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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



Pathogenesis

MICR570/ZMR/F12 11/12

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




Pathogenesis

MICR570/ZMR/F12 11/12

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



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


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



Pathogenesis

MICR570/ZMR/F12 11/12


Exposure & Entry

Attachment


Effects on host


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



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



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



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



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


Effects on host


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



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



Pathogenesis

MICR570/ZMR/F12 11/12-

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



Pathogenesis

MICR570/ZMR/F12 11/12-

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



Pathogenesis

MICR570/ZMR/F12 11/12-

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



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



Pathogenesis

MICR570/ZMR/F12 11/12-


Exposure & Entry

Attachment


Effects on 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

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



Pathogenesis

MICR570/ZMR/F12 11/12-

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


Effects on host




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?

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