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Pathogenesis
MICR570/ZMR/F12 11/12
Bacterial Pathogenesis
some Fun al
Lectures 11 & 12
OBJECTIVES: LECTURES 11 & 12
• Discuss basic concepts of virulence factors
• Discuss basic princip les of pathogenesis
• xp a n o e s eps – With examples of micr obial strategies (virulence
factors)
• Discuss pathogenesis wrt contri bution todisease
VIRULENCE FACTORS
• Influence the microbes ability t o cause disease:
– Promote colonization of host
– Cause damage to host
• re om nan y enco e y assoc a e w mo e
genetic elements:
– phages, plasmids, insertion sequences, transposons
• Large proportion located within
– Pathogenicity Islands (PAI’s)
EXAMPLES VIRULENCE FACTORS
ENCODED BY PAI
• Iron uptake systems
• Adhesins
• Pore-forming toxins
• Superantigens
• Secreted lipases
• Secreted proteases
• Proteins transported by type I, III, IV & V secretion systems
• Antibiotic resistance phenotype
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Pathogenesis
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4 KEY CONCEPTS
1. Infective dose; host defence
2. Infection vs Intoxication
3. Pathogenic cycle
4. Intracellular vs Extracellular Pathogens
STEPS OF THEPATHOGENIC CYCLE
Exposure & Entry
Attachment
Evasion of immune response
Effects on host
Dissemination/Shedding
INTRACELLULAR vs EXTRACELLULAR
PATHOGENSIntracellular
Extracellular Obligate Facultative
Bacteria
Chlamydia spp.Mycobacterium leprae
Rickettsia spp.
Brucella spp.Bordetella pertussis
Campylobacter spp.(Some) E. coli
Group B Streptococcus
Leigonella spp.
Bacillus spp.Borrelia spp.
Clostridiumspp.Corynebacterium diphtheriae
(Most) E. coli
Group A StreptococcusListeria monocytogenes
Neisseria gonorrhoeae (meningitides)Salmonella spp.
Shigella spp.
Yersinia spp.
Haemophilus spp.
Klebsiella spp.Helicobacter spp.
Proteus spp.
Pseudomonas spp.Staphylococcus spp.
Treponema spp.Vibrio cholerae
Fungi
None Blastomyces dermatitidisCandida albicans
Coccidioides immitisHistoplasma capsulatum
Cryptococcus neoformansPneumocystis jirovecii
Adapted from McClane, B.A. & Mietzner,T.M. (1999) Microbial Pathogenesis.Fence Creek Publishing. p5
2nd STEP OF PATHOGENIC
PROCESS
Exposure & Entry
Attachment
InvasionEvasion of immune response
Effects on host
Dissemination/Shedding
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Pathogenesis
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ATTACHMENT/ADHERENCE
Necessary for colonization (particular site)
• REQUIRES
Host cell receptors (CH2O’s, proteins, cytokine/hormone)
Microbial surface com onents adhesins/li ands
Defined by:
• Ability to adhere (interaction)
• Favourable environment (pH, nutrients, etc)
• Presence/absence of normal microflora (commensals)
See Murray et al., Table 18-2, p181
Tissue Tropism/Specificity
• Streptococcus mutans – enamel
• Streptococcus salivarius – tongue epithelial cells
Other examples
– H. pylori – gastric mucosa
(Dental plaque)
– Campylobacter spp. – intestinal mucosa
– N. gonorrhoeae – urethral epithelium
– B. pertussis – upper respiratory tract epithelium
– S. aureus – nasal membranes
Absence of appropriate bacterial ligand = colonize
Absence of appropriate cell receptor = colonize
Occupied cell receptor = colonize
Adhesins
Pili & Fimbriae (Bacteria)
• May be coordinated with
other virulence factors
– Cholera toxin
– TcpA (Toxin Coregulated Pilus A)
E.g., E. coliLigand: Type I pili (CFA)
Receptor: GM1 ganglioside (intestinal epithelium)
Image from www.cdc.gov
Afimbr ial adhesins (Bacter ia & Fungi)
Typ e o f adh es in Or ganis m Sub stratu m
i i i i i li l ll
Adhesins ARE Virulence factors
E.g., B. pertussis Ligand: Filamentous haemagglutinin (FHA)
Prevention: FHA in vaccine
i i i i .
S. pyogenes
i li l ll
Buccal epithelial cells
LPS or LOS H. pylori
S. typhimurium
Gastric epithelial cells
Macrophages
Mannans C. albicans Epithelial mucosa
Image from www.cdc.gov
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Pathogenesis
MICR570/ZMR/F12 11/12
NEXT STEP OF PATHOGENICPROCESS
Exposure & Entry Attachment
Not essential
for all bacteria
nvas on
Evasion of immune response
Effects on host
Dissemination/Shedding
INVASION
Bacterial Mechanisms
• Phagocytic cells: via phagocytosis
• Non-phagocytic cells: variety of possible mechanisms
• nvas ns
– Bacterial & Fungal
– interact with specific cell receptors, induce endocytosis
• Host cell cytoskeleton rearrangement
– Internalin A: L. monocytogenes
– Usually injected into host cell Æ membrane ruffling
Figure 1: Scanning electron micrograph (taken by Roger Wepf, Philippe Sansonetti and ArielBlocker at the EMBL) of the rod-like Shigella flexneri entering a HeLa cell.
From: http://users.path.ox.ac.uk/~ablocker/introduction.html
POST-INVASION CONSEQUENCES
Possible fates of an invasive pathogen
• Restricted at site of entry
• Spread from epithelium to immediate underlying tissue, nofurther dissemination
• Spread to other body sites (Dissemination - discussed later)
Enzyme Action
Hyaluronidase Breaks down hyaluronic acid of connective
tissue
Streptokinase Breaks down fibrin clots
(Converts plasminogen Æ plasmin)
Col laginase Breaks down col lagen
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Pathogenesis
MICR570/ZMR/F12 11/12
NEXT STEP OF PATHOGENICPROCESS
Exposure & Entry
Attachment
InvasionEvasion of immune response
Effects on host
Dissemination/Shedding
Reminder of the Immune response to Bacteria
Component Function
Complement Opsonization
Killing of G-ve bacteria
B cell activation
Neutrophils Phagocytosis
Summarised from: Murray et al., Box 12-1, p.126
Macrophages Phagocytosis
Antigen presentation
Activation of inflammation
Antibody Block attachment
Toxin & enzyme neutralization
Opsonization
Component to be
evaded
Mechanism(s) used for evasion
Complement Capsules
Complement-binding proteins
Proteases
Host cell mimicry
Phagocytic kil ling Capsules
Type III Secretion systems
Intracellular parasitism
Antigen processing Interfere with MHC function &
antigen processing
Antibodies Ig-binding/inactivating proteins
Antigenic variation
EVASION OF COMPLEMENT &
PHAGOCYTES
Bacterial (Fungal) solution: Capsules
• Composition varies: enablesserotyping; used for vaccines
Box 18-5. Examples of Encapsulated
MicroorganismsStaphylococcus aureus
Streptococcus pneumoniaeStreptococcus pyogenes (group A)Streptococcus agalactiae (group B)
• Polysaccharide
• Polyribose ribitol phosphate
• Hyaluronic acid
– “host cell mimicry”
• Not “seen” as foreignÆ nophagocytosis/opsonization
See Murray et al. p.186
Bacillus anthracisBacillus subtilis
Neisseria gonorrhoeae
Neisseria meningitidisHaemophilus influenzaeEscherichia coli
KlebsiellapneumoniaeSalmonella spp.
Yersinia pestisCampylobacter fetus
Pseudomonas aeruginosaBacteroides fragilisCryptococcus neoformans (yeast)
Murray et al. Examples of Encapsulated Microorganisms. p186
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Pathogenesis
MICR570/ZMR/F12 11/12
Unencapsulated bacteriaPathogen-associated
Molecular patterns (PAMPS)
LTA, LPS
Picture Used with permission from: http://www.rit.edu/~gtfsbi/imm/parhamimages.htm/JPEG/CHAP01/1-13.JPG
Encapsulated bacteria
Phagocyte cannot attach
Modified from http://www.rit.edu/~gtfsbi/imm/parhamimages.htm/JPEG/CHAP01/1-13.JPG
Encapsulated bacteria/Opsonizing
Antibodies
Fc
Modified from http://www.rit.edu/~gtfsbi/imm/parhamimages.htm/JPEG/CHAP01/1-13.JPG
receptor
Solution: Secrete extracellular protease
• Pseudomonas aeruginosa (Gram -ve)
– ElastaseÆ prevents opsonization/phagocytosis
EVADE COMPLEMENT & PHAGOCYTOSIS
WITHOUT USING CAPSULES
– inactivates C3b & C5a
• Streptococcus pyogenes (Gram +ve)
– C5a peptidase Æ inhibits phagocyte chemotaxis
– degrades C5a
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Pathogenesis
MICR570/ZMR/F12 11/12
XX
X
EVASION OF PHAGOCYTE DESTRUCTIONSolution: Replicate (& live) in phagocytic cells
3. Inactivate oxygen
radicals or
degradative
enzymes
(S. aureus)
(H. capsulatum)
macrophage
X
1. Inhibit phagosome-
lysosome fusion(L. pneumophilia)
2. Escape phagolysosome(L. monocytogenes)
4. Production of
surface components
Solution: Toxins to kill macrophages & leukocytes
• Leukocidins Æ kill neutrophils and macrophages
EVASION OF MACROPHAGES &
LEUKOCYTES
• Alters phospholipid metabolism by ADP-ri bos ylation of a
protein controlling phosphatidylinositol
Æ disruption of cellular activities
• Typical producers = highly invasive bacteria, E.g.,
Staphylococci
– PVL: Panton-Valentine Leukocidin
EVADING IMMUNOLOGICAL RESPONSES Ant igen processi ng & presentation
Dendritic cell
Intracellular
antigen lysosome
(Redrawn from Fig. 10-2, Lippincott’s Illustrated Reviews of Immunology, p.122)
pMHC Class II
proteasome
pMHC Class Iphagolysosome
EVASION OF ANTIBODIES
• Ig-binding/inactivating proteins
– Direct binding of cell wall protein to Fc domain of IgG
– Bacterial IgA proteases (secreted); inactivate sIgA by
proteolysis
• Ant igenic /phase vari ation
– Phase variation = protein expression switchable
on/off
– Antigenic variation = multiple antigenic forms
expressed at different times
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Pathogenesis
MICR570/ZMR/F12 11/12
N. gonorrhoeae pili variation
Expressed pilin genes pilE
Recombination
Silent pilS genes
Expressed pilin genes
Silent pilS genes
event
ADDITIONAL CHALLENGEIRON SEQUESTRATION
• Iron = necessary for microbial growth in body fluids or onbody surfaces
• Limited amounts available in the body:
– is chelated, E.g., as transferrin, lactoferrin or haem
Bacterial strategies:
1. Specific surface receptors (Neisseria spp.)
“grab” host’s iron, chelators
2. Secretion siderophores (E. coli; K. pneuomoniae)
Low mw compounds; High affinity for iron
Siderophore-iron complex
GENERAL EVASION MECHANISM
Solution: Biofilm formation
Definition of a biofilm:
“ Collection of microorganisms that are attached to a surface,covered by a microbially-produced exopolymeric substance
(EPS)”
Hall-Stoodley, L. & Stoodley, P. (2009) Evolving concepts in biofilm infections. Cellular Microbiology 11(7) p1034-1043
. .
Dental caries
Peridontitis
Otitis media
Necrotizing fasciitis
Osteomyelitis
Examples of infections involving biofilms
Pneumonia
Cystic fibrosis (P. aeruginosa)Endocarditis
CDC data indicates that >60% of ALL bacterial
infections are growing as biofilms
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Pathogenesis
MICR570/ZMR/F12 11/12
WHY ARE BIOFILMS IMPORTANT?
1. Is changing the field of medical microbiology
2. Attachment up-regulates some genes and down
regulates others (including some virulence genes E.g.,
toxin production)
3. Free-floating “planktonic” bacteria behave,
communicate and respond very differently to attached
bacteria (i.e., biofilm)
4. Biofilms are different in terms of their: growth rates,
community interactions, susceptibilities to antimicrobial
agents and components of the immune response, etc
Addi tional take-home message
• Bacteria growing as biofilms can be SIGNIFICANTLY
more resistant to antimicrobial agents
100 – 1000 x higher concentrations required
• And this is in the ABSENCE of additional resistance
mechanisms such as β –lactamases or plasmids….
NEXT STEP OF PATHOGENIC
PROCESS
Exposure & Entry
Attachment
InvasionEvasion of immune response
Effects on host
Dissemination/Shedding
POSSIBLE
OUTCOMES OF
INFECTION
Infection & Damage
Host mediated
Immune response
Microbial mediated
ToxinsInvasion
Virulence factors
Replication
i l
1
Permanent relationship
Inapparent/subclinical
hysical growth
Prevention/Clearance
4
3
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Pathogenesis
MICR570/ZMR/F12 11/12-
OUTCOME: DAMAGEDirect Damage Mechanisms
1. Degradative enzymes 3. Toxic structures
2. Physical growth through tissues
4. Toxin sDamage membranes
Inhibit cellular function
(1) Degradative Enzymes
Enzyme Action
Hyaluronidase Breaks down hyaluronic acid of
connective tissue z y m e s ”
Streptokinase Breaks down fibrin clots
(Converts plasminogen Æ plasmin)
Collaginase Breaks down collagen “ s p r e a d i n g e
(2) Physical Growth Through Tissues
• No known degradative enzymes or exotoxins
i.e., Aspergillosis
• Mechanism of damage is unclear
– probably due to fungal grow th through tissues
• Displacement/destruction of vital structures
– E.g., angioinvasive Aspergillus
(3) Toxic Structures
• Gram -ve
bacterial LPS(Endotoxin)
Fig. 18-4, p.185.
Murray et al. 6th Ed
Fig 18-4, p185
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Pathogenesis
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Fungal toxins – A. fumigatius: Gliotoxin
• -
(4) Toxins
,& induces apoptosis
– A. flavus: Aflatoxin
• Carcinogenic
1. Protein tox ins
– EXOTOXINS
– Secreted into extracellular environment
– Specificity varies
– Ver otent
Bacterial Toxins
– Gram +ve and Gram -ve bacteria
2. Lipopolysaccharide toxins
– ENDOTOXINS
– Only acts as toxin under certain circumstances
– LPS of Gram -ve bacteria
Characteristic EXOTOXIN ENDOTOXINS
Toxicity Minute amounts High doses
Effects on body Specific to cell type Systemic, fever,
i l i
Overview of differentiating characteristics
COMPARISON OF
EXO/ENDOTOXINS
i l i
Chemical
composition
Polypeptide LPS of cell wall
Toxoid formation Yes No
Fever stimulation Not usually Yes
Manner of release Secreted from live cells Cell lysis
Examples of Exotoxins
Microorgan isms Key toxin(s) Cont ribu tion
Vibrio cholerae Cholera toxin Excessive watery
diarrhoeae
Clostridium botulinum Botulism toxin Muscle paralysis (flaccid)
Clostridium tetani Tetanus Toxin Muscle paralysis (rigid)
Staphylococcus aureus TSST-1 System-wide shock
Corynebacteriumdiphtheriae Diphtheria toxin Pseudomembrane,systemic effects
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Pathogenesis
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Nomenclature & Classes/Groups
Classified according to:
• Target s ite/cells: neurotoxins, enterotoxins
• Producing bacterium: Staphylococcal α –toxin
• Mechanism of action: ADP-ribosylators, pore-forming
toxins
• Associated di seases : cholera toxin, diphtheria toxin
Genes encoding for toxin production
• Plasmids
• Bacteriophage
Control via environmental regulators (pH, iron)
CLASSES OF EXOTOXINS
Class IMembrane acting/bind to host cell surface Class II
Membrane
damaging
E.g., TSST-1
E.g.
Phospolipases
Class III
Intracellular E.g., Diphtheria
ore- ormers
CLASS I EXOTOXINS
Superantigens
Powerful T-cell mitogens
• Interfere directly with host immune response coordination
Æ divert/confuse host defenses
• Interact directly with T-cells & APC’s
• Important contributor to damage
– E.g., Staphylococci: Toxic Shock Syndrome Toxin
(TSST-1); Streptococci: (SPE’s) Rheumatic fever
T cell
T cell
T cell
Antigen-
presenting
cell
SUPERANTIGEN
Bind directly to T cell receptor Æ 20% of T-cells activated
(normal pathway = 0.01% of T cells activated)
T cell
T cell
IL-2
IFN-γTNF-α
TNF-α
TNF-αTNF-α IL-2
IL-2
IL-2
IFN-γ
IFN-γ
IFN-γIFN-γ
Fever & shock
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Pathogenesis
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i) Phospholipid membrane destabilisation
Example: Lecithinase of C. perfringens
(α toxin (Phospholipase C))
CLASS II EXOTOXINSMembrane-acting
Target cells : various
Effect: indiscriminate lysis
Mechanism: Destabilize cell membrane by
removing polar head group from
phospholipids
Advantages to bacterium: eliminates host defences; createsnutritionally rich environment
ii. Pore formation
Example: Listeriolysin O
Target cells: Various
Effect: Cell death occurs due to
osmotic lysis or apoptosis
H20
K+
K
+
K
+
K+
ro e n pores es a ze
membrane
Often responsible for cytolytic activity
Also referred to as CFT’s = Channel Forming Toxins
CLASS III EXOTOXINS
Intracellular
A = catalytic ( Active) domain
• Enzymatically attacks a particular host cell function or structure
B = Binding subunitS S
Basic Structure
• Specific host cell surfaceglycoprotein or glycolipid
A portion
B portion
Catalytic activities:
ADP-ribosyltransferase (diphtheria toxin, cho lera toxin)Zinc metalloendoprotease (tetanus & botulismneurotoxins)DeamidaseGlucosyltransferase
Most common toxin structures:
AB• Single gene encodes for single peptide; post-translationally
modified to A&B fragments which are covalently linked
– E. . di htheria toxin. .,
A5B• Two genes encode A&B subunits; noncovalently associated
in stable complex
– E.g., cholera toxin
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Pathogenesis
MICR570/ZMR/F12 11/12-
1
2
1. Bacteria secrete exotoxin
2. Toxin enters host cell
3. Toxin removes ADP-ribose
Mechanism of Action: ADP-ribosylator
NAD
ADP-ribose
Protein ProteinX
ADP-ribose
4
3
5
group from NAD
= ADP-ribosylation
4. Transfers it to host cell protein
5. Host cell protein inactivated
A
B
A
B
A
B
InternalizationH
o
Diphtheria toxinReceptor = heparin-
binding epidermal growth
factor - like precursor
Acidification of endosome
NAD+ + EF-2 Æ ADPR-EF-2 + nicotinamide + H-
Active Inactive
A
st
c
e
l
l
Zinc metalloendoprotease
Tetanus toxin Botulinum toxin
Produced by Clostridium tetani Clostridium botulinum
Proteolytic cleavage of toxin Æ 2 linked fragments
a) Light chain (LC) – enzymatically active; zinc metalloprotease
b) Heavy chain – binding & translocation
Site of action Presynaptic
membrane of motor
neurons
Gangliosides
Mechanism Inhibition of inhibitory
neurotransmitters
Inhibition of
acetylcholine release
Effect “Spastic” paralysis Flaccid paralysis
TOXIN-BASED VACCINES
• Toxoid = inactivated toxin
• Methods for inactivation:
– heat
– chemicals, E.g., formaldehyde
• Still antigenic but do not cause damage
• Currently in use:
– Diphtheria toxoid (as part of DTaP vaccine) – Tetanus toxoid
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Pathogenesis
MICR570/ZMR/F12 11/12-
NEXT STEP OF PATHOGENICPROCESS
Exposure & Entry
Attachment
InvasionEvasion of immune response
Effects on host
Dissemination/Shedding
a) Direct: In surface fluids, by flow of intestinal contents
• Facilitated by toxin production (E.g., hyaluronidase, etc.)
• Growth through tissues, E.g., Aspergillus
DISSEMINATIONto a different site within the host
b) Via CSF
• Cross blood-CSF junction (choroid plexus)
– E.g., H. influenzae
c) Blood (hematogenous)
• Can be in the blood or blood components, E.g.
– Plasma: B. anthracis
– Mononuclear cells: Listeria
d) Lymphatics or immune system cells
• From tissue fluids into lymphatic capillaries
– E.g., Yersinia pestis
TRANSMISSION
Transmissibility = number of microorganisms shed andperiod of time shed for
Microbial stability/survival in an environment influenced by:
•
– Sensitivity to UV damage
– Ability to resist drying
• Extrinsic factors
– Humidity – Temperature
– Exposure to disinfectants
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Pathogenesis
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INFECTIOUS DOSEvia oral route of infection
Organism Infectious or disease
producing dose
Vibrio cholerae
8
Oral plus bicarbonate 104
Shigella dysenteriae 10 bacteria
Mycobacterium tuberculosis 1-10 bacteria
Modified from Mims et al., Medical Microbiology. Table 11.1, p. 362.
12
34
ROUTES OF TRANSMISSION
67 Infections/diseases can be:
Communicable
Non-communicable
(“dead-end”)
ROLE OF
VEHICLES & VECTORS
• Vehicle: food, water, contaminated soil
• Vectors are usually animals or insects (lice, fleas, ticks,
Vector-borne bacterial infections include:
• Lyme disease (ticks)
• Plague (fleas)
• RMSF (ticks)
Exposure & Entry
Attachment
COMPLETON OF THE
PATHOGENESIS CYCLE
InvasionEvasion of immune response
Effects on host
Dissemination/Shedding
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Pathogenesis
Questions w e can ask (and should be able to
answer!) regarding microbial pathogenesis:
1. How does exposure to the microorganism occur?
– Routes and mechanisms of entry (also Lecture 2)
2. How does the microorganism attach to/enter the host cell?
3. Do they remain localised or spread systemically and establish a
secondar siteof re l icat ion?i l i i
4. What effects occur as a result of the infection & microbial replication
a. Damage
b. Direct damage
c. Latent/persistent infections
5. How does the microorganism evade the host immune responses?
6. How it is shed from the infected individual and transmitted to a new
host?