1

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

2

Exam 1

Lecture 2: Discussed intracellular killing mechanisms

Rapid development into a microbicidal organelle

Important maturation

steps

3

Choreographed fusion and fission events involving endocytic pathway compartments.

Early, Intermediate and Late maturation stages.

Fcγ receptor pathway best understood

Fc receptors

bacterium Ig molecules

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).

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

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?

Intermediate events

5

Late events - preparation for lysosomal fusion

• 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

Late events

Mature phagolysosome - the ultimate microbicidal organelle!

• Highly acidic environment • Dramatic increase in V-ATPase • Lysosomal markers LAMP1 & LAMP2 • Increase in Cathepsins and Hydrolases

Phagolysosome

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

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

Down-regulate

Escape endosomal trafficking

Escape phagosome Maturation

Defense factors

Induction of apoptosis Uptake

Green: host Orange: bacterial

7

Should I stay or should I go?

These are just a couple of representatives

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

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