1

Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Chapter 13
Complement
Competed Covered
Chapter Preview

▰ General Properties
▰ Complement Pathways
▰ Effector Functions of Complement
▰ Regulation of Complement Pathways
▰ Complement Deficiencies

3
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
COMPLEMENT

▰ Represents a group of proteins normally found in serum in inactive

form, but when activated they augment the immune responses.
▰ Complements constitute about 5% of normal serum proteins.

▰ Their level does not increase following either infection or

vaccination.

4
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
General properties

▰ Bind to Fc region of antibody

▰ Role of antigen

▰ Species nonspecific

▰ Heat labile

5
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Complement Components
▰ Complement system comprises of about 30 serum proteins grouped
into complement components, the properdin system and the
regulatory proteins.
▰ Complement components are named by numerals. There are nine
components; C1 to C9. C1 has three subunits- C1q, C1r and C1s.
▰ Properdin system and the regulatory proteins are named by letter
symbols, e.g., factor-B

6
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Synthesis

▰ Liver is the major site of synthesis of complement proteins.

▰ Minor sites include blood monocytes, tissue macrophages, and

epithelial cells of GIT and genitourinary tract.

7
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Complement Activation

▰ All the complement proteins are synthesized in inactive form (e.g.

zymogens) and are activated by proteolysis.
▰ Complements have two unequal fragments (large and small
fragment).
▰ The larger fragments are usually designated as ‘b’ (e.g. C3b) and the
smaller fragments are designated as ‘a’ (e.g. C3a). An exception is C2a
which is larger fragment.

8
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Complement Activation (Cont..)

▰ During proteolysis, the smaller fragment is removed exposing the

active site of the larger fragment.
▰ The larger fragment participates in the cascade reaction of
complement pathway and the smaller fragment diffuses away to
mediate other functions.

9
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Complement Activation (Cont..)

▰ Cascade reaction- Fragments of complements interact in a definite

sequential manner with a cascade like effect, which leads to
formation of complex. Such complex having enzymatic activity is
designated by putting a bar over the number or symbol

(e.g. C 3bBb).

10
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
COMPLEMENT PATHWAYS
11
COMPLEMENT PATHWAYS

▰ Classical pathway- Antibody dependent pathway. Pathway is triggered

by the antigen antibody complex formation.
▰ Alternative pathway- Antibody independent pathway, triggered by the
antigen directly.
▰ Lectin pathway - recently described pathway. It resembles classical
pathway but it is antibody independent.

12
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Stages of complement activation

▰ There are four main stages in the activation of any of the complement
pathways.
➢ Initiation of the pathway
➢ Formation of C3 convertase
➢ Formation of C5 convertase
➢ Formation of membrane attack complex (MAC)

13
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Stages of complement activation (Cont..)

▰ All the three pathways differ from each other in their initiation till
formation of C3 convertase. Then, the remaining stages are
identical in all the pathways.

14
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Classical Pathway

▰ Antibody dependent
▰ Not all antibodies can bind to complements of classical pathway.
▰ Decreasing order of ability of antibodies to fix complement is- IgM
(most potent) > IgG3> IgG 1> IgG2.
▰ The other classes of antibodies do not fix complements. CH2
domain on IgG, CH4 on IgM participate in complement binding.
▰ The classical pathway begins with activation of C1 and binding to
antigen-antibody complex.
15
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Initiation
▰ The first step is the binding of C1 to the antigen- antibody complex.
▰ The first binding portion of C1 is C1q, which reacts with the Fc portion of IgM or IgG
bound to antigen.
▰ C1q is a hexamer having six globular heads each acting as a combining site.
▰ Effective activation of classical pathway begins only when C1q is attached to the Fc
portion of antibody by at least two of its globular binding sites.

16
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Initiation (Cont..)
▰ C1q binding in the presence of calcium ions, in turn activates sequentially
C1r followed by C1s.

17
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Formation of C3 Convertase

▰ Activated C1s acts as an esterase (C1s esterase), which can cleave C4

to produce C4a (an anaphylatoxin), and C4b which binds to C1 and
participates further in complement cascade.
➢ C14b in the presence of magnesium ions cleaves C2 into C2a,
which remains linked to complement complex, and C2b (has kinin
like activity), which is released outside.
➢ C14b2a is referred to as C3 convertase of the classical
pathway.
18
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Formation of C5 Convertase

▰ C3 convertase hydrolyses many C3 molecules into two fragments:

➢ C3a (an anaphylatoxin)
➢ C3b which remains attached to C14b2a to form C14b2a3b complex
which acts as C5 convertase of classical pathway.

19
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Formation of Membrane Attack Complex
▰ Begins with C5 convertase cleaving C5 into C5a (an anaphylatoxin,
released into the medium) and C5b, which continues with the
cascade.
➢ C5b is extremely labile, gets stabilized by binding soon with C6
and C7 to form C5b67 followed by addition of C8.
➢ Hydrophobic regions on C7 and C8 help in penetration into the
target cell membrane.

20
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Formation of Membrane Attack Complex (Cont..)

➢ This inserted membrane complex (C5b678) has a catalytic property to

bind to C9 molecule and then it polymerizes the C9 into a tubular channel
of 10 nm diameter.
➢ Penetration of C9 - channels or pores on the target cell membrane

➢ Each tubular channel - hydrophobic outside, hydrophilic inside - free

passage of ions and water into the cell - cellular swelling - lysis.

21
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Formation of Membrane Attack Complex (Cont..)

➢ C5b6789 destroys the target cell by attacking the cell membrane – MAC.

➢ Process of cytolysis is referred to as complement-mediated cytotoxicity.

22
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
 23
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Alternative Pathway

▰ Independent of antibody; hence is considered as a part of innate immunity.

▰ Four stages.
▰ Differs from the classical pathway in first two stages.
▰ Three complement components C1, C4 and C2 are not involved. Requires
three other complement proteins present in serum named factor B, factor
D and properdin.

24
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Initiation

25
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Initiation (Cont..)

▰ First complement component to be involved in alternative pathway is

free C3 in the serum.

▰ C3 hydrolyzes spontaneously, to generate C3a which diffuses out and

C3b fragment which attaches to foreign cell surface antigen.

26
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Formation of C3 Convertase

▰ Factor B binds to C3b coated foreign cells.

▰ Factor D - acts on factor B, and cleaves it into Ba (diffuses out) and Bb
(remains attached).
▰ C3bBb - C3 convertase.

▰ C3bBb has a very short half-life of 5 minutes.

▰ Stabilized by properdin (half-life is increased to 30 minutes).

27
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Alternative Pathway (Cont..)
▰ Formation of C5 convertase and formation of membrane attack
complex - identical to that of classical pathway.

28
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Lectin Pathway
▰ Complement pathway of innate immunity -works independent of
antibody.
▰ Mediated through lectin proteins of the host that interact with mannose
residues present on microbial surface.
▰ Lectin pathway involves all complement components used for classical
pathways except C1.
▰ Instead of C1, host lectin protein called mannose binding lectins
mediate the first ‘initiation’ stage.
29
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Initiation

▰ Activation - Mannose carbohydrate residues of glycoproteins present on

microbial surfaces.
▰ Mannsoe binding lectins (MBL) bind to mannose residues on microbial
surface.
▰ MBL is an acute phase reactant protein, similar to C1q in structure.

30
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Initiation (Cont..)

▰ After binding of MBL to microbial surface, another host protein called

MASP (MBL associated serine protease) gets complexed with MBL.

▰ MASP is similar or C1r and C1s and mimics their functions.

▰ MBL-MASP complex cleaves C4 which in turn splits C2.

▰ MBL/MASP-C4b2a acts as C3 convertase.

31
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Differences between the three complement pathways

32
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
EFFECTOR FUNCTIONS OF
COMPLEMENT
33
EFFECTOR FUNCTIONS OF COMPLEMENT

▰ MAC and other complement by-products produced during the activation

augment the immune response in many ways.
➢ Target cell lysis by MAC
➢ Inflammatory response
➢ Opsonization
➢ Removing the immune complexes from blood-
➢ Viral neutralization
34
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Target cell lysis by MAC

▰ MAC makes pores or channels in the target cell

membrane.

▰ Allows the free passage of various ions and water

into the cell leading to cell swelling, lysis and death.

▰ E.g. Bacteria, enveloped viruses, damaged cells,

tumor cells, etc
35
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Inflammatory response

▰ C3a, C4a and C5a - Anaphylatoxins.

▰ Bind to surface receptors of mast cells and
induce their degranulation leading to release of
histamine and other inflammatory mediators.
▰ Cause vasoconstriction, and increased vascular
permeability.

36
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Opsonization
▰ C3b and C4b - major opsonins - coat the immune complexes and
particulate antigens.
▰ Phagocytic cells express complement receptors (CR1, CR3 and CR4)
for complement components (C3b, C4b).
▰ Bind to complement coated antigens and enhance phagocytosis.

▰ C5a - enhances the CR1 expression on phagocytes by 10 folds.

37
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Opsonization (Cont..)

38
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Removing the immune complexes from blood

▰ C3b - important role.

▰ C3b bound immune complexes - Recognized by

complement receptor CR1 present on RBCs.

▰ Immune complexes bound to RBCs are taken to

liver and spleen where they are phagocytosed
after being separated from the RBCs.
39
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
Viral neutralization

▰ Complements coated on virus surfaces neutralize the viral infectivity

by blocking their attachment sites.
▰ C3b mediated opsonization of viral particles
▰ Lysis of the enveloped viruses by:
➢ Activation of classical pathway (most viruses)
➢ Alternative or lectin pathways (viruses like Epstein Barr virus,
rubella etc)

40
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
COMPLEMENT
RECEPTORS
41
COMPLEMENT RECEPTORS
▰ Play an important role in mediating the activities of complement
products as well as in regulating their activities.
▰ There are many complement receptors (CR1 to CR5) - distributed on
various cell types and bind to specific ligands to mediate specific
function.
▰ Example - CR2 is present on B cells and is involved in humoral immune
response - also acts as receptor for Epstein-Barr virus.

42
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
EVASION OF COMPLEMENT SYSTEM
BY MICROORGANISMS
43
EVASION OF COMPLEMENT SYSTEM
BY MICROORGANISMS

44
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
REGULATION OF COMPLEMENT
PATHWAYS
45
REGULATION OF COMPLEMENT PATHWAYS

▰ Antigen non-specific.

▰ Capable of attacking microorganisms as well as host cells.

▰ Several regulatory mechanisms have evolved to restrict complement

activity only to the designated target cells.
▰ There are a series of regulatory proteins, which inactivate various
complement components at different stages.

46
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
REGULATION OF COMPLEMENT PATHWAYS
(Cont..)

Examples:
▰ C1 inhibitor (or C1 esterase inhibitor): soluble glycoprotein, inhibits the
action of C1q by splitting C1qrs into C1rs and C1q - whole classical
pathway is inhibited.
▰ DAF (Decay accelerating factor):CD55 molecule present on cell
membrane, accelerates dissociation of C3 convertase - inhibiting all
three pathways.

47
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
COMPLEMENT DEFICIENCIES
48
COMPLEMENT DEFICIENCIES

49
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
EXPECTED QUESTIONS

I. Write essay on:

▰ 1. What is complement? Explain in detail about classical
▰ complement pathway. List various effector functions of complement.
II. Write short notes on:
▰ 1. Alternative complement pathway.
▰ 2. Various mechanisms of microbial evasion of complement system.
▰ 3. Complement deficiency diseases

50
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
III. Multiple Choice Questions (MCQs):

1. C-3 convertase in alternative complement pathway is:

▰ a. C14b2a b. C3bBb
▰ c. MBL/MASP-C4b2a d. C3b
2. Which of the following acts as an anaphylatoxin?
▰ a. C3a b. C3b
▰ c. C4b d. C2a
3. Endotoxin acts by:
▰ a. Classical pathway b. Lectin pathway
▰ c. Alternative pathway d. None
51
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
4. Disseminated Neisseria infection is commonly associated with deficiency of:
▰ a. Properdin
▰ b. Factor D
▰ c. C1 inhibitor deficiency
▰ d. Membrane attack complex (MAC)
5. Complement (classical pathway) is best fixed by:
▰ a. IgA b. IgD
▰ c. IgE d. IgM
52
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
6. Decreasing order of IgG in complement fixation:
▰ a. IgG1>IgG2>IgG3>IgG4
▰ b. IgG4>IgG3>IgG2>IgG1
▰ c. IgG3>IgG1>IgG2>IgG4
▰ d. IgG2>IgG1>IgG3>IgG4
7. Early complement deficiency is a predisposing factor for all, except:
▰ a. Systemic lupus erythematosus (SLE)
▰ b. Disseminated Neisseria infection
▰ c. Glomerulonephritis
▰ d. Pyogenic infections 53
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
8. C1 esterase inhibitor deficiency leads to:
▰ a. Paroxysmal nocturnal hemoglobinuria
▰ b. Hereditary angioneurotic edema
▰ c. Immune complex disease
▰ d. Recurrent pyogenic infections
9. The first complement component to be involved in alternative complement
pathway is:
▰ a. C1 b. C2
▰ c. C3 d. C4
54
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers
10. Complement mediated cell lysis is done by:
▰ a. Anaphylatoxins
▰ b. Activation of apoptosis
▰ c. Membrane attack complex
▰ d. Inhibition of protein synthesis

▰ Answers 1. b, 2. a, 3. c, 4. d, 5. d, 6. c, 7. b, 8. b, 9. c, 10.
c
55
Essentials of Medical Microbiology, 4/e by Apurba S Sastry © Jaypee Brothers Medical Publishers