General aspects of this review - specific examples were addressed in class.

Transcription

1 General aspects of this review - specific examples were addressed in class. 1

2 Exam 1 Lecture 2: Discussed intracellular killing mechanisms Important maturation steps Rapid development into a microbicidal organelle 2

3 Choreographed fusion and fission events involving endocytic pathway compartments. Early, Intermediate and Late maturation stages. Fcγ receptor pathway best understood bacterium Ig molecules Fc receptors Exposure to Ig induces clustering of receptors initiates signaling to tyrosine kinases for further downstream signaling. Leads to the remodelling of actin for pseudopod extension. Maturation begins early! Considerable amount of plasma membrane is internalizes. Paradox: during phagocytosis plasma membrane surface area increases! Recycling of endosomeal compartment (exosomal). 3

4 Early events may begin as early as during phagosome sealing Early events Early phagosome gains properties of early endosomes (fusion event). Mildly acidic Marker Rab5 (GTPase) Uses effector proteins VPS34, EEA1 - tethering bridge Increase of PI(3)P rounds of fusion with vesicles (endomembrane) - but no real increase in phagosome size. continual recycling of endosomes Phosphoinositides are phosphorylated lipids produced at cell membranes during signaling events. Prominent role as a second messenger (direct or indirect signaling) such as diacylglycerol or inositol trisphosphate and phosphatidylinositol 3,4,5 -trisphosphate. Prominent role in cytoskeletal rearrangements - absolutely necessary for phagocytosis. Possess features not present on early phagosome, but also lacks features of late phagosomes Intermediate events Similar to Multivesicular body - transit vesicle between early and late endosomes. internal vesicles rich in phosphoinositides. sorting of some proteins destined for degradation. Notice the ph of the vesicle is decreasing? Why? 4

5 Late events - preparation for lysosomal fusion Late events Much more acidic environment Increase of V-ATPase Enriched in LAMP1 - lysosomal - associated membrane protein Rab7 is a characteristic marker RILP recruitment by Rab7 RIPL bridging between phagosome and lysosomes via microtubule motor complex MHC II preentation Increase in Cathepsins and Hydrolases Mature phagolysosome - the ultimate microbicidal organelle! Phagolysosome Highly acidic environment Dramatic increase in V-ATPase Lysosomal markers LAMP1 & LAMP2 Increase in Cathepsins and Hydrolases Build on information from Lecture 2 Concept: phagosome needs to acquire a full arsenal of antimicrobial features. Acidification of Phagosome Reactive Oxygen & Nitrogen Antimicrobial peptides and proteins 5

6 Acidification of Phagosome V-ATPases - H+ Favors hydrolytic enzymes Reactive Oxygen & Nitrogen Inducible Nitric Oxide synthase Increase in Reactive Oxygen species (ROS) Increase in Reactive Nitrogen species (RNS) Refer to Lecture 2 regarding respiratory burst enzymes Evasion: Nature 3:11, 2002 editorial Induction of apoptosis Uptake Green: host Orange: bacterial Down-regulate Escape endosomal trafficking Escape phagosome Maturation Defense factors 6

7 Should I stay or should I go? These are just a couple of representatives 7

8 Early endosome Multiple Levels of Evasion Arrest phagosome maturation PIM - phosphatidylinositol mannoside LAM - lipoarabinomannin SapM - phosphatase Counteract inflammatory response ZmpA - zinc metalloprotease that inhibits IL-1b processing Reprograms Maturation Pathway Creates intracellular niche for replication avoids interaction with endosomal pathway promotes fusion with ER-derived vesicles DrrA and LidA effector molecules recruit host cel Rab1 Clearly many other of the T4SS effector molecules (~80) are also required. DrrA and RalF mutants can still surive and replicate in the vacuole - suggesting redundant pathways are present 8

9 Phagosomal Escape Artist Modify and escape from phagosome Combination of effectors LLO - cholesterol-dep. Listeriolysin O Activated by acidification in phagosome Can create pores within 5 min Leakage of Ca2+ and H+ PlcA - phospholipase - PI specific PlcB - braod range phospholipase Three effectors causes breakdown of phagosome membrane Cellular motility required host cell actin ActA polymerizes actin Weathering the storm Resides in the acidified compartment! Interacts with default endocytic pathway fusion with APV (autophagocytic vesicles) Delayed maturation (fusion w/lysosomes) Allows time to start replication program Acidic environment required for replication. Induces SOS DNA repair 9