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Pathogenesis

OBJECTIVES: LECTURES 11 & 12


Discuss basic concepts of virulence factors

Bacterial Pathogenesis (some Fungal)


Lectures 11 & 12

Discuss basic principles of pathogenesis Explain E l i 5 of f the th 6 PATHOGENESIS steps t


With examples of microbial strategies (virulence factors)

Discuss pathogenesis wrt contribution to disease

VIRULENCE FACTORS
Influence the microbes ability to cause disease: Promote colonization of host Cause damage to host P Predominantly d i tl encoded d d by/associated b / i t d with ith mobile bil genetic elements: phages, plasmids, insertion sequences, transposons Large proportion located within Pathogenicity Islands (PAIs)

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

4 KEY CONCEPTS
1. Infective dose; host defence 2. Infection vs Intoxication 3. Pathogenic cycle 4. Intracellular vs Extracellular Pathogens

STEPS OF THE PATHOGENIC CYCLE


Exposure & Entry Attachment Invasion Evasion of immune response Effects on host Dissemination/Shedding

INTRACELLULAR vs EXTRACELLULAR PATHOGENS


Intracellular Obligate
Bacteria

Facultative
Brucella spp. Bordetella pertussis Campylobacter spp. (Some) E. coli Group B Streptococcus Leigonella spp. Listeria monocytogenes Neisseria gonorrhoeae (meningitides) Salmonella spp. Shigella spp. Yersinia spp.

Extracellular

2nd STEP OF PATHOGENIC PROCESS


Exposure & Entry Attachment I Invasion i Evasion of immune response Effects on host Dissemination/Shedding

Chlamydia spp. Mycobacterium leprae Rickettsia spp.

Bacillus spp. Borrelia spp. Clostridium spp. Corynebacterium diphtheriae (Most) E. coli Group A Streptococcus Haemophilus spp. Klebsiella spp. Helicobacter spp. Proteus spp. Pseudomonas spp. Staphylococcus spp. Treponema spp. Vibrio cholerae Cryptococcus neoformans Pneumocystis jirovecii

Fungi

None

Blastomyces dermatitidis Candida albicans Coccidioides immitis Histoplasma capsulatum

Adapted from McClane, B.A. & Mietzner, T.M. (1999) Microbial Pathogenesis. Fence Creek Publishing. p5

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Pathogenesis

ATTACHMENT/ADHERENCE
Necessary for colonization (particular site) REQUIRES Host cell receptors (CH2Os, proteins, cytokine/hormone) Microbial surface components (adhesins/ligands) Defined by: Ability to adhere (interaction) Favourable environment (pH, nutrients, etc) Presence/absence of normal microflora (commensals)

Tissue Tropism/Specificity
Streptococcus mutans enamel (Dental plaque) Streptococcus salivarius tongue epithelial cells Other examples H. py pylori g gastric mucosa 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

See Murray et al., Table 18-2, p181

Adhesins
Pili & Fimbriae (Bacteria) May be coordinated with other virulence factors Cholera toxin TcpA (Toxin Coregulated Pilus A)

Afimbrial adhesins (Bacteria & Fungi)

Type of adhesin Organism Lipoteichoic acid LPS or LOS Mannans S. aureus S S. pyogenes

Substratum Epithelial cells Buccal epithelial cells

H. pylori Gastric epithelial cells S. typhimurium Macrophages C. albicans Epithelial mucosa

E.g., E. coli Ligand: Type I pili (CFA) Receptor: GM1 ganglioside (intestinal epithelium)
Image from www.cdc.gov

Adhesins ARE Virulence factors E.g., B. pertussis Ligand: Filamentous haemagglutinin (FHA) Prevention: FHA in vaccine
Image from www.cdc.gov

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Pathogenesis

NEXT STEP OF PATHOGENIC PROCESS Not essential


for all bacteria Exposure & Entry Attachment In asion Invasion Evasion of immune response Effects on host Dissemination/Shedding

INVASION
Bacterial Mechanisms Phagocytic cells: via phagocytosis Non-phagocytic cells: variety of possible mechanisms

I Invasins i Bacterial & Fungal interact with specific cell receptors, induce endocytosis Host cell cytoskeleton rearrangement Internalin A: L. monocytogenes Usually injected into host cell membrane ruffling

POST-INVASION CONSEQUENCES
Possible fates of an invasive pathogen Restricted at site of entry Spread from epithelium to immediate underlying tissue, no further dissemination Spread to other body sites (Dissemination - discussed later)
Enzyme Hyaluronidase Streptokinase
Figure 1: Scanning electron micrograph (taken by Roger Wepf, Philippe Sansonetti and Ariel Blocker at the EMBL) of the rod-like Shigella flexneri entering a HeLa cell. From: http://users.path.ox.ac.uk/~ablocker/introduction.html

Action Breaks down hyaluronic acid of connective tissue Breaks down fibrin clots (Converts plasminogen plasmin) Breaks down collagen

Collaginase

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Pathogenesis

NEXT STEP OF PATHOGENIC PROCESS


Exposure & Entry Attachment I Invasion i Evasion of immune response Effects on host Dissemination/Shedding

Reminder of the Immune response to Bacteria


Component Complement Function Opsonization Killing of G-ve bacteria B cell activation Phagocytosis Cytokine production Phagocytosis Antigen presentation Activation of inflammation Block attachment Toxin & enzyme neutralization Opsonization

Neutrophils Dendritic cells Macrophages

Antibody

Summarised from: Murray et al., Box 12-1, p.126

Component to be evaded Complement

Mechanism(s) used for evasion Capsules Complement-binding proteins Proteases Host cell mimicry Capsules C l Type III Secretion systems Intracellular parasitism Interfere with MHC function & antigen processing Ig-binding/inactivating proteins Antigenic variation

EVASION OF COMPLEMENT & PHAGOCYTES


Bacterial (Fungal) solution: Capsules
Box 18-5. Examples of Encapsulated Microorganisms Composition varies: enables Staphylococcus aureus serotyping; used for vaccines Streptococcus pneumoniae Streptococcus pyogenes (group A) Streptococcus agalactiae (group B) Chemical compositions include: B ill anthracis Bacillus th i Bacillus subtilis Polysaccharide Neisseria gonorrhoeae Neisseria meningitidis Polyribose ribitol phosphate Haemophilus influenzae Hyaluronic acid Escherichia coli Klebsiella pneumoniae host cell mimicry Salmonella spp. Not seen as foreign no Yersinia pestis Campylobacter fetus phagocytosis/opsonization Pseudomonas aeruginosa Bacteroides fragilis Cryptococcus neoformans (yeast) Murray et al. Examples of Encapsulated Microorganisms. p186 See Murray et al. p.186

Ph Phagocytic ti killing killi

Antigen processing Antibodies

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Pathogenesis

Unencapsulated bacteria
Pathogen-associated Molecular patterns (PAMPS) LTA, LPS

Encapsulated bacteria
Phagocyte cannot attach

Picture Used with permission from: http://www.rit.edu/~gtfsbi/imm/parhamimages.htm/JPEG/CHAP01/1-13.JPG

Modified from http://www.rit.edu/~gtfsbi/imm/parhamimages.htm/JPEG/CHAP01/1-13.JPG

Encapsulated bacteria/Opsonizing Antibodies

EVADE COMPLEMENT & PHAGOCYTOSIS WITHOUT USING CAPSULES


Solution: Secrete extracellular protease Pseudomonas aeruginosa (Gram -ve) Elastase prevents opsonization/phagocytosis inactivates C3b & C5a Streptococcus pyogenes (Gram +ve) C5a peptidase inhibits phagocyte chemotaxis degrades C5a

Fc receptor

Modified from http://www.rit.edu/~gtfsbi/imm/parhamimages.htm/JPEG/CHAP01/1-13.JPG

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Pathogenesis

EVASION OF PHAGOCYTE DESTRUCTION


Solution: Replicate (& live) in phagocytic cells
3. Inactivate oxygen radicals or degradative enzymes (S. aureus)

EVASION OF MACROPHAGES & LEUKOCYTES


Solution: Toxins to kill macrophages & leukocytes

X X

Leukocidins kill neutrophils and macrophages Alters phospholipid metabolism by ADP-ribosylation of a protein controlling phosphatidylinositol disruption of cellular activities Typical producers = highly invasive bacteria, E.g., Staphylococci PVL: Panton-Valentine Leukocidin

macrophage X

4. Production of surface components (H. capsulatum) 2. Escape phagolysosome (L. monocytogenes)

1. Inhibit phagosomelysosome fusion (L. pneumophilia)

EVADING IMMUNOLOGICAL RESPONSES


Antigen processing & presentation
Dendritic cell

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 Antigenic/phase variation Phase variation = protein expression switchable on/off

Intracellular antigen
proteasome

lysosome

pMHC Class II
phagolysosome

Antigenic variation = multiple antigenic forms expressed at different times

pMHC Class I
(Redrawn from Fig. 10-2, Lippincotts Illustrated Reviews of Immunology, p.122)

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Pathogenesis

N. gonorrhoeae pili variation


Expressed pilin genes pilE

ADDITIONAL CHALLENGE IRON SEQUESTRATION


Iron = necessary for microbial growth in body fluids or on body 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 hosts iron, chelators 2. Secretion siderophores (E. coli; K. pneuomoniae) Low mw compounds; High affinity for iron Siderophore-iron complex

Silent pilS genes

Recombination event Expressed pilin genes

Silent pilS genes

GENERAL EVASION MECHANISM


Solution: Biofilm formation

Examples of infections involving biofilms


Dental caries Peridontitis Otitis media

Osteomyelitis
Taken by Dr Dr. J J. Rayner

Necrotizing fasciitis

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

Pneumonia Cystic fibrosis (P. aeruginosa) Endocarditis

CDC data indicates that >60% of ALL bacterial infections are growing as biofilms

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Pathogenesis

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

Additional 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 lactamases or plasmids.

NEXT STEP OF PATHOGENIC PROCESS


Exposure & Entry Attachment I Invasion i Evasion of immune response Effects on host Dissemination/Shedding POSSIBLE OUTCOMES OF INFECTION

Infection & Damage

Microbial mediated Host mediated Toxins Immune response Invasion Virulence factors Replication 2 Ph i l growth Physical th

3 4 Prevention/Clearance

Permanent relationship

Inapparent/subclinical

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Pathogenesis

OUTCOME: DAMAGE
Direct Damage Mechanisms

(1) Degradative Enzymes


Enzyme Action

spreading en nzymes

1. Degradative enzymes

3. Toxic structures

Hyaluronidase Breaks down hyaluronic acid of connective tissue Streptokinase Breaks down fibrin clots (Converts plasminogen plasmin) Collaginase Breaks down collagen

4. Toxins 2. Physical growth through tissues


Damage membranes Inhibit cellular function

(2) Physical Growth Through Tissues


No known degradative enzymes or exotoxins i.e., Aspergillosis Mechanism of damage is unclear probably due to fungal growth through tissues Displacement/destruction of vital structures E.g., angioinvasive Aspergillus

(3) Toxic Structures


Gram -ve bacterial LPS (Endotoxin)

Murray et al. 6th Ed Fig 18-4, p185


Fig. 18-4, p.185.

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Pathogenesis

(4) Toxins
Fungal toxins
A. fumigatius: Gliotoxin Inhibits macrophage phagocytosis, phagocytosis T-cell T cell activation & induces apoptosis A. flavus: Aflatoxin Carcinogenic

Bacterial Toxins
1. Protein toxins EXOTOXINS Secreted into extracellular environment Specificity varies Very potent Gram +ve and Gram -ve bacteria 2. Lipopolysaccharide toxins ENDOTOXINS Only acts as toxin under certain circumstances LPS of Gram -ve bacteria

COMPARISON OF EXO/ENDOTOXINS
Overview of differentiating characteristics Characteristic
Toxicity Effects on body Chemical composition Toxoid formation Fever stimulation Manner of release

Examples of Exotoxins
Microorganisms
Vibrio cholerae

EXOTOXIN
Minute amounts Specific to cell type Polypeptide Yes Not usually Secreted from live cells

ENDOTOXINS
High doses Systemic, fever, inflammation LPS of cell wall No Yes Cell lysis

Key toxin(s) Contribution


Cholera toxin Botulism toxin Tetanus Toxin TSST-1 Excessive watery diarrhoeae Muscle paralysis (flaccid) Muscle paralysis (rigid) System-wide shock

Clostridium botulinum Clostridium tetani Staphylococcus aureus Corynebacterium diphtheriae

Diphtheria toxin Pseudomembrane, systemic effects

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Pathogenesis

Nomenclature & Classes/Groups


Classified according to: Target site/cells: neurotoxins, enterotoxins Producing bacterium: Staphylococcal toxin Mechanism of action: ADP-ribosylators, pore-forming toxins Associated diseases: cholera toxin, diphtheria toxin Genes encoding for toxin production Plasmids Bacteriophage Control via environmental regulators (pH, iron)

CLASSES OF EXOTOXINS
Class I E.g., TSST-1 Membrane acting/bind to host cell surface Class II Membrane damaging
E.g. Phospolipases P Pore-formers f

Class III Intracellular


E.g., Diphtheria

CLASS I EXOTOXINS Superantigens


Powerful T-cell mitogens Interfere directly with host immune response coordination divert/confuse host defenses Interact directly with T-cells & APCs

Bind directly to T cell receptor 20% of T-cells activated (normal pathway = 0.01% of T cells activated)
SUPERANTIGEN

T cell T cell

Antigenpresenting cell

T cell T cell

T cell

Important contributor to damage E.g., Staphylococci: Toxic Shock Syndrome Toxin (TSST-1); Streptococci: (SPEs) Rheumatic fever

TNF- Fever & shock

TNF-

TNF- IL-2 TNF- IFN- IFN- IFN- IFN- IFN-

IL-2 IL-2

IL-2

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Pathogenesis

CLASS II EXOTOXINS Membrane-acting


i) Phospholipid membrane destabilisation Example: Target cells: Effect: Mechanism: Lecithinase of C. perfringens ( toxin (Phospholipase C)) various indiscriminate lysis Destabilize cell membrane by removing polar head group from phospholipids

ii. Pore formation Example: Target cells: Effect: Mechanism: Listeriolysin O Various Cell death occurs due to osmotic lysis or apoptosis Protein pores destabilize membrane

Often responsible for cytolytic activity


H20 K+ K+ K+ K+

Advantages to bacterium: eliminates host defences; creates nutritionally rich environment

Also referred to as CFTs = Channel Forming Toxins

CLASS III EXOTOXINS Intracellular


Basic Structure A = catalytic (Active) domain Enzymatically attacks a particular host cell function or structure B = Binding subunit Specific host cell surface glycoprotein or glycolipid

Most common toxin structures:

AB
Single gene encodes for single peptide; post-translationally modified to A&B fragments which are covalently linked E.g., diphtheria toxin

SS

B portion

A5B
Two genes encode A&B subunits; noncovalently associated in stable complex E.g., cholera toxin

A portion

Catalytic activities: ADP-ribosyltransferase (diphtheria toxin, cholera toxin) Zinc metalloendoprotease (tetanus & botulism neurotoxins) Deamidase Glucosyltransferase

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Pathogenesis

Mechanism of Action: ADP-ribosylator


1

Diphtheria toxin
A B A B

Receptor = heparinbinding epidermal growth factor - like precursor

1. Bacteria secrete exotoxin 2. Toxin enters host cell


2 A

NAD
3

3. Toxin removes ADP-ribose group from NAD = ADP-ribosylation ADP-ribose


4

H o s t c e l l

Internalization
B

ADP-ribose

4. Transfers it to host cell protein 5. Host cell protein inactivated

Acidification of endosome

Protein X
5

Protein

NAD+ + EF-2 ADPR-EF-2 + nicotinamide + HActive Inactive

Zinc metalloendoprotease
Proteolytic cleavage of toxin 2 linked fragments a) Light chain (LC) enzymatically active; zinc metalloprotease b) Heavy chain binding & translocation Tetanus toxin Produced by y Site of action Clostridium tetani Presynaptic membrane of motor neurons Inhibition of inhibitory neurotransmitters Spastic paralysis Botulinum toxin Clostridium botulinum Gangliosides

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

Mechanism

Inhibition of acetylcholine release Flaccid paralysis

Effect

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Pathogenesis

NEXT STEP OF PATHOGENIC PROCESS


Exposure & Entry Attachment I Invasion i Evasion of immune response Effects on host Dissemination/Shedding

DISSEMINATION
to a different site within the host
a) Direct: In surface fluids, by flow of intestinal contents Facilitated by toxin production (E.g., hyaluronidase, etc.) Growth through tissues, E.g., Aspergillus

b) Via CSF Cross blood-CSF junction (choroid plexus) E.g., H. influenzae

TRANSMISSION
c) Blood (hematogenous) Can be in the blood or blood components, E.g. Plasma: B. anthracis Mononuclear cells: Listeria Transmissibility = number of microorganisms shed and period of time shed for Microbial stability/survival in an environment influenced by:

d) Lymphatics or immune system cells From tissue fluids into lymphatic capillaries E.g., Yersinia pestis

Microbial factors Sensitivity to UV damage Ability to resist drying Extrinsic factors Humidity Temperature Exposure to disinfectants

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Pathogenesis

INFECTIOUS DOSE
via oral route of infection Organism Vibrio cholerae Oral Oral plus bicarbonate Shigella dysenteriae Mycobacterium tuberculosis Infectious or disease producing dose
1

ROUTES OF TRANSMISSION
3 4

108 104 10 bacteria 1-10 bacteria

Infections/diseases can be: Communicable Non-communicable (dead-end)

Modified from Mims et al., Medical Microbiology. Table 11.1, p. 362.

ROLE OF VEHICLES & VECTORS


Vehicle: food, water, contaminated soil Vectors are usually animals or insects (lice, fleas, ticks, mosquitoes) Vector-borne bacterial infections include: Lyme disease (ticks) Plague (fleas) RMSF (ticks)

COMPLETON OF THE PATHOGENESIS CYCLE


Exposure & Entry Attachment Invasion Evasion of immune response Effects on host Dissemination/Shedding

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Pathogenesis

Questions we 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 secondary site of replication? 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?

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