This document discusses various virulence factors of bacteria. It describes components like capsules, cell walls, adhesins, invasins, toxins, plasmids, bacteriophages, infecting dose, route of infection and communicability. It explains concepts like adaptation, adhesion, invasion, toxigenicity, and dissemination. It provides examples of specific virulence factors for different bacteria like Yersinia enterocolitica, Aeromonas hydrophila and their role in pathogenesis.

1. Major advisor
Parampal Sahota
Vainy Garg
M.Sc Microbiology
L-2011-BS-255-M

4.  Capsule
 Lipid A
 Adhesins
 Invasins
 Toxins
 Plasmids
 Bacteriophages
 Infecting dose
 Route of infection
 Communicability
 Bacterial appendages

7.  CAPSULE
 produce inflammatory cytokines.
e.g. Streptococcus pneumoniae, Neisseria
meningitidis, and Pseudomonas
aeruginosa.
 differences in capsular polysaccharide
chemical structure determine the
meningococcal serogroups.
 CELL WALL :
 contain toxic components involved in
bacterial septic shock, collapse of the
circulatory system and multiple organ
system failure.
 Acts via the initiation of an inflammatory
response through the stimulation of
monocytes and macrophages.

8.  ADHESINS :
 Adherence of the pathogen to host surfaces.
MECHANISMS OF ADHERENCE TO CELL OR
TISSUE SURFACES
 Possible interactions and forces involved
are:
 hydrophobic interactions
 electrostatic attractions
 atomic and molecular vibrations resulting from
fluctuating dipoles of similar frequencies
 Brownian movement
 recruitment and trapping by biofilm polymers
interacting with the bacterial glycocalyx (capsule)

9.  Receptor-ligand
interactions promotes
adherence involves protein-
protein and protein-
carbohydrate interactions.
 molecules serve as host
receptors for microbes
include membrane-spanning
proteins, surface
immunoglobulin, glycolipids,
glycoproteins, and
extracellular matrix proteins.

10. Enterotoxigenic E.
coli
Type-I fimbriae
Intestinal
epithelium
Diarrhea
Uropathogenic
E. coli
Type I fimbriae
Urethral
epithelium
Urethritis
Uropathogenic
E. coli
P-pili (pap) Upper urinary tract Pyelonephritis
Bordetella pertussis
Fimbriae (filamentous
hemagglutinin)
Respiratory
epithelium
Whooping
cough
V. cholerae
N-methylphenyl-
alanine pili
Intestinal
epithelium
Cholera
Treponema
pallidum
Peptide in outer
membrane
Mucosal
epithelium
Syphilis

11.  INVASION :
 Pathogens gain deeper access into the host to perpetuate the infection
cycle.
 Extracellular invasion : allows pathogens to proliferate tissues,
disseminate to other sites in the body, express toxins, and initiate
inflammatory responses.
 E.g. : β hemolytic streptococcus and S. aureus- secrets hyaluronidase
streptokinase and staphylokinase, lipase, and nuclease .
 Intracellular invasion : microbe penetrates the cells of host tissue and
survives within this environment.
 E.g. : Chlamydia spp, Rickettsia spp and Mycobacterium leprae : obligate
intracellular lifecycle that requires mammalian cell for growth.

12. Invasin Activity
Hyaluronidase Degrades hyaluronic of connective tissue
Collagenase Dissolves collagen framework of muscles
Neuraminidase Degrades neuraminic acid of intestinal mucosa
Coagulase Converts fibrinogen to fibrin which causes clotting
Kinases Converts plasminogen to plasmin which digests fibrin
Leukocidin
Disrupts neutrophil membranes and causes discharge of
lysosomal granules
Streptolysin
Repels phagocytes and disrupts phagocyte membrane and
causes discharge of lysosomal granules
Hemolysins
Phospholipases or lecithinases that destroy red blood cells
(and other cells) by lysis
Lecithinases Destroy lecithin in cell membranes
Phospholipases Destroy phospholipids in cell membrane
Anthrax EF
One component (EF) is an adenylate cyclase which causes
increased levels of intracellular cyclic AMP
Pertussis AC
One toxin component is an adenylate cyclase that acts
locally producing an increase in intracellular cyclic AMP
Extracellular bacterial proteins that are considered invasins

13.  Toxigenicity :
 Exotoxins and Endotoxins
 Classification of exotoxins:
a. Membrane damaging toxins:
destroy the host membranes by
inducing pore formation or by
destabilising cytoplasmic
membranes.
b. Superantigens: bind non-
specifically, activate large numbers
of T cells leading to shock.
c. A-B component toxins : The B
component binds to specific host
cell receptors and A is
enzymaticcally active portion of the
toxin.

16.  PLASMIDS: Gene coding for virulent characteristics can be
plasmidborne.
 E.g. surface antigens responsible for colonisation of intestinal
mucosa by E. coli and enterotoxin production by E. coli and S.
aureus.
 BACTERIOPHAGES : In diphtheria bacilli, gene for toxic
production is present in beta or other toxic corynephages.
 COMMUNICABILITY : Ability of parasite to spread from
one host to another.
 Highly virulent parasite may not exhibit high degree of
communicability due to rapid lethal effect on host. E.g.
respiratory and intestinal diseases.
 BACTERIAL APPENDAGES: Bacterial surface antigens
such as Vi antigen of S typhi and K antigens of E coli : prevents
bacteria from phagocytosis and lytic activity of compliments.

17.  INFECTING DOSE :
Dosage estimated as
minimum infecting dose
(MID) or minimum lethal
dose (MLD)
 Route of infection : Modes
by which different bacteria
able to initiate tissue
damage.
 Cholera vibrios infect orally
and unable to cause infection
subcutaneously.

18. • SECRETED OR SURFACE-EXPOSED
BACTERIAL PROTEINS IN BACTERIAL-
HOST INTERACTIONS.
• Proteins secreted by the Type I system cross
directly from the cytoplasm to the cell surface,
bypassing the general secretory pathway
completely.
• Type II-secreted proteins use the general
secretory pathway to reach the periplasm and
traverse the outer membrane through distinct
channel proteins.
• Type III system : effector molecules move to the
external surface of the bacterium, facilitate the
pathogen’s ability to survive and replicate.

19.  colonization of the
intestinal tract, and
penetrate M cells of
Peyer’s patches.
 replicate in extracellular
form within micro-
abscesses.
 form microcolonies and
resistant to phagocytosis
by macrophages and
neutrophils (Fabrega et al
2011).

21.  six biotypes differentiated by physiochemical and
biochemical tests (1A, 1B, 2, 3, 4, and 5)
 more than 50 serotypes differentiated by antigenic
variation in cell wall lipopolysaccharide.
 virulent biotypes 1B and 2–5 has highly conserved
70-kb virulence plasmid, termed pYV/pCD and
certain chromosomal genes.

22.  The biotype 1A lack pYV plasmid encodes virulence
factors {Yersinia adhesin A (YadA) and Ysc-Yop type
III secretion system (TTSS)} and chromosomally
borne virulence genes {ail, myfA, ystA, ysa, high
pathogenicity island- (HPI-)}
 Biotype 1B carry high-pathogenicity island
(HPI),facilitates the uptake and utilization of iron by
bacterial cells, promote growth under iron-limiting
conditions in host tissues.

23.  Virulence-associated determinants of pYV-negative
strains includes:
 cell surface lipopolysaccharide
 SodA (a superoxide dismutase) : bacterial survival
in tissues.
 urease, enhances bacterial resistance to stomach
acid and in nitrogen assimilation.

24.  pYV, antihost plasmid, resist
phagocytosis and complement-
mediated lysis, to proliferate
extracellularly in tissues.
 virulence factors, outer
membrane protein adhesin,
YadA, and type III protein
secretory apparatus
translocates effector proteins
(Ysc-Yops) from bacterial cell
to the cytoplasm of susceptible
host cells.

26.  Adaptation : Yersinia adapt surface antigenic structures
(outer membrane proteins) to colonize in the intestines of
humans at temperature of 37◦C through 70-kb virulence
plasmid (pYV).
 Adhesion : allow intimate attachment to the epithelial
cells.
 YadA, a pYV plasmid-encoded protein, adhesion for
attachment, induction of disease (e.g., inflammation and
necrosis in the liver).
 mediates adherence to epithelial cells, phagocytes and
extracellular matrix components, and protects the
bacterium killed by neutrophils.

27.  Fimbriae present in biotype1A :
 MR/Y-HA : 8 nm in diameter, agglutinates
erythrocytes in the presence of mannose and
expressed in vitro at low temperature.
 MR/K like HA : 4 nm in diameter and mediates
mannose resistant hemagglutination of chicken
erythrocytes.
 Y. enterocolitica produces Myf (for mucoid Yersinia
fibrillae), bestows mucoid appearance on bacterial
colonies. (Sabina et al 2011)

28.  Invasion :
 Ail (attachment-invasion locus) :
 Localized in the OM.
 Eight transmembrane β-sheets and four cell surface-exposed loops,
and the extracellular loop for Ail-mediated binding to host cells.
 Promotes Yop delivery into the primary target of T3SS--the
phagocytic cells and into epithelial cells.
 Invasin :
 Located in middle of gene cluster encoding the flagella proteins
 ~100 kDa, anchored by its amino-terminal region
 Receptor is β1 integrin, intergrins couple extracellular adhesion
events to numerous signaling pathways, and the bacterium is taken
up by zipper mechanism.

29.  Mechanisms of bacterial
epithelial cell internalization
:
 “zippering” process : tight
enclosing of the bacterial cell
by the mammalian cell
membrane.
 involving surface bound
bacterial protein invasin (Inv)
binding an integrins of the β1
family of mammalian cell
surface.

30.  TOXINS :
 ystA, ystB :
 heat-stable enterotoxin.
 causes Yersinia associated diarrhea.
 stimulate cGMP synthesis in the intestinal brush border,
leading to fluid loss and lack of fluid absorption.
 Pathogenicity islands in Yersinia : HPI
 capture the iron molecules for systemic dissemination of
the bacteria in the host via yersiniabactin.

32.  LOCAL AND SYSTEMIC DISSEMINATION
 Cross the intestinal epithelium through FAE (follicle associated
epithelial cell), in the Peyer’s patches of the ileum.
 Invasin (Inv), a 103 kDa outer membrane protein of Yersinia binds
β1 integrins that are expressed apically on M cells.
 Yersinia surface proteins (Ail, PsaA, and YadA ) account for
residual invasion of inv mutants.
 Yersinia defend attack by resident macrophages by expressing an
antiphagocytic strategy.
 mediated by plasmid encoded type III secretion, of three protein
effectors, YopH, T, and E, disrupt cytoskeletal assembly required
for phagocytosis process.
 Extracellular in infected Peyer’s patches and mesenteric lymph
nodes and disseminate to cause local and systemic infection.

33.  When bacteria bind to tissue
culture cells, 10 different
effector molecules secreted
and three injected into cells.
 YopE and YopH, modify
macrophage proteins to
destroy the cells abilities to
engulf and kill bacteria.
 immune cells neutralized by
effector molecules, enables
Yersinia spp to flourish in the
reticuloendothelial
environment.

34. • capture the iron molecules for systemic dissemination
in the host.
• Yeriniabactin, sub-group of phenolate siderophores and
has affinity for ferric iron.
•FyuA/Psn-Irp system uses yersiniabactin, a siderophore
that remove iron from mammalian proteins.
• YbtA, AraC-like regulator required for transcription
of fyuA/psn, irp2 and ybtP, downregulate its own
transcription.
• In the presence of iron, Fur, a cytosolic protein, bind
ferrous iron, changes conformation and binds DNA at
specific site called Fur box, preventing transcription,
downregulates transcription of fyuA/psn, irp2 and other
iron-regulated genes.

35.  classified into 96 serogroups and the O-antigen
LPS of A. hydrophila 0:34 strains in adhesion to
HEp-2 cells.
 attaches and enters into host cells through
production of flagella, pili and adhesins.
 multiplication in host tissue by production of
siderophores and outer membrane proteins
 enterotoxins, proteases, phospholipases, and
hemolysins cause damage to host cells leading to
cell death.

36.  directed locomotion
 attachment to
gastrointestinal epithelium
 biofilm formation
 colonization
 elaboration of virulence
factors
 infection (Janda et al

37.  Enterotoxins—cytotonic and cytotoxic.
 Cytotonic enterotoxins (heat-labile (Alt) and heat-
stable (Ast)), donot degenerate crypts and villi of
small intestine.
 Cytotoxic enterotoxin (Act) result in extensive
damage to epithelium.
 Aerolysin, extracellular, soluble, hydrophilic protein
exhibiting hemolytic and cytolytic properties.
 The mature form of Act is 49 kDa in size and
involved in hemolytic, cytotoxic, enterotoxic and
lethality of mice.

38.  The capsular gene cluster 17,562 bp long.
 include 13 genes assembled into three distinct regions.
 Regions I and III: four- and two-capsule transport
genes
 region II : five genes.
 type IV pili (bundle-forming pili (Bfp) and Tap (type
IV Aeromonas pili)) associated with gastroenteritis.
 Bfp promote colonization by forming bacterium-to-
bacterium linkages.

39.  The Tap biogenesis gene cluster : four genes
(tapABCD) -
 tapA gene encode subunit protein
 tapB and tapC genes involved in pilus biogenesis
 tapD gene encoded type IV prepilin peptidase/N-
methyltransferase.
 Nine lateral flagellar genes
(lafA,B,C,E,F,S,T,U,X) : lateral flagella distinct
from the polar flagellum and involved in swarming
motility

40.  Entry of water from the external milieu into
erythrocytes through the pores, resulting in cell
swelling and subsequent lysis.
 Preincubation of the toxin with cholesterol result in dose-
dependent reduction in hemoglobin release from
erythrocytes.
 Act interact with cholesterol on the membranes of
erythrocytes
 aggregation occurred
 resulting in transmembrane pore formation and cytolysis
of erythrocytes.

41.  production in two precursor forms (pre-
protoxin)
 conversion to an active toxin by removal of a 23-
aa-long NH2-terminal signal peptide (protoxin)
 proteolytic cleavage of the protoxins at their
carboxy-terminal end (removal of 4–5 kDa
peptide) to form a mature, biologically active
toxin.
 their characteristics of punching holes in the
membranes.

42.  divert some of the metal ions to
microbial metabolism.
 produce siderophores,
enterobactin or amonabactin.
 amonabactin producers
-siderophore-dependent and
independent means for iron
acquisition.
 enterobactin producers-
nonsiderophore heme
utilization.

43. The ligand exchange step occurs at the cell surface and involves the exchange of iron
from a ferric siderophore to an iron-free siderophore bound to the receptor.

44.  CRYSTAL VIOLET BINDING
TEST
 Virulent plasmid bearing
colonies (P+
) : dark violet by
binding of crystal violet dye
 Plasmidless (P-
) colonies
remained white as they could not
bind the crystal violet dye.
 CONGO-RED BINDING TEST
 Plasmid bearing strains : red
colonies
 Plasmidless strains : colourless
to pale pink colonies.
Bhaduri et al (1987).
Riley and Toma (1989).

45.  LIPASE TEST
 egg yolk agar medium.
 Colonies iridescent and pearl like
 surrounded by a precipitation
ring and a clear zone.
 AUTOAGGLUTINATION
TEST
 Test organisms grown in MR-VP
tubes.
 Agglutination positive (Agg+
) :
Flocculation of irregularly edged
layer of a agglutinated bacteria at
the bottom of the tube with clear
supernatant fluid.
 Agg-
: smooth round pellet at
bottom.
Autoagglutination test
Laird and Cavanaugh (1989).

46.  DEOXYRIBONUCLEA
SE (DNase) TEST
The test and positive
control organism
(Staphylococcus aureus)
were examined for
appearance of clear zone
around the colonies within
5 min of adding HCl
which indicated positive
test.

47.  PROTEASE PRODUCTION
 The protease production was estimated by the formation of a
clear zone caused by casein degradation.
 HEMOLYSIN PRODUCTION
 Young test culture (3-4 hour old) was streaked on to the sheep
blood agar plates (5%) and observe for hemolysis zone.
 ESCULIN HYDROLYSIS
Protease production Esculin producion

48.  PYRAZINAMIDASE ACTIVITY
 Pink colour indicate the presence of
pyrazinoic acid and were
pyrazinamidase positive (Pyz+
).
 Pyz-
strains were positive for
virulence.
 SIDEROPHORE PRODUCTION
 Succinate media with CAB dye was
prepared and yellow to orange color
zone showed positive for
siderophore production. Yellow colored zones showing
siderophore production
Kandola and Wauters (1985).

49. Plasmid based phenotypic
tests
Aeromonas Yersinia
Crystal violet binding test Violet coloured colonies Violet coloured colonies
Congo red binding test Red coloured colonies Red coloured colonies
Lipase test Clear zone formed around
colonies
Clear zone formed around
colonies
Autoagglutination test An irregular edged layer
of aggglutinated bacteria
which formed flocculate
covering at the bottom of
tube with clear
supernatant fluid.
An irregular edged layer of
aggglutinated bacteria which
formed flocculate covering at
the bottom of tube with clear
supernatant fluid.
Deoxyribonuclease
(Dnase) test
No clear zone formed. No clear zone formed.

50. Aeromonas Yersinia
Capsular polysaccharide Positive Positive
Cell surface
hydrophobicity
Positive Positive
Protease production Positive Positive
Hemolysin production Positive Positive
Lipopolysaccharide
production
Positive Positive
Siderophore production Positive Positive
Esculin hydrolysis Positive Positive