Killing by Phagocytes Flashcards
explain Intracellular Bacterial Killing
- Phagosome
- oxygen independent by lysosomal enzymes
- Oxygen dependent killing by NO and Reactive Oxygen Intermediates
what are the Two main types of Lysosomes in Neutrophils?
Specific granules (Specific to mature neutrophils) Azurophil granules
what are Specific granules ?
- made late in PMN development
- Contain alkaline phosphatase, lysozyme, and lactoferrin
- Function best at neutral or alkaline pH
what are Azurophil granules?
- made early in PMN development
- Contain peroxidase, and the”acid hydrolase” enzymes, defensins, also lysozyme
- Most have pH optima of pH4.5 and lower
who discovered Sequential fusing of different lysosomes is linked to the pH optima of their contents?
Dorothy F. Bainton, 1973
what is the pH optima of Specific granules?
- Function best at neutral or alkaline pH
what is the pH optima of Azurophil granules?
- pH optima 4.5 or lower
- e.g. peroxidase, ”acid hydrolase” enzymes, lysozyme,
- Azurophilic granules fuse with phagosomes later
- pH of phagocytic vacuoles drops to 6.5 within 3 min and to 4 within 7–15 min.
- The pH drop is due to a proton pump (V Type H+ ATPase) in the azurophilic membrane
what is Oxygen independent killing?
Destruction of microbes by lysosomal contents, i.e:
1) Anti-microbial peptides
2) Enzymes
3) Nutrient starvation (prevention of growth)
name 2 Lysosomal anti-microbial peptides
Defensives and Bactericidal permeability increasing protein (BPI)
what are Defensins?
4 types Highly cationic (+ve charge) peptides/proteins Adhere to -ve charged bacterial cell membranes and punch holes
what are BPIs?
Highly cationic
Binds Gram -ve bacteria (via LPS)
name the 2 types of Lysosomal enzymes
Lysozyme and “Acid Hydrolases”
what the action of Lysozyme?
- Splits peptidoglycan in bacterial cell walls
- Constitutively expressed by macrophages
- Stored in lysosomes and secreted
name 4 Acid Hydrolases?
Proteases
DNAses
Lipases
Glycopeptideases
what do acid hydrolases do?
Digest dead organisms
name 2 Lysosomal nutrient binding proteins
Lactoferrin- Deprives proliferating bacteria of iron
Vitamin B12 binding protein- Deprives proliferating bacteria of cobalt
what do Lysosomal nutrient binding proteins do?
Bind growth limiting metal ions required for microbial enzyme functionality
describe Oxygen-dependent killing
- Enzymes add electrons to oxygen to make radical “Superoxide”
- Enzymes react to form Reactive Oxygen Species (ROS)
what are Free Radicals?
- a cluster of atoms, one of which contains an unpaired electron in its outermost shell of electrons.
- extremely unstable configuration, and radicals quickly react with other molecules or radicals to achieve a stable configuration
what is NADPH?
- an electron carrier.
- molecule exists in two forms that vary in whether or not they are carrying electrons.
- NADPH is the reduced form
what does NADPH oxidase do?
- assembly stimulated by microbes
- produces oxygen radicals inside the phagolysosome
- converts o2 to superoxide o2-
- 2nd enzyme superoxide dismutase convert superoxide to hydrogen peroxide.
- peroxidase enzymes and Fe convert h2o2 to hypochlorite ions and hydroxyl ions.
what is the affect of Hypochlorous acid and chloramines?
- increase bactericidal power of ROI system by destroying biologically important proteins through chlorination and bromination.
what are Myeloperoxidases
- MPO is not found in macrophages, which lose MPO within 2 days of entering the tissues.
- MPO synthesized during promyelocyte stage, prior to final granulocyte/ monocyte commitment.
- Peroxidases contain iron in haem-like groups, which means neutrophils or e.g. nasal mucus containing them appear green
how does OH (hydroxyl radical) cause damage?
- short-lived, powerful oxidant with high antibacterial activity, causing damage to DNA, membrane lipids, and proteins.
- hydroxyl radical is most reactive radical known.
- can attack and damage every molecule found in living cells
what is the “Washing machine” analogy
- Alkaline cycle
Lysozyme
2. Acid cycle Enzymes (protease, lipase, glycopeptidase, DNAse) Detergent (cationic peptides) Superoxide, Hydrogen Peroxide Hydroxyl Radicals, Singlet oxygen Bleach (Hyperchlorous acid) Nitric oxide
what are Reactive Nitrogen Species
- principal RNI is nitric oxide (NO) derived from terminal guanidino-nitrogen atom of L-arginine.
- reaction catalyzed by inducible form of nitric oxide synthase (iNOS), leading to formation of NO.
what up-regulate iNOS
Microbes and Interferon gamma
what are the Oxygen-dependent killing pathways?
Parallel and interacting metabolic pathways generate reactive oxygen intermediates (*ROI) and nitrogen intermediates in phagocytes
Summarise phagocyte killing mechanisms
- Acidification- bactericidal/static
- toxic o2 derived- o2-, H2O2, 1o2, OH, OCL-
- toxic nitrogen oxides- NO
- AMP- defusing
- Enzyme- lysozyme and acid hydrolases
- competitors- lactorferin
summarise Oxygen independent killing
- Fusion with lysosomes
Defensins
Degradative enzymes e.g., lysozyme, proteases
Binding of metal cations - Late phagosomal membranes contain ATPases that pump protons into vacuole (pH ~ 4)
- Lower pH activates acid hydrolase enzymes
summarise Oxygen dependent Respiratory Burst killing
- Activated following phagocytosis
- Stimulated by Pathogen Recognition Receptors
- Requires increased oxygen consumption
- Produces substances that are directly toxic to the bacteria
what are the substances directly toxic to bacteria?
- Oxygen-derived products
O2-, H2O2 , Hydroxyl radicals (. OH) - Nitrogen-derived products
NO (nitrogen oxide)
Produced by inducible NO synthase (iNOS) enzyme
Enzyme is induced by cytokines (Interferon )
name a Respiratory burst deficiencies
chronic granulomatous disease
