Complement

Learning objectives
At the end of the session, the students will be able to
understand:

▰ Definitions

▰ Classical, alternate and lectin pathway

▰ Role of complement

▰ Complement deficiencies
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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

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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.
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▰ Properdin system and the regulatory proteins are named by
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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 C2aof Medical
Essentials which is larger fragment.
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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.

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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).
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COMPLEMENT
PATHWAYS
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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 - 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.
 Initiation of the pathway
 Formation of C3 convertase
 Formation of C5 convertase
 Formation of membrane attack complex (MAC)

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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.

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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 15
Essentials of Medical Microbiology
binding to antigen-antibody complex.
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

Essentials pathway begins only when
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Initiation (Cont..)
▰ C1q binding in the presence of calcium ions, in turn
activates sequentially C1r followed by C1s.

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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.
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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.
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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
Essentials C7 and
of Medical C8 help in penetration
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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 passageEssentials
of ions and
of Medical water into the cell - cellular
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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.

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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
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in serum named factor B, factor D and properdin.
Initiation
Antigens from pathogen Non microbial initiators

Endotoxin or LPS Human antibodies in

(lipopolysaccharide) from Gram complexes- IgA, IgD
negative bacteria
Teichoic acid from Gram Tumor cells
positive bacteria
Fungal cells- Yeast cells Cobra venom factor

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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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.

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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 (Cont..)

▰ Formation of C5 convertase and formation of membrane

attack complex - identical to that of classical pathway.

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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 30

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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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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.

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Differences between the three
complement pathways
Features Classical pathway Alternative pathway Lectin pathway
Activator (initiator) Antigen antibody Endotoxin Carbohydrate residue
complex IgA, IgD, Cobra venom, of bacterial cell wall
Nephritic factor (mannose binding
protein) that binds to
host lectin antigen.
First 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 34
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EFFECTOR FUNCTIONS
OF COMPLEMENT
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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.
 Target cell lysis by MAC
 Inflammatory response
 Opsonization
 Removing the immune complexes from blood-
 Viral neutralization
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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,
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of Medical
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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 Essentials

CR1 expression
of Medical Microbiologyon phagocytes by 10
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Opsonization (Cont..)

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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
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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
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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

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EVASION OF COMPLEMENT
SYSTEM
BY MICROORGANISMS

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

Shown by Gram positive bacteria

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

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REGULATION OF
COMPLEMENT PATHWAYS

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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.
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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.
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COMPLEMENT
DEFICIENCIES

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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 Common deficiency Disseminated Neisseria
(C5-C9) infection

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COMPLEMENT DEFICIENCIES
(Cont..)

Complement regulatory Pathway(s) involved Disease/pathology

protein deficiencies
C1 esterase inhibitor Overactive classical Hereditary angioneurotic
pathway edema
DAF (Decay accelerating Deregulated C3 convertase PNH (Paroxysmal nocturnal
factor) & CD59 Increased RBC lysis hemoglobinurea)

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Questions:
▰ Q1. C-3 convertase in alternative complement pathway is:

a. C14b2a

b. C3bBb

c. MBL/MASP-C4b2a

d. C3b

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Questions:
▰ Q2. Endotoxin acts by:

a. Classical pathway

b. Lectin pathway

c. Alternative pathway

d. None

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Questions:
▰ Q3. Early complement deficiency is a predisposing factor for
all, except:

a. Systemic lupus erythematosus (SLE)

b. Disseminated Neisseria infection

c. Glomerulonephritis

d. Pyogenic infections
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