1
Q
Complement
A
- innate immune killing mechanism of blood
- pre-formed effector exploited by adaptive system
- intrinsic antibacterial killing mechanism of blood
- free bacteria in blood are unusual because they engage a key immediate innate response
2
Q
Complement and humoral defense
A
historically cell free serum was observed to kill many bacteria
- important features: facilitates by antibody but does not require specific antibody
- some bacteria are resistant
3
Q
Complement Naming
A
- C1-C9 are classical complement components
- B, H, D, I are regulatory factors (accessory factors)
- when complement factors are cleaved the products get a letter appended (C3 becomes C3a and C3b)
4
Q
Alternative complement pathway
A
- complement component C3 present at high concentration in serum
- C3 is a central protein
5
Q
C3
A
- has a sequence of residues forming an internal covalent cysteine-glutamine thioester bond (unstable but shielded from water)
- at some small frequency the thioester is hydrolysed
- hydrolysis generates a conformational change (water leaks in and hydrolyses the bond)
- C3(H2O) binds a circulating serine protease zymogen, factor B
- C2(H2O)B is cleaved by another serine protease factor D generating C3(H2O)Bb and Ba
- Ba floats away
- C3(H2O)Bb is able to cleave other C3 molecules (convertase serine protease enzyme)
- ** see notes ***
6
Q
C3 Cleavage
A
- critical event in complement activation
- C3 is cleaved into C3a and C3b
- C3a is a signalling molecule: it drives smooth muscle contraction, macrophage recruitment, mast cell degranulation, vascular permeabilization
- in C3b the thioester bond is exposed and reactive - reacts with water or with local hydroxyl or amine groups
- if C3b reacts to a group on the bacterial surface it recruits B, D factors
- a factor is proinflammatory and C3b is a structural component carrying the bond
7
Q
Bacterial Reaction
A
- biological surfaces react with the thioester of C3b and Bb (active convertase)
- bound to the microbe surface
- amplification of C3b bound to the bacterium surface
- C3b is an opsonin : surface becomes covered with C3b and are recognised as foreign by phagocytes and targeted for phagocytosis
8
Q
Higher order complexes
A
- at high concentrations of C3 there is competition for binding to factor B
- multiple C3 bind to a single factor B
9
Q
C5
A
- cleaved and activated by C3bBbC3b
- splits into C5a and C5b
- C5a is a signalling molecule and potent inflammatory signal to dendrites
10
Q
C5 fragments
A
- each C5 fragment has a function
- C5a is an anaphylotxoin but more potent than C3a
- signals to immature dendritic cells to enhance production of antibacterial helper T cells
- C5b nucleates the membrane attack complex (C5b,C6,C7,C8,C9)
- C9 multimerizes to form a pore in biological membranes directly lysing foreign bodies
11
Q
MAC Formation
A
- C5b binds C6 and C7
- C7 has membrane affinity and recruits C8
- C8 inserts into the cell membrane
- C9 molecule binds and polymerises to the complex (pore forms)
- 10-16 molecules of C9 bind to form the pore
12
Q
Phagocytosis
A
- via macrophages requires recognition by phagocytic receptors
- C3b is an opsonin
- C3b bound on microbial surfaces is recognised by C3R, a phagocytic receptor, to drive uptake of labeled microbes
13
Q
Classical complement Pathway
A
- role for antibody
- alternative pathway (initiated by spontaneous C3 hydrolysis) is 80-90% of successful complement activation
- classical pathway affords a mechanism for complement to be activated by specific antibodies (not so common)
- example of adaptive immunity using innate immune mechanisms to promote microbial killing (antibody recruitment of complement system)
14
Q
Antibodies
A
- mainly IgG
- bind a bacterium in serum
- variable region binds antigen and constant region (Fc region) is bound by other recognition proteins of the immune system such as C1
15
Q
C1
A
- C1 is a complex containing 5 peptides (C1qC1r2C1s2)
- C1q binds Fc constant antibody region
- C1r and C1s are serine protease zymogens
- C1q binding to Fc activates C1r activity : C1r cleaves and activates C1s
16
Q
C4
A
- C1s cleaves C4
- C4 is like C3 : contains a thioester bond (C4b) that is revealed by cleavage
- C4 cannot have spontaneous cleavage like C3
- C4a is an anaphylotoxin : C4b binds microbial surfaces (like the C3b reaction with hydroxyl groups of bacterial surfaces)
- triggers inflammatory signalling pathways
- C4b binds C2 and cleaves by C1s to form C4bC2a
- C4bC2a is a C3 convertase (serine protease) just like C3bBb
- bacterium is opsonized with C3b and lysed
17
Q
General Mechanisms of Classical Pathways
A
- generalise to other circulating receptors
- example is the mannose binding lectin pathway
- MBL binds mannose and recruits MASP1 and MASP2 (MBL associated serine proteases like C1r/s)
- MASP1/2 cleave and activate C4 then C2 as in the classical pathway
- therefore: circulating receptors utilise/recruit complement systems
18
Q
Effector Cassette
A
- detection mechanisms funnel into a consistent effector cassette
- antibody activates C1 and then C4bC2a
- MBL activates MASP1/2 and then C4bC2a
- C3 spontaneous activation
- all funnel into C3 activation and opsonization/phagocytosis or the C5 activated membrane attack complex
19
Q
Host Control of Complement
A
- C3a is a signalling molecule driving smooth muscle contraction, macrophage recruitment, etc, etc
- in C3b the thioester bond is exposed and reactive reacting with water or with local hydroxyl/amine groups
- host cells are covered in these groups and anaphylotoxins such as C3a, C4a, C5a have symptoms of septic shock
- control pathways prevent overactivity and host targeting
20
Q
Complement Regulatory Factors
A
- H, I, P, CD46
- H binds glycosaminoglycans (surface of host cells) and recruits factor I
- CD46 is a TM protein produced in host cells recruiting factor I
- I is a serine protease cleaving and inactvating C3b and C4b preventing opsonization and killing the amplification step of complement activation (terminates the process of host cell complement activation)
- P is an activating factor binding biological surfaces and increasing the half life of C3bBb (released by neutrophils)
- enhance stability of complement activating systems
21
Q
Complement Deficiencies
A
- people who lack C1q or C2 have autoimmune disease (failure to clear complexes of cell debris with antibodies)
- people who lack C3 are susceptible to infections with pyogenic bacteria (gram negative as MAC is good at targeting gram negative bacteria)
- people who lack MAC components (C5-C9) are similar
22
Q
Bacterial Evasion
A
General mechanisms disrupting general immune recognition
- polysaccharide capsules
- inhibition of antibody binding
Complement specific effects
- mimickry of host cell surfaces
- hiding inside host cells
23
Q
Polysaccharide Capsule
A
- escape from complement
- layer is slimy forming diffusion barrier
- impairs complement mediated killing in several ways
- capsule excludes efficient diffusion of C3 to bacterial surface - so that activated C3 is mostly exposed to capsule (not the membrane)
- MAC can’t get to membrane making opsonisation less efficient
- because C5 convertase is bound at capsule surface, efficiency of membrane attack is severely diminished
- diffusion within the capsule is inefficient : distance between activated C3 convertase complexes is high reducing amplification of effectiveness
- capsule inhibits phagocytosis by increasing effective size of bacterium
24
Q
Capsular Targets
A
- capsules can still be targets for Abs and complements
- capsular polysaccharides are recognized as non host due to antibodies
- diversity of capsules indicates selective pressure of bacteria
25
Streptococcus pyogenes
- mimics the host
- uses more targeted mechanisms
- capsule : glycosaminoglycan binds host factor H to impair complement activation
- M protein recruits factor H
- ultimate goal is to recruit endogenous protective mechanism
26
Neisseria meningitidis
- uses mimickry and interference
- LPS is sialylated to recruit factor H and facilitate interaction with C3
- secretes factors that bind and degrades C3
- impairment and degradation of complement amplification
27
Staphylococcus aureus
- uses many mechanisms
- Efb: secreted to bind and inhibit C3
- Protein A: binds Fc and inhibits C1q binding (inhibition of classical complement system)
- SdrE: TM protein binding C4BP to recruit H
ICB
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