Notes From Feb 19th Handout

1) Phagocytosis Overview
Engulfment of microbe by a cell. Receptors on cell surface
i) Professional phagocytes – cells that are specialized in phagocizing
A. neutrophils, dendritic cells, macrophages, anything with –phagos in the name.
B. Non – professtional phagocytes are capable of engulfing but is not their main job.
Ex: epithelial cells
ii) Phagocytosis – receptor mediated engulfing of a large particle stuck on surface is the to engulf it.
A. Any type of phagocytosis (receptor mediated) will involve cytoskeleton rearrangements.
iii) Macropinocytosis – drinking cells by pulling in droplets into a vacuole.
A. requires rearrangement of cytoskeleton to engulf droplets from surrounding environment, does not involve receptors or interaction with anything else.
B. Not receptor mediated
C. Bigger droplets.
2) Steps of phagocytosis
i) Binding of receptors on phagocytes
A. phagocytes bind to opsonins that have been attached to microbes.
B. Receptors on phagocytes called fc receptors would be on phagocyte interacting with conserved region of antibodies to engulf microbes.
Ex: mannose binding lectorsmannose binding lectorscarbohydrate motifs on microbes.
C. Phagocytosis of opsonin with no microbes attached to them. Harder to bind.
Ex: Mannose receptor on surface of professional phagocytes interacts with mannose containing molecules on microbes.
D. Dectin-1 binds to other beta-glucan carbohydrates on microbe cell walls.
ii) Actin polymerization
A. Caused by acation of Rho-family GTPases which are triggered by internal events or binding of receptor on phagocytes.
B. More receptors on phagocytes that are bound to a microbe, the more rapid the engulfment of the microbe will be.
a. CR3 (phagocyte receptor) binds to C3B and can also interact with microbe specific carbohydrate motifs if CR3 is binding to CB3 the internalization of interaction will go slow, but if CR3 interacts with C3b and beta-glucans it will go really fast.
b. Example of a receptor that binds both an opsonin and directly to microbe = CR3: binds antibody conserved region; binds β-glucan in bacterial cell wall.
iii) Phagolysosome formation – destroys the microbe after the engulfment of it.
A. Vacuoles containing microbe fuses with other vacuoles containing antimicrobial chemicals
B. Neutrophils engulfing and killing bacteria
a. involved in defense of bacteria
b. granuocytes
(b) Primary granules – granules in neutrophils that contain antimicrobial peptides
—pokes holes in membrane because of peptides amphipathic and cationic properies
1. Antimicrobial peptides
a. Lysosomes breaks peptoglycagen
b. BPI protein (pokes holes in membrane)
2. Proteases – primary granules can contain proteases (any kind of enzyme that breaks down proteins)
iv) Secondary granules and Reactive Oxygen Species (ROS)
A. fuses with vacuole in microbe
B. Contains enzyme (NADPH oxidase) creates reactive oxygen intermediates which create free radicals. Free radicals destroy DNA and denature proteins.
(a) Why toxic?
ROS leads to creation of free Oxygen radical with are free to bounce around and cause problems in DNA and proteins.
(b) Reactive nitrogen species similarly lead to the free radicals which can damage DNA and proteins.
***iNOS(inducible nitric oxide synthase)
*transcription, translation of iNOS turned on in response to phagocytosis
*iNOS transcription activated by LPS, other bacterial products, IFNγ (interferon-gamma
(c) Hypochlorous acid- reduces pH inside vacuole containing microbe  denaturing of microbial proteins.
v) Defects in ROS mediated killing of microbes
1. Lack gene for NADPH oxidase have: chromic granulomatous infections
a. Granuloma: tumor-ish granulocyte growth; whole bunch of granulocytes surrounding a bacterial infection
b. Pus= lots of dead neutrophils + bacteria
2. Lack gene for NADPH oxidase and iNOs: killed by normal gut flora in a few weeks
vi) Parasite strategies based on phagolysosome action
1. Diphtheria: respiratory infections; symptoms caused by exotoxins(proteins secreted by bacteria that damage your cells)
2. Pertussis: respiratory infection, symptoms caused by exotoxins
3. Both: toxins are activated by low pH in pagolysosome
4. Viral replication relies on degradation of its capsid by phagolysosome proteases
3) Variation on phagocytosis
Macropinocytosis-like mechanism of intracellular parasites
i. Rho-GTPases actin rearrangement
ii. Yersinia pestis(black plaque): uses Rho-GTPases to force host cell to engulf it (not
lead to primary or secondary granuolocytes)
iii. Salmonella use same method
iv. Some respiratory pathogens (S. pyogees, S.pneumoniae) bind to epithelial cell
receptors, trigger phagocytosis by non-prof. phagocytes that can’t kill them.

i) Macrophinocytosis-like mechanism of intracellular parasites
ii) Phagocytosis of apoptotic cells
iii) Microbial evasion of killing by phagolysosome

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