Complement

Learning objectives
At the end of this session students should be able to
• Define complement
• Understand classical, alternate and lectin pathway
• Role of complement

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 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.

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 GENERAL PROPERTIES

• Bind to Fc region of antibody

• Role of antigen
• Species nonspecific
• Heat labile

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 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

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 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.

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 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.
• During proteolysis, the smaller fragment is removed exposing the
active site of the larger fragment.
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 Complement activation

• The larger fragment participates in the cascade reaction of

complement pathway and the smaller fragment diffuses away to
mediate other functions.
• 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).

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 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 is a recently described pathway. It resembles
classical pathway but it is antibody independent.

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 Stages of complement activation

• There are four main stages in the activation of any of the

complement pathways.
o Initiation of the pathway
o Formation of C3 convertase
o Formation of C5 convertase
o Formation of membrane attack complex (MAC)
• 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.
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 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.
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 CLASSICAL PATHWAY -
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.

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 CLASSICAL PATHWAY -
Initiation

• IgM being pentameric, has five Fc regions, hence one molecule of

IgM can initiate the pathway.
• Whereas IgG is monomeric, therefore two IgG molecules are
needed to initiate the process. Hence IgM is much efficient
stimulator of classical pathway.
• C1q binding in the presence of calcium ions, in turn activates
sequentially C1r followed by C1s.

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 CLASSICAL PATHWAY -
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.
o 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.
o C14b2a is referred to as C3 convertase of the classical pathway.

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 CLASSICAL PATHWAY -
Formation of C5 convertase

• C3 convertase hydrolyses many C3 molecules into two fragments:

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

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 CLASSICAL PATHWAY –
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.
o C5b is extremely labile, gets stabilized by binding soon with C6 and C7
to form C5b67 followed by addition of C8.
o Hydrophobic regions on C7 and C8 help in penetration into the target
cell membrane.
o 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

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 CLASSICAL PATHWAY –
Formation of Membrane attack complex

• 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.
• C5b6789 destroys the target cell by attacking the cell membrane –
MAC,
• Process of cytolysis is referred to as complement-mediated
cytotoxicity.

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Essentials of Medical Microbiology © 2018, 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.

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 ALTERNATIVE PATHWAY-
Initiation

Antigens from pathogen Non microbial initiators

Endotoxin or LPS Human antibodies in complexes-
(lipopolysaccharide) from Gram IgA, IgD
negative bacteria
Teichoic acid from Gram positive Tumor cells
bacteria
Fungal cells Cobra venom factor
Yeast cells Heterologous RBCs from mouse,
rabbit and chicken
Parasites like Trypanosomes Anion polymer like dextran
sulphate
Virus infected cells Pure carbohydrates like agar, inulin

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 ALTERNATIVE PATHWAY

• 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.

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 ALTERNATIVE PATHWAY-
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).

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 ALTERNATIVE PATHWAY

• Formation of C5 convertase. Identical to that of

• Formation of MAC classical pathway.

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Essentials of Medical Microbiology © 2018, 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.

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 LECTIN PATHWAY-
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.

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 LECTIN PATHWAY-
Initiation

• 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 and the MBL/MASP-C4b2a acts as
C3 convertase.
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 ALTERNATIVE PATHWAY

• Formation of C5 convertase. Identical to that of

• Formation of MAC classical pathway.

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Essentials of Medical Microbiology © 2018, Jaypee Brothers Medical Publishers
 Differences between the three complement pathways

Classical Alternative pathway Lectin pathway

pathway
Activator (initiator) Antigen antibody Endotoxin Carbohydrate residue of bacterial cell
complex IgA, IgD, Cobra venom, wall (mannose binding protein) that
Nephritic factor binds to host lectin antigen.

1st complement C1 C3b C4

activated
C3 convertase C14b2a C3bBb MBL/MASP-C4b2a
C5 convertase C14b2a3b C3bBb3b MBL/MASP-C4b2a3b
(C3 convertase + 3b)

Complement level in All C1-C9: Low C1,C4,C2- Normal C1- Normal

the serum Others- Low Others- Low
Immunity Acquired Innate Innate

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 EFFECTOR FUNCTIONS OF COMPLEMENT

• MAC and other complement by-products produced during the

activation augment the immune response in many ways.
o Target cell lysis by MAC
o Inflammatory response
o Opsonization
o Removing the immune complexes from blood-
o Viral neutralization

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 Target cell lysis by MAC-
complement mediated cell lysis

• 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

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 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.

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 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.
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 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.

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 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)

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 COMPLEMENT RECEPTORS

CR Ligands Distribution Function

CR1 C3b, RBCs, phagocytes 1. Regulates complement pathway by
(CD35) C4b All blood cells inhibiting C3 convertase
2.Helps in removal of immune complexes

CR2 (CD21) C3d, C3dg B cells, T cells, 1. Forms a part of B cell co-receptor-
Follicular dendritic involved in humoral responses
cells 2. Acts as EBV receptor

CR3, CR4 iC3b Phagocytes 1. Opsonization

2. Binding and extravasation of
neutrophils
CR3a, CR4a, C3a, C4a, C5a Mast cells, Basophils Degranulation of mast cells and basophils
CR5a

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 EVASION OF COMPLEMENT SYSTEM BY MICROORGANISMS

Mechanisms Examples
Shown by Gram negative bacteria
Long polysaccharide side chain of bacteria can prevent MAC Escherichia coli
insertion Salmonella
Non covalent interactions between bacterial cell wall Neisseria gonorrhoeae
components can prevent MAC insertion
Elastases destroy C3a & C5a Pseudomonas

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 EVASION OF COMPLEMENT SYSTEM BY MICROORGANISMS
Mechanisms Examples
Shown by Gram positive bacteria
Thick peptidoglycan cell wall prevents MAC insertion Staphylococcus
Streptococcus
Bacterial capsule forms a physical barrier between C3b and Streptococcus pneumoniae
CR1 interaction
Shown by other microbes
Proteins mimicking complement regulatory proteins Vaccinia virus,
Herpes simplex virus,
Epstein-Barr virus, Trypanosoma cruzi,
Candida albicans

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 REGULATION OF COMPLEMENT PATHWAYS

Regulatory proteins Pathway affected Type of Regulatory function

protein
C1 regulator
C1 inhibitor Classical only Soluble It is a glycoprotein, inhibits the action of C1q
(C1 Inh, or C1 esterase inhibitor) by splitting C1qrs into C1rs and C1q. Thus the
whole classical pathway is inhibited
C3 convertase regulators
C4b-binding protein (C4bBP) Classical and lectin Soluble It blocks formation of C3 convertase by
binding C4b;
It acts as cofactor for cleavage of C4b by factor
I

CR-1(Complement-receptor-1) All three pathways Membrane Blocks formation of C3 convertase by

MCP(Membrane-cofactor protein) bound binding C3b or C4b
Factor H Alternative only

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 REGULATION OF COMPLEMENT PATHWAYS

Regulatory proteins Pathway affected Type of Regulatory function

protein
C3 convertase regulators..
DAF (Decay accelerating factor) All three pathways Membrane Accelerates dissociation of C3 convertase
or CD55 bound
Factor-I All three pathways Soluble Cleaves C4b or C3b by using C4b-binding
protein
MAC formation regulators
S protein Soluble Binds soluble C5b67 and prevents its
insertion into cell membrane
Membrane inhibitor of reactive Membrane Inhibit MAC formation by blocking C9 binding
lysis (MIRL or CD59) All three pathways bound

Homologous restriction Membrane Inhibit MAC formation by blocking C9 binding

factor bound
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 COMPLEMENT DEFICIENCIES

Complement protein Pathway(s) involved Disease/pathology

deficiencies
C1, C2, C3, C4 C1, C2,C4-Classical SLE, glomerulonephritis &
pathway pyogenic infections
C3- Common deficiency
Properdin, Factor D Alternative pathway Neisseria and pyogenic
infection
Membrane attack complex (C5-C9) Common deficiency Disseminated Neisseria infection

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 COMPLEMENT DEFICIENCIES

Complement regulatory protein deficiencies Diseases

C1 esterase inhibitor Overactive classical pathway Hereditary angioneurotic edema

DAF (Decay accelerating factor) Deregulated C3 convertase PNH (Paroxysmal nocturnal

& CD59 Increased RBC lysis hemoglobinurea)

Factor I Deregulated classical pathway Immune complex disease;

with over consumption of C3 recurrent pyogenic infections
Factor H Deregulated alternative Immune complex disease;
pathway with increased C3 pyogenic infection
convertase activity

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