Infection

Virulence factors of
bacteria.
 Infection
• Char ac teris tic of infec tio us
di seas e
• 1.Inf ecti vit y
• 2.P resenc e of i ncub at ion p eriod
• 3.Cycl ic d evel opme nt
• 4.Forma ti on of post inf ec tion immu ni ty
 The Nature of Host-parasite
Interactions in Humans
• Bacteria are associated with the body surfaces of
animals. These bacteria have a full range of symbiotic
interactions with their animal hosts. In biology,
symbiosis is defined as "life together
• Types of Symbiotic Associations
• 1. Mutualism. Both members of the association benefit.
• 2. Commensalism. There is no apparent benefit or harm
to either member of the association.
• 3. Parasitism. One member of the association lives at
the expense of the other member.
For many parasites are or can become pathogens,
microorganisms the cause disease.
 Bacterial Pathogenesis

• A pathogen is a microorganism (or virus)

that is able to produce disease.
Pathogenicity is the ability of a
microorganism to cause disease in
another organism, namely the host for the
pathogen. Рathogenicity is a manifestation

of a host- parasite interaction.

 Bacterial Pathogenesis
• In humans, some of the normal bacterial
flora (e.g. Staphylococcus aureus,
Haemophilus influenzae) are potential
pathogens that live in a commensal or
parasitic relationship without producing
disease. They do not cause disease in their
host unless they have an opportunity
brought on by some compromise or
weakness in the host's anatomical
barriers, tissue resistance or immunity.
 Bacterial Pathogenesis

• There are some pathogens that do not

associate with their host EXCEPT in
the case of disease. These bacteria
are obligate pathogens, even though
some may rarely occur as normal flora
 Opportunistic Pathogens
• Bacteria which cause a disease in a compromised
host which typically would not occur in a healthy
(noncompromised) host are acting as
opportunistic pathogens. A member of the normal
flora can such as Staphylococcus aureus or E. coli
can cause an opportunistic infection, but so can
an environmental organism such as Pseudomonas
aeruginosa. When a member of the normal flora
causes an infectious disease, it might be referred
to as an endogenous bacterial disease.
 Determinants of Virulence
• The term virulence is best interpreted as
referring to the degree of pathogenicity.)
The sum of the characteristics that allow
a bacterium to produce disease are the
pathogen's determinants of virulence.
 Properties of the Host

• The host in a host-parasite interaction is

the animal that maintains the parasite.
The host and parasite are in a dynamic
interaction, the outcome of which
depends upon the properties of the
parasite and of the host. The bacterial
parasite has its determinants of virulence
that allow it to invade and damage the
host and to resist the defenses of the
host.
 Virulence factor of bacteria.
• The first stage of microbial infection is
colonization: the establishment of the
pathogen the portal of entry. Sites of
entry in human hosts include the
urogenital tract, the digestive tract, the
respiratory tract and the conjunctiva.
Organisms that infect these regions
have usually developed tissue
adherence mechanisms and some
ability to overcome.
 Bacterial Adherence to Mucosal
Surfaces.
• Bacterial adherence or attachment to a
eukaryotic cell or tissue surface requires the
participation of two factors: a receptor and
an adhesin. The receptors are usually
specific carbohydrate or peptide residues on
the eukaryotic cell surface. The bacterial
adhesin is typically a macromolecular
component of the bacterial cell surface
which interacts with the host cell receptor.
Adhesins and receptors usually interact in a
complementary.
The Importance of the Bacterial Surface

• All of the various surface components of

a bacterial cell are important in its
ecology since they mediate the contact of
the bacterium with its environment.
• The surface properties of a bacterium are
determined by the molecular
composition of its membrane and cell
wall, including LPS, and the other surface
structures such as flagella, fimbriae and
capsules.
Streptococcus pyogenes.
Cell surface fibrils
 Bacterial surface structures

• . Bacterial surface structures may act as

• (1) permeability barriers that allow selective
passage of nutrients
• (2) adhesins used to attach or adhere to
specific surfaces or tissues;
• (3) enzymes to mediate specific reactions on
the cell surface important in the survival of the
organism;
 The Structure of the Bacterial Surface
• (4) protective structures against phagocytic
engulfment or killing;
• (5) antigenic disguises;
• (6) "sensing proteins" that can respond to
temperature, osmolarity, light, oxygen,
nutrients, etc., resulting in a molecular
signal to the genome of the cell that will
cause expression of some determinant of
virulence (e.g. an exotoxin).
The Structure of the Bacterial Surface
• Flagella are filamentous protein
structures attached to the cell surface
that provide movement for most motile
bacterial cells.
• The flagellar filament is rotated by a
motor apparatus in the plasma
membrane allowing the cell to swim in
fluid environments.
The Structure of the Bacterial Surface
Flagella
Flagella
Flagella
Flagella
 Fimbriae and Pili

• Fimbriae and Pili are short, hair-like

structures on the surfaces of bacterial
cells. Fimbriae are shorter and stiffer
than flagella, and smaller in diameter.
Like flagella, they are composed of
protein. A specialized type of pilus
(always called a pilus), the F or sex
pilus, mediates the transfer of DNA
between bacteria.
 Common pili (fimbriae)
• Common pili (fimbriae) are usually involved in
adherence (attachment) of bacterial cells to
surfaces in nature.Тhey are determinants of
bacterial virulence because they allow
pathogens to attach to (colonize) tissues and to
resist attack by phagocytic white blood cells.
G- bacteria posses “common pili”, which help
them adhere to mucosal surface.Most E.coli
strains that cause pyelonephritis produce a
fimbrial adhesin termed P fimbrial.
N.gonorrhoeae pili
 Common pili (fimbriae)

• Fimbriae are also antigenic and

secretory antibodies (IgA) will often
block bacterial colonization, while
circulating antibodies (IgG or IgM)
will opsonize bacterial cells for
phagocytosis.
• Pili can be antiphagocytic.
The Structure of the Bacterial Surface

• Teichoic acids and lipoteichoic acids (LTA)

• Cell wall components of Gram-positive
bacteria that may be involved in nonspecific
or specific adherence
• The membrane proteins –determined
• By plasmed
• F- protein ( binds to fibronectin) – a host
protein that coats the epithelial cells of
oropharynx
Teichoic acids and lipoteichoic
acids (LTA)
 LTA

Peptidoglican

ЦП
М
 a capsule

• Most bacteria contain some sort of a polysaccharide

layer outside of the cell wall or outer membrane, this
layer is called a capsule
 Mechanisms of Adherence to Cell or
Tissue Surfaces

• Nonspecific adherence
• 1. hydrophobic interactions
• 2. electrostatic attractions
• 4. Brownian movement
 Mechanisms of Adherence to Cell or
Tissue Surfaces

• Specific adherence involves

permanent formation of many specific
lock-and-key bonds between
complementary molecules on each cell
surface.
Specific Adherence of Bacteria to Cell
and Tissue Surfaces

• 1. Tissue tropism: particular bacteria are

known to have an apparent preference for
certain tissues over others, e.g. S. mutans is
abundant in dental plaque but does not
occur on epithelial surfaces of the tongue;
the reverse is true for S. salivarius which is
attached in high numbers to epithelial cells
of the tongue but is absent in dental plaque.
 Specific Adherence of Bacteria to
Cell and Tissue Surfaces
• 2. Species specificity: certain pathogenic
bacteria infect only certain species of
animals, e.g. N. gonorrhoeae infections
are limited to humans; Enteropathogenic
E. coli K-88 infections are limited to pigs;
Group A streptococcal infections occur
only in humans.
Phagocytosis
 The factorie preven phagocytosis
• Bacteria can avoid the attention of phagocytes
in a number of ways.
• 1. Pathogens may invade in regions
inaccessible to phagocytes. Certain internal
tissues (e.g. the lumens of glands, the urinary
bladder) and surface tissues (e.g. the skin) are
not patrolled by phagocytes.
• 2. Some pathogens are able to avoid
provoking an overwhelming inflammatory
response. Without inflammation the host is
unable to focus the phagocytic defenses.
 The factorie preven phagocytosis

• 3. Some bacteria inhibit phagocyte

chemotaxis. For example, Streptococcal
streptolysin (which also kills phagocytes)
suppresses neutrophil chemotaxis, even in
very low concentrations.
 The factorie preven phagocytosis
• 4. Some pathogens can cover the surface of the
bacterial cell with a component which is seen
as "self" by the host phagocytes and immune
system. Such a strategy hides the antigenic
surface of the bacterial cell. Phagocytes cannot
recognize bacteria upon contactsubstance
(tissue cement) in connective tissue

• For example, Group A streptococci are able to

synthesize a capsule composed of hyaluronic
acid. Hyaluronic acid is the ground
 Inhibition of Phagocytic Engulfment
• Many important pathogenic bacteria
bear on their surfaces substances that
inhibit phagocytic adsorption or
engulfment. Resistance to phagocytic
ingestion is usually due to a
component of the bacterial cell
surface (cell wall, or fimbriae, or a
capsule).
 Classical examples of antiphagocytic
substances on the bacterial surface include:

• 1. Polysaccharide capsules of S.
pneumoniae, Haemophilus influenzae,
Treponema pallidum and Klebsiella
pneumoniae.
• 2. M protein and fimbriae of Group A
streptococci
• 3. Surface slime (polysaccharide)
produced as a biofilm by Pseudomonas
aeruginosa
 Classical examples of antiphagocytic
substances on the bacterial surface include:

• . O 4polysaccharide associated with LPS of E. coli

• 5. K antigen (acidic polysaccharides) of E. coli or
the analogous Vi antigen of Salmonella typhi
• 6. Cell-bound or soluble Protein A produced by
Staphylococcus aureus. Protein A attaches to
the Fc region of IgG and blocks the cytophilic
(cell-binding) domain of the Ab. Thus, the ability
of IgG to act as an opsonic factor is inhibited,
and opsonin-mediated ingestion of the bacteria
is blocked.
M protein and fimbriae of Group A
streptococci
Polysaccharide capsules
 Survival Inside of Phagocytes

• Some bacteria survive inside of

phagocytic cells, in either neutrophils or
macrophages. Bacteria that can resist
killing and survive or multiply inside of
phagocytes are considered intracellular
parasites.
 The factorie preven phagocytosis
• 1. Inhibition of fusion of the phagocytic
lysosomes (granules) with the phagosome.
The bacteria survive inside of phagosomes
because they prevent the discharge of
lysosomal contents into the phagosome
environment. –
• With M. tuberculosis, bacterial cell wall
components (sulfatides) are thought to be
released from the phagosome and modify
lysosomal membranes to inhibit fusion.
 The factorie preven phagocytosis

• --InLegionella, it is known that a single

gene is responsible for the inhibition of
phagolysosomal fusion.
• -In Salmonella typhimurium, the pH that
develops in the phagosome after
engulfment actually induces bacterial
gene products that are essential for their
survival in macrophages.
 The factorie preven phagocytosis
• Survival inside the phagolysosome. With some
intracellular parasites, phagosome-lysosome
fusion occurs, but the bacteria are resistant to
inhibition and killing by the lysosomal
constituents. Also, some extracellular
pathogens can resist killing in phagocytes
utilizing similar resistance mechanisms.
Mycobacteria (including M. tuberculosis) have
waxy, hydrophobic cell wall and capsule
components (mycolic acids), which are not
easily attacked by lysosomal enzymes.
The factorie preven phagocytosis

• The outer membrane and capsular

components of Gram-negative bacteria
(e.g. Salmonella, Yersinia, Brucella, E.
coli) can protect the peptidoglycan layer
from the lytic activity of lysozyme.
 Products of Bacteria that Kill or Damage
Phagocytes
• Most of extracellular enzymes or toxins that kill
phagocytes. Phagocytes may be killed by a
pathogen before or after ingestion.
• Killing Phagocytes Before Ingestion
• -Pathogenic staphylococci produce leukocidin,
which also acts on the neutrophil membrane and
causes discharge of lysosomal granules.
• -Extracellular proteins that inhibit phagocytosis
include the Exotoxin A of Pseudomonas
aeruginosa which kills macrophages, and the
bacterial exotoxins that are adenylate cyclases
(e.g. anthrax toxin EF and pertussis toxin AC)
which decrease phagocytic activity.
Products of Bacteria that Kill or
Damage Phagocytes
Products of Bacteria that Kill or
Damage Phagocytes
 Sp read in g F ac tors
• Spreading Factors" is a term for a family of
bacterial enzymes that affect the physical
properties of tissue matrices and intercellular
spaces, thereby promoting the spread of the
pathogen. Hyaluronidase. is the original
spreading factor It is produced by
streptococci. staphylococci, and clostridia.
The enzyme attacks the interstitial cement
("ground substance") of connective tissue by
depolymerizing hyaluronic acid.
 Spreading Factors
• Collagenase is produced by Clostridium
histolyticum and Clostridium perfringens. It
breaks down collagen, the framework of
muscles, which facilitates gas gangrene due
to these organisms.
• Neuraminidase is produced by intestinal
pathogens such as Vibrio cholerae and
Shigella dysenteriae. It degrades neuraminic
acid (also called sialic acid), an intercellular
cement of the epithelial cells of the
intestinal mucosa.
 Spreading Factors
• Streptokinase and Staphylokinase are
produced by streptococci and
staphylococci.
• Kinase enzymes convert inactive
plasminogen to plasmin which digests
fibrin and prevents clotting of the blood.
The relative absence of fibrin in spreading
bacterial lesions allows more rapid
diffusion of the infectious bacteria.
 Enzymes that Cause Hemolysis and/or
Leucolysis
• These enzymes act on the animal cell
membrane by insertion into the membrane
(forming a pore that results in cell lysis), or by
enzymatic attack on phospholipids, which
destabilizes the membrane. They may be
referred to as lecithinases or phospholipases,
and if they lyse red blood cells they are
sometimes called hemolysins. Leukocidins,
produced by staphylococci and streptolysin
produced by streptococci specifically lyse
phagocytes and their granules.
lecithinases
 Staphylococcal coagulase

• Coagulase, formed by Staphylococcus

aureus, is a enzyme that converts
fibrinogen to fibrin which causes clotting
and helps prevent the phagocytosis.
Coagulase activity is almost alway
associated with pathogenic S. aureus and
almost never associated with
nonpathogenic S. Epidermidis.
 Extracellular Digestive Enzymes

• proteases, lipases, glycohydrolases,

nucleases, which are not clearly shown to
have a direct role in invasion or
pathogenesis. These enzymes have other
functions related to bacterial nutrition or
metabolism, but may aid in invasion either
directly or indirectly.
 Toxins With Short-Range Effects
Related to Invasion

• Bacterial protein toxins which have adenylate

cyclase activity, are thought to have immediate
effects on host cells that promote bacterial
invasion. One component of the anthrax toxin
(EF = Edema Factor) is an adenylate cyclase that
acts on cells to cause increased levels of cyclic
AMP and disruption of cell permeability. These
toxins may contribute to invasion through their
effetcts on macrophages or lymphocytes in the
vicinity which are playing an essential role to
contain the infection.
 Bacterial Toxigenesis

• There are two types of bacterial toxins,

lipopolysaccharides, which are associated
with the cell walls of Gram-negative
bacteria, and proteins, which are released
from bacterial cells and may act at tissue
sites removed from the site of bacterial
growth. The cell-associated
lipoplysaccharide (LPS) toxins are
referred to as endotoxins and the
extracellular diffusible toxins are referred
to as exotoxins.
 Bacterial Toxigenesis
• Endotoxins are cell-associated substances that
are structural components of the outer membrane
of Gram-negative bacteria.
• Endotoxins may be released from growing
bacterial cells or from cells which are lysed.
• The biological activity of endotoxin is associated
with the lipopolysaccharide (LPS). Toxicity is
associated with the lipid component (Lipid A) and
immunogenicity is associated with the
polysaccharide components. The cell wall
antigens (O antigens) of Gram-negative bacteria
are components of LPS. LPS elicits a variety of
inflammatory responses in an animal.
 Structural components of the outer
membrane of Gram-negative bacteria.
 Structural components of the outer
membrane of Gram-negative bacteria.
 The O polysaccharide and virulence

• How are the polysaccharide side chains

involved in the expression of virulence?
There are a number of possibilities:
• 1. Smooth antigens could allow organisms to
adhere specifically to certain tissues
• Smooth antigens probably allow resistance
to phagocytes, since rough mutants are more
readily engulfed and destroyed by
phagocytes.
• 3. The hydrophilic O polysaccharides could
act as water-solubilizing carriers for toxic
Lipid A.
 The O polysaccharide and virulence

• The O antigens could provide protection

from damaging reactions with antibody
and complement
• The O-polysaccharide or O antigen is the
basis of antigenic variation among many
important Gram-negative pathogens
including
 Lipid A and virulence

• The injection of living or killed Gram-negative

cells, or purified LPS, into experimental
animals causes a wide spectrum of
nonspecific pathophysiological reactions such
as: fever, changes in white blood cell
counts, disseminated intravascular
coagulation, hypotension, shock and
death.
 Lipid A and virulence

• LPS released into the bloodstream by lysing

Gram-negative bacteria is first bound by
certain plasma proteins identified as LPS-
binding proteins. The LPS-binding protein
complex interacts with CD14receptors on
monocytes and macrophages and other types
of receptors on endothelial cells. In
monocytes and macrophages three types
of events are triggered during their interaction
with LPS:
 Lipid A and virulence

• Production of cytokines, including IL-1, IL-6,

IL-8, tumor necrosis factor (TNF) and platelet-
activating factor. These in turn stimulate
production of prostaglandins and
leukotrienes. These are mediators of
inflammation and septic shock that
accompanies endotoxin toxemia. LPS
activates macrophages to enhanced
phagocytosis and cytotoxicity. Macrophages are
stimulated to produce and release lysosomal
enzymes, IL-1 ("endogenous pyrogen"), and
tumor necrosis factor (TNFalpha), as well as
other cytokines and mediators.
 Lipid A and virulence
• 2. Activation of the complement cascade. C3a
and C5a cause histamine release (leading to
vasodilation) and effect neutrophil chemotaxis and
accumulation. The result is inflammation.
• 3. Activation of the coagulation cascade. The
net effect is to induce inflammation, intravascular
coagulation, hemorrhage and shock.
• LPS also acts as a B cell mitogen stimulating the
polyclonal differentiation and multiplication of B-
cells and the secretion of immunoglobulins,
especially IgG and IgM.
 Bacterial Toxigenesis
• Exotoxins are usually secreted by bacteria but
in some cases they are released by lysis of the
bacterial cell. Exotoxins are typically soluble
proteins secreted by living bacteria during
growth. bacterial exotoxins are denatured by
heat, acid and proteolytic enzymes; they have a
high biological activity (most act catalytically);
and they exhibit specificity of action.
 Bacterial Toxigenesis
• Protein toxins have very specific cytotoxic
activity (i.e., they attack specific types of cells).
protein toxins have very specific cytotoxic
activity (i.e., they attack specific types of cells).
For example, tetanus or botulinum toxins attack
only neurons.
• Some toxins (as produced by staphylococci,
streptococci, clostridia, etc.) have fairly broad
cytotoxic activity and cause nonspecific death
of all sorts of cells and tissues.
 Bacterial Toxigenesis
• Bacterial protein toxins are strongly antigenic.
In vivo, specific antibody (antitoxin) neutralizes
the toxicity of these bacterial proteins.
• Protein toxins are inherently unstable: in time
they lose their toxic properties but retain their
antigenic ones.
• Toxoids are detoxified toxins which retain their
antigenicity and their immunizing capacity.
Bacterial Toxigenesis
 A plus B subunit Arrangement of Protein
Toxins
• Many protein toxins, consist of two components:
one component (subunit A) is responsible for the
enzymatic activity of the toxin; the other
component (subunit B) is concerned with
binding to a specific receptor on the host cell
membrane and transferring the enzyme across
the membrane. The enzymatic component is not
active until it is released from the native (A+B)
toxin. Isolated A subunits are enzymatically active
but lack binding and cell entry capability. Isolated
B subunits may bind to target cells (and even
block the binding of the native toxin), but they are
nontoxic.
 Attachment and Entry of Toxins
• There are at least two mechanisms of toxin entry
into target cells.
• In one mechanism called direct entry, the B subunit
of the native (A+B) toxin binds to a specific receptor
on the target cell and induces the formation of a pore
in the membrane through which the A subunit is
transferred into the cell cytoplasm.
• In an alternative mechanism, the native toxin binds to
the target cell and the A+B structure is taken into the
cell by the process of receptor-mediated
endocytosis (RME). The toxin is internalized in the
cell in a membrane-enclosed vesicle called an
endosome.
 Attachment and Entry of Toxins

• The specific receptors for the B subunit of

the toxin on target cells or tissues are
usually sialogangliosides (glycoproteins)
called G-proteins on the cell membrane.
For example, the cholera toxin utilizes the
ganglioside GM1, and tetanus toxin
utilizes ganglioside GT1 and/or GD1b as
receptors on host cells.
 Mechanisms of actioon exotoxin
• Toxins cause damage in various ways:
• 1. Toxins that block protein synthesis in
target cells.
• The diphtheria toxin is a two component
bacterial exotoxin synthesized as a single
polypeptide chain containing an A (active)
domain and a B (binding) domain. Proteolytic
nicking of the secreted form of the toxin
separates the A chain from the B chain.
The diphtheria toxin

• AB toxin. The receptor

for the B-portion is
found on
• The membrane of all
cell susceptible
species. A protease
sensitive site between
the A and B portion?
Which remain
covalenly associated
by disulfide linkage.
 Mechanisms of actioon diphtheria
toxin
• The toxin binds to a specific receptor on susceptible cells and
enters by receptor-mediated endocytosis. Acidification of the
endosome vesicle results in unfolding of the protein and
insertion of a segment into the endosomal membrane.
Apparently as a result of activity on the endosome membrane,
the A subunit is cleaved and released from the B subunit as it
inserts and passes through the membrane. Once in the
cytoplasm, the A fragment regains its conformation and its
enzymatic activity. Fragment A catalyzes the transfer of ADP-
ribose from NAD to the eukaryotic Elongation Factor 2 which
inhibits the function of the latter in protein synthesis.
Ultimately, inactivation of all of the host cell EF-2 molecules
causes death of the cell. Attachment of the ADP ribosyl group
occurs at an unusual derivative of histadine called
diphthamide.
 Cholera Toxin

• Cholera toxin activates the adenylate cyclase

enzyme in cells of the intestinal mucosa leading to
increased levels of intracellular cAMP, and the
secretion of H20, Na+, K+, Cl-, and HCO3- into the
lumen of the small intestine. The effect is dependent
on a specific receptor, monosialosyl ganglioside
(GM1 ganglioside) present on the surface of intestinal
mucosal cells. The bacterium produces an invasin,
neuraminidase, during the colonization stage which
has the interesting property of degrading gangliosides
to the monosialosyl form, which is the specific receptor
for the toxin.
 Cholera Toxin
• The toxin has been characterized and contains 5 binding
(B) subunits of 11,500 daltons, an active (A1) subunit of
23,500 daltons, and a bridging piece (A2) of 5,500
daltons that links A1 to the 5B subunits. The A1 subunit
enzymatically transfers ADP ribose from NAD to a protein
(called Gs or Ns), that regulates the adenylate cyclase
system which is located on the inside of the plasma
membrane of mammalian cells.
• Enzymatically, fragment A1 catalyzes the transfer of the
ADP-ribosyl moiety of NAD to a component of the
adenylate cyclase system. The process is complex.
Adenylate cyclase (AC) is activated normally by a
regulatory protein (GS) and GTP; however activation is
normally brief because another regulatory protein (Gi),
hydrolyzes GTP. The normal situation is described as
follows.
Toxins that block nerve function

• Tetanus toxins
produce irreversible
muscule contraction.
• Consist of single
polypeptide chains.
 Toxins that block nerve function

• Activated by proteolysis and disylfide

reduction, A+B structure is taken into the cell
by the process of receptor-mediated
endocytosis (RME). The toxin is internalized
in the cell in a membrane-enclosed vesicle
called an endosome.
• Zn++ dependent protease that Inhibits
neurotransmission at inhibitory synapses
resulting in spastic paralysis
 Toxins that block nerve function

• Botulinum Toxins consist of singl

polypeptide chains that contain A and B
region.
• Toxins are activated by proteolysis and
disulfide reduction.
• Inhibits presynaptic acetylycholine release
from peripheral cholinergic neurons
resulting in flaccid paralysis