BACTERIAL VIRULENCE FACTORS

- DR.VINOTHANANDAN
I ST YEAR POST GRADUATE
Introduction:
• The words “virulence” and “virulent” are derived from the
Latin word
Virulentus = “full of poison.”
Virus(poison) & lentus(fullness).

• Virulence is a measure of a microbe’s ability to cause

disease.

• It is a quantitative measure of pathogenicity and is measured

by the number of organisms required to caused disease.
 Definition:
• 'Pathogenicity‘ - refer to the ability of a
microbial species to produce disease.
• ‘Virulence' - relative degree of
pathogenesis (tissue damage), which may
vary between different strains of the same
organism depending upon the expression of
the virulence factors.
The virulence of a strain may undergo spontaneous or induced
variation ,

• Exaltation :
Enhancement of virulence.
Induced by serial passage in susceptible host .
• Attenuation :
Reduction of virulence.
It is achieved by
- passage through unfavourable host ,
- repeated culture in artificial media ,
- growth in high temperatures .
What is virulence factor?
• Some bacteria have certain cell parts that
help them to invade other organisms better.
These are called virulence factor.
• Virulence factors depends on
- Number of infecting microorganisms,
- Route of entry into body ,
- Response of the host immune system.
 Infective dose of the organism
• Low Infective dose:
Shigella : very low (as low as 10 bacilli);
Cryptosporidium parvum : very low (10 to 30 oocysts);
Escherichia coli O157:H7 (< 10 bacilli);
Entamoeba coli and Giardia : few cysts;
Campylobacter jejuni (500 bacilli)
• Large Infective dose:
Escherichia coli (106 - 108 bacilli);
Salmonella (102 – 105 bacilli);
Vibrio cholerae (106 - 108 bacilli)
• Higher is the virulence ,lower the infective dose.
• Virulence factors are the molecular components
expressed by a pathogen that increases its ability
to cause disease .

Virulence factors

cause damage Donot damage to the host but

to the host promote colonization and survival
VIRULENCE FACTORS OF BACTERIA
 Adherence /colonization :
- Fimbriae & pili
- Non-pilus adhesions ( Mprotein , lipoteichoic acid )
- Flagella and outer membrane proteins .
 Infectious process :
- Intracellular growth (Invasion )
- Toxin production (Exotoxin &
Endotoxin).
 Production against Host defense :
- Antiphagocytic factors
- Antigenic variation
- Biofilm formation .
Factors that contribute to Microbial colonization:

• Survival against Environmental conditions :

- Localization in moist areas,
- Protection in ingestion and inhaled debris ,
- Expression of specific metabolic characteristics .
• Achieving attachment and Adherence to host cell surfaces :
- Pili
- Adherence proteins
- Biofilm formation
• Other factors
- Motility
- Acquisition of essential nutrients
- Ability to coexist with other colonising microorganisms.
MECHANISM OF BACTERIAL PATHOGENECITY :
• ADHESION:
• When bacteria enter the body of the host , they must
adhere to cells of a tissue surface .
• Adherence requires participation of two factors :
- Bacterial adhesins
- Receptor on host cell
 BACTERIAL ADHESINS

• The various molecules that mediate adherence to the cell

surface are called adhesins .
• These adhesins allow the bacteria to adhere to the cell
surface of human cell, thereby promoting their ability to
cause disease.

Bacterial adhesins

Pili (fimbriae ) Non pilius

(afimbrial adhesins )
Pili Adhesins Non Pilus Adhesins
Pili:
• These are the main mechanisms by which
bacteria adhere to human cells.

• They are the fibers that extend from the bacterial

surfaces and mediate attachment of bacteria to
specific receptor on the host cells.

• The pili of many Gram-negative bacteria bind

directly to sugar residues.
Nonpilus adhesins:
• These include glycocalyx and other adhesins present on the bacterial surfaces.

• Glycocalyx is a polysaccharide “slime layer” secreted by some strains of bacteria

that mediates strong adherence to certain structures, such as catheters,
prosthetic implants, and heart valves.

• For example, the glycocalyx of Staphylococcus epidermidis and that of certain

viridans streptococci allows the bacteria to adhere strongly to the endothelium
of the heart valve.

• The matrix formed by these adhesins forming proteins is called a biofilm.

• The biofilms are important in pathogenesis because they protect the bacteria by
host defense and antibiotics.
• Streptococcus pyogenes makes use of nonpilus
adhesins (such as lipoteichoic acid, protein F, and
M protein) to bind to epithelial cells.

• The lipoteichoic acid and protein F cause

adherence of the streptococci to buccal epithelial
cells.

• M protein acts as an antiphagocytic molecule.

 Flagella
• Flagellated bacteria are more virulent than
non- flagellated ones.
• Motility towards the host cell is for adhesion
and invasion.
• Colonization by bacterial motility.
 INVASION
• Once surface attachment has been secured , microbial
invasion into subsurface tissues and organs is accomplized by
disruption of the skin and mucosal surfaces by several
mechanisms .
• Factors that contribute the disruption of skin and mucus
surface:
Trauma Inhalation Diseases Others
Abrasions Toxic gases Malignancies Child birth
Burns (chemical Particulate Alcoholism and Implantation of
and fire) matter other chemical Medical devices
Surgical wounds dependencies
Needle sticks smoking Diabetes Overuse of
Antibiotics
Two types of Bacterial mediated invasion:
Zippering
Bacteria present ligands on their
surface allow them to bind to host cells
and initiate the entry process
Triggering
Bacteria inject effectors into host cells via
T3SS to regulate phagocytosis.
• Invasion of tissues followed by inflammation
is enhanced by many factors, which include:

(a) Enzymes,
(b) Antiphagocytic factors,
(c) Inflammation,
(d) Intracellular survival.
(e) Biofilms,
 1. Enzymes: Invasion of bacteria is enhanced by many enzymes.

■ Hyaluronidases and collagenase:

• Hyaluronidases and collagenase are the enzymes
that hydrolyze hyaluronic acid and degrade collagen,
respectively; thereby allowing the bacteria to spread
through subcutaneous tissues.
• Examples :
Hyaluronidase Collagenase
Streptococci Clostridium perfingens
Staphylococcci Clostridium histolyticum
 Coagulase:
• Staphylococcus aureus produces the enzyme coagulase,
which in association with blood factors coagulates the
plasma.

• Coagulase contributes to the formation of fibrin walls around

staphylococcal lesions, which protects bacteria from
phagocytosis by walling off the infected area.

• The enzyme also causes deposition of fibrin on the surfaces

of individual staphylococci, which may help protect them
from phagocytosis or from destruction within phagocytic
cells.
■ Streptokinase ( fibrinolysin):
• Many hemolytic streptococci produce enzyme
streptokinase, which activates a proteolytic enzyme
of plasma.

• This enzyme is then able to dissolve coagulated

plasma and thereby possibly aids in the rapid spread
of streptococci through tissues

• Streptokinase has been used in the treatment of

acute myocardial infarction to dissolve fibrin clots.
■ IgA1 proteases:

• Certain pathogenic bacteria produce enzymes IgA1 proteases

that split IgA1 at specific proline– threonine or proline–serine
bonds in the hinge region and inactivate its antibody activity.

• Production of IgA1 protease allows the pathogens to

inactivate the primary antibody found on mucosal surfaces
and thereby facilitates the attachment of these bacteria to the
mucous membrane.
• Eg:

Neisseria gonorrhaea Haemophilus influenza

Neisseria meningitidis Streptococcus pyogenes
2. Antiphagocytic factors:
• Many bacterial pathogens are rapidly killed once
they are ingested by polymorphonuclear cells or
macrophages.

• Some pathogens evade phagocytosis or leukocyte

microbicidal mechanisms by several antiphagocytic
factors:
(a) capsule,
(b) cell wall proteins,
(c) cytotoxins, and
(d) surface antigens.
■ Capsule:

• The capsule surrounding bacteria, such as S. pneumoniae and

N. meningitidis, is the most important antiphagocytic factor.

• It retards the phagocytosis of bacteria by preventing the

phagocytes from adhering to the bacteria.
• There are two types of capsule.
Macrocapsule Microcapsule
Thickness of 0.2µm or more Thickness less than 0.2µm
visible under light microscope visible under Electron microscope
■ Cell wall proteins:
• cell wall proteins, such as the protein A and
protein M, of S. aureus and S. pyogenes
especially are antiphagocytic.
• For example,
Protein A of S. aureus binds to IgG and
prevents the activation of complement protein
 Important bacterial surface virulence:
Virulence factors Bacteria
Capsule
Polysaccharide capsule Streptococcous pneumoniae ,
Klebsiella pneumoniae,
Haemophilus influenza,
Salmonella typhi,
Neisseria meningitidis .
Polypeptide capsule Bacillus anthracis
Pili protein Escherichia coli
Protein A Staphylococcus aureus
M protein Streptocoocus pyogenes
V & W protein Yersinia pestis
■ Cytotoxins:
• Certain bacteria produce cytotoxins that interfere
with chemotaxis or killing of phagocytes.
• For example, S. aureus produces hemolysins and
leukocidins that lyse and damage RBCs and WBCs.

■ Surface antigens:
• Surface antigens of bacteria, such as Vi antigen of S.
typhi and K antigen of E. coli make the bacteria
resistant to phagocytosis and lytic activity of
complement.
3. Inflammation:
• Inflammation is an important host defense induced by the
presence of bacteria in the body.
• It is of two types: pyogenic and granulomatous.
-Pyogenic inflammation is the host defense seen primarily
against pyogenic or pus producing bacteria, such as
S.pyogenes.
It typically consists of neutrophils and the production of
specific antibodies and elevated level of complement.
-Granulomatous inflammation is the host defense seen
primarily against intracellular granuloma-producing bacteria,
such as Mycobacterium tuberculosis, Mycobacterium leprae, etc.
• The response consists of production of macrophages and CD4+
T cells.
4. Intracellular survival:

• A few mechanisms that are suggested for

intracellular survival of bacteria include
(a) Inhibition of phagolysosome fusion,
(b) resistance to action of lysosomal enzymes,
(c) adaptation to cytoplasmic replication .
Mechanism of intracellular Organism
survival

Inhibition of phagolysosome Legionella species,

Fusion Mycobacterium tuberculosis,
Chlamydia species

Resistance to lysosomal Salmonella typhimurium,

Enzymes Coxiella species,
Mycobacterium leprae,
Leishmania species

Adaptation to Listeria monocytogenes &

cytoplasmic replication Rickettsia
 Obtaining nutrients
• Pathogenic bacteria have intricate methods to obtain all
essential nutrients.
• Obligate intracellular bacteria have complex nutrient
requirements and parasitize the living cell for an extended
period.
• Host cytoplasm is a very nutrient rich environment.
i). Extracellular pathogens often lyse cells to obtain
nutrients.
ii). Intracellular pathogens will either escape from
phagosomes to enter the nutrient rich cytoplasm or modify
the vacuole so they can get nutrients from the cytoplasm.
Iron:
• Host tissues are very low in iron because it is bound to
transferrin, lactoferrin, ferritin, and heme.
• Bacterial strategies for obtaining iron (often induced by
low iron conditions):
1) Siderophores--low molecular weight compounds
that chelate iron with very high affinity; secreted and
taken up by bacterial surface receptors.
2) Direct binding of host transferrin, lactoferrin,
ferritin, or heme by bacterial surface receptors.
Bacterial Toxins:
• Toxins are products of a pathogen that destroy
& inactivate one or more vital component of
the host.
1. Exotoxin 2. Endotoxin
[ Protein molecule liberated from [Toxin- released when cells die]
intact living bacterium]
 Exotoxins
• They are heat labile proteins; secreted by certain species of both Gram
positive & Gram negative bacteria and diffuse readily into the surrounding
medium.
• High potency- Exotoxins are highly potent even in minute amounts.
Botulinum toxin is the most potent, it has been estimated that 39.2g of
botulinum toxin would be sufficient to eradicate the entire humankind.

• Used for vaccine-Exotoxins can be converted into toxoids by treatment

with formaldehyde. Toxoids lack toxicity but retain antigenicity and thus
induce protective immunity when used as vaccines.

• Specific action- They are highly specific for a particular tissue e.g. tetanus
toxin for CNS. They have specific pharmacological activities.
Types of Exotoxins:
i). A-B toxins (intracellular acting)
1) Composed of two parts: A and B portions
2) The B portion mediates binding to a specific host cell
receptor.
3) After binding to the host cell, the A portion is
translocated into host cells and has biological activity against
an intracellular target
ii). Membrane disrupting (surface damaging)
• Cause damage or disruption of plasma membranes, which
leads to osmotic lysis and cell death. Many were originally
termed “hemolysins” because they lyse RBCs.
• Three types of membrane disrupting toxins:
a) Enzymes that hydrolyze phospholipids: phospholipase,
sphingomyelinase
b) Toxins with detergent-like surfactant activity that disrupt
by membrane solubilization
c) Pore forming toxins (the most common): proteins that
insert in the host membrane and form a hydrophilic pore.
iii).Super antigens
• Toxins that bind directly to MHC II on macrophages (without
being processed) and form a crosslink with T cell receptors.
• Crosslinking causes stimulation of up to 1 in 5 T cells in the
body (normal antigens cause stimulation of 1 in 10,000).
• Excessive IL-2 production results from the massive
stimulation of T helper cells & stimulation of other cytokines
by IL-2 lead to shock.
Eg: staphylococcal toxic-shock syndrome
Diff. b/w Normal & Super antigen
Antigen Super antigen

Specific Nonspecifc
activation of T- activation of T
cells cells

Binds with Binds to

particulate variable beta
epitope of TCR chain of TCR

Activate 1 in Activate 1 in 5
10000 T cells T cells
Examples of Exotoxin:
Organism
Staphylocoocus aureus
Streptococcus pyogenes
Corynebacterium
diphtheriae
Bacillus anthracis
Clostridium tetani
Clostridium botulinum
Vibrio chlorae
Shigella dysenteriae type 1
Pseudomonas
Exotoxin Mechanism
Enterotoxin TSS Toxin Act as super antigen
Pyrogenic Exotoxin Inhibit protein synthesis
Diphtheria toxin cAMP in target cell ,
edema
Anthrax toxin Lecithinase and
phopholipase activity
(Myonecrosis)
Tetanus toxin Inhibits GABA and glycine
(Spastic paralysis )
Botulinum toxin Inhibit acetylcholine
(flaccid paralysis)
Cholera toxin Activation of adenylate
cyclase
Shiga toxin Inhibit ribosomes
Exotoxin -A Inhibit elongation factor -2
 Endotoxins:
• They are the lipid A portion of lipopolysaccharide. They are
present as an integral part of the cell wall of Gram negative
bacteria.
• Macrophages and monocytes release cytokines (IL-1, IL-6, IL-
8, TNF alpha, Platelet Activating Factor), which subsequently
trigger prostaglandin and leukotriene release , complement
and coagulation cascades are activated.
• Endotoxins are released from the bacterial surface by natural
lysis of the bacteria & endotoxic shock occurs, i.e Circulatory
system collapse followed by multiple organ system failure .
Mechanism of Endotoxin activity:
 FEATURE ENDOTOXINS EXOTOXINS
Nature LPS Proteins
Location Chromosome Plasmid & Bacteriophage
Source Part of cell wall of Gram Secreted both by Gram
Negative Bacilli. Negative Bacilli & Gram
Positive Cocci.
Released by Cell lysis, not by secretion Actively secreted & diffuse
into surrounding medium
Heat stability Highly stable Heat labile destroyed at
60oC.

Mode of action Mediated by Interleukins Mostly enzyme like action.

(IL -1 ) and Tumour
necrosis factor .
Effect Non-specific (fever, shock, Specific action on
etc particular tissues.
Contd..,

FEATURE ENDOTOXINS EXOTOXINS

Tissue affinity No Specific affinity for tissues.

Fatal dose Only large doses are fatal. More potent,even smaller
doses fatal.
Antigenecity Poorly antigenic Highly antigenic

Neutralization by Ineffective Neutralized by specific

Antibodies antibodies.
Used for vaccine No effective vaccine Toxoid forms are used as
vaccine eg: tetanus toxin.
Diseases Gram negative septicemia , Botulism ,Staphlococcal
Meningococcemia Toxic shock syndrome .
5.BIOFILM
• Biofilm is an aggregate of microorganisms in which the
bacterial cells are adhere to eachother on a surface and are
embedded within the layer(slime layer )of a self- produced
extracellular polymeric substance called ‘glycocalyx’.
• Stages of Biofilm :
 Biofilm a great threat to Implants
• A Significant number of people are affected by biofilm infections
which develop on medical devices implanted in the body such as
- Intravascular cathetars
- Artificial joints
- Mechanical heart valves .
• When implanted material becomes colonized by microorganisms, a
slow developing but persistant infection results .
• Bacteria growing in a biofilm are highly resistant to antibiotics .
• Stranded ANTIBIOTIC THERAPY is often useless and only recourse
may be to remove the contaminated implant.
 BIOFILMS AND HUMAN INFECTION:
• These pathogenic organisms have been associated with biofilm formation in
human infections.
• Artificial prosthetics and Indwelling Devices
- Candida albicans
- Staphylococcus
- Streptococcus
- Coagulase negative Staphylococci
- Enterococcus spp.
- Klebsiella pneumoniae
- Pseudomonas aeruginosa .
• Food – borne contamination
- Listeria monocytogenes
PATHOGENECITY ISLANDS:(PAI)
•Large genomic islands located in the chromosomal regions of some bacteria
containing sets of genes encoding numerous virulence factors.

• Genes encoded in pathogenecity island are expressed in a co-ordinated way

to initiate the virulence process.

• Major Properties :
- They have one or more virulence factors.
- Present in the genome of pathogenic members of a species but
absent in non pathogenic members.
- Typically have different (G+C) content than rest of bacterial genome.
- Commonly associated with t- RNA genes.
FEW EXAMPLES OF PAI:

Genus and species PAI name Virulence characteristics

Escherichia coli PAI I 536 II 536 Alpha hemolysin, fimbiae
Staphylococcus aureus SaPI 1 Toxic shock syndrome -1
Staphylococcus aureus SCC mec Methicillin and other
antibiotic resistance
Yersinia pestis HPI Enhance iron uptake
Enterococcus faecalis NP Biofilm formation
Vibrio chlorae EL Tor O1 VPI -1 Utilization of amino sugars.
Salmonella typhimurium SPI-1 Invasion and damage to
host cells , diarrhoea.
 Reference
• Jawetz , Melnick & Adelberg ‘s Medical
Microbiology .
• Essentials of Medical microbiology,
Aburva S Sastry .
• Bailey & Scott’s Diagnostic Microbiology.
• www.ncbi.nlm.nih.gov
• Textbook of Microbiology , S C Parija.
 THANK YOU
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