due to efffectors secreted by Salmonella When salmpnella replicates, it forms filaments of actin (due to SifA) |
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Why is the assembly of the phagosomal NADPH oxidase important?
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Key microbicidal mechanism of the innate IS
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Why would this be stopped?
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Must avoid exposure to superoxide and other O2 species .: must block NADPH oxidase
Act on assembly of complex at phagosomal mb |
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What is required to evade the NADPH oxidase of the phagocyte?
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SPI-2
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What does SPI-2 interfere with?
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Trafficking of oxidase containing vesicles to the SCV
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What normally happens during phagocytosis?
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Vesicles normally recruited to mb and phagosome can make radicals
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What hapens when Salmonella infection?
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If SPI-2 there, vesicles diffuse with phagosome with S
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What normally happens to bact that are SPI-2 mutants?
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They can't block the recruitment of oxidase to the mb .: they die
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What doSPI-2 mutants cause in gp91phox KO mice?
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These mice can't make oxidase
.: the bacteria cuase lethal infections in these mice |
