Pathogenesis

Question 1

Pathogen - ?

Answer 1

Organism that, by its actions, causes harm to its host

Question 2

Commensal - ?

Answer 2

Benefits from the interaction with host
Host neither benefits nor is harmed

Question 3

Opportunistic pathogen - ?

Answer 3

Organism that can cause disease in its host given right set of circumstances
- Otherwise commensal

Question 4

Zoonotic pathogen - ?

Answer 4

Organism with animal reservoir that can be transmitted to humans and cause disease

Question 5

Define purpose of Koch’s postulates?
What are the postulates? (4 postulates)

Answer 5

1. The microorganism must be found in abundance in all organisms suffering from the disease but not in healthy organisms
2. The microorganism must be isolated from a diseased organism and grown in pure culture
3. The cultured microorganism should cause disease when introduced into a healthy organism
4. The microorganism must be re-isolated from the diseased experimental host and identified as being identical to the original causative agent

Question 6

In what cases does Koch’s postulates not work? (4 cases)

Answer 6

When pathogen secretes something that causes disease symptoms; Organism itself doesn’t need to be present to see disease

When the organism cannot be grown in pure culture; Usually because of complex or unknown nutritional requirements

When growth in the lab leads to loss of virulence; Disease cannot be recapitulated in a model organism

When no animal model is available so in the absence of human experimentation, disease cannot be reproduced in the lab

Question 7

Traits of strict (professional) pathogens? (5 traits)

Answer 7

Highly adapted organisms for which the pathogenic lifestyle is their main or only option often can’t survive outside of host

Nutritional requirements only satisfied through pathogenesis

Access to a unique niche in the host with little competition from other microorganisms

Transmit very efficiently between hosts

Some are so adapted to the in-host lifestyle that their genome has reduced in size to the point that they can’t live independently

Question 8

Traits of opportunistic pathogens? (3 traits)

Answer 8

Only cause disease when host defences are compromised e.g. barrier breach, immunocompromisation etc.

When not being a pathogen, they exist as commensals

Inefficiently spread between hosts; Some exceptions

Question 9

Traits of facultative pathogens? (3 traits)

Answer 9

Majority of pathogens

Well-adapted for multiple lifestyles; Equally at home in niches and when causing disease

Flexibility usually means a larger genome

Question 10

What is strain variation in context of pathogenesis?
- Use E. coli as example

Answer 10

Not all strains of a particular species will be equally pathogenic, or even pathogenic at all

Most E. coli are harmless commensals
Some have requisite virulence factors and cause human disease; Opportunistic and facultative

Question 11

Define:
- Pathogenesis
- Pathogenicity
- Virulence

Answer 11

Pathogenesis - Mechanism of disease
Pathogenicity - Ability to cause disease
Virulence - Degree of pathogenicity

Question 12

What is ID50?
Why is it not necessarily a measure of a pathogens severity?

Answer 12

Dose of bacteria, viruses, or other infective agents that produces infection in 50% of test subjects

A pathogen can have a higher ID50 but cause a more severe disease than one with lower ID50

Question 13

Virulence factors - ?
Virulence factors of Salmonella enterica? (3 key ones)

Answer 13

Any product of bacterium that contributes to pathogenicity

Colonises mucosal surface
Invades intestinal epithelial cells
Transits to and colonises liver

Question 14

What 2 factors are bacterium virulence based on?
- What does each rely on?

Answer 14

Invasiveness - Bacterium must get into particular niche and survive there
- Transmissibility
- Adherence/Colonisation
- Evasion of host defences
- Nutrient acquisition

Damage - Bacterium needs to damage host
- Toxins
- Degradative enzymes

Question 15

Reasons for studying pathogenesis?
Give some examples

Answer 15

Can exploit mechanisms of pathogenicity to treat or prevent infection

e.g. Vaccines, anti-virulence factor therapeutics

Question 16

How do we study pathogenesis?
2 step process?

Answer 16

Study role of individual proteins/genes and their amino acids and nucleotides

1. Phenotypic read out - Measure some property that contributes to virulence
2. Study contribution of individual genes/proteins to that phenotype

Question 17

What does phenotypic read out rely on and why? (hint - strain comparison)

Answer 17

Relies on ability of a pathogen to cause disease in a model species
Animal models allows us to identify and study virulence factors through comparison of different strains with varying pathogenicity

Question 18

Things to consider when choosing animal models? (3 things)

Answer 18

Ethics, practicality and cost

Question 19

Alternatives to using animal models (2 alternatives)

Answer 19

Cell lines - Immortalised or primary cell cultures
Organoids - ‘Mini organs’

Question 20

Traits of Cell lines? (2 traits
Cons? (4 cons)

Answer 20

Less ethical issues
Simpler, cheaper, less variability, easily scalable

Only model a single cell type
Will not mimic disease; Although can model aspects
Immortalised cells often display difference to original tissue
Specialised growth conditions can affect bacterial behaviour

Question 21

Traits of organoids (3 traits)
Cons? (3 cons)

Answer 21

Less ethical issue
Simpler, cheaper, less variability, easily scalable
Multiple cell types and 3D architecture

Will not mimic disease; Disease is multi-system
Immortalised cells can be different to original tissue
Specialised growth conditions can affect bacterial behaviour

Question 22

Koch’s Postulates for molecular era (3 postulates) (hint - genes)

Answer 22

1. Phenotype under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species
2. Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence
3. Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity

Question 23

How can induction of Gain/loss of function in genes be used to study virulence?

Answer 23

Gain - Over express a gene of interest to see if it enhances or introduces a new phenotype

Loss - Removing or disabling gene to see if pathogen behaves differently without encoded protein

Question 23

Targeted methods of mutagenesis? (3 methods)

Answer 23

Specific disruption (via insertion) or deletion of target gene

Subtle changes e.g. alter DNA sequence to change structure or function of encoded protein

Homologous recombination is most common tool, but CRISPR-based methods are becoming more common

Question 24

Random methods of mutagenesis (2 methods)

Answer 24

Chemical mutagen or radiation
Transposons - Jumping genes that insert randomly into genes (disruption)

Question 25

After transmission to host, what are the 2 main features that determine virulence of pathogen?

Answer 25

Ability to colonise host and survive
Ability to cause damage

Question 26

How does a bacteria colonise a host? (4 steps)
- How many virulence factors?

Answer 26

Target niche
Keep themselves there
Feed themselves while there
Survive attack by the host immune system

1 more virulence factors pre step

Question 27

How can bacteria move towards a nutrient source or safety etc? (2 ways)

Answer 27

Movement can be facilitated by swimming through liquid environment; Flagella for directional swimming

Bacteria can move via twitching motility (crawling) via type IV pili

Question 28

Describe type IV pili (hint - Proteinaceous arm)

Answer 28

Proteinaceous arm sticks out and minor pilins on the end adhere to the surrounding environment to allow for movement

Question 29

Why is adherence of bacteria important in infection?
How does it occur?

Answer 29

Infection often happens under flow so adherence is needed to prevent pathogens being washed away

Adherence is done by either single proteins (adhesins) or complex multiprotein (pili or flagella)
These bind to a complementary receptor on the host

Question 30

What are the virulence factors, fimbriae/pili?
2 types?

Answer 30

Rod structure on bacterial cell surface that interacts with host cell receptor via adhesin at the tip

Type I - Recognises mannose glycoproteins
Type P - Recognises some glycolipids

Question 31

What are non-fimbrial adhesins?
- Gram +ve vs Gram -ve
What do they do?

Answer 31

Bacterial cell surface protein that recognise specific host receptors
Attached to gram +ve cell wall or gram -ve outer membrane

Mediate intimate attachment to a surface

Question 32

How do bacteria acquire resources/nutrients in the host? (3 ways)
Give example of rare but essential resource

Answer 32

Iron is essential for bacterial growth but there is little free iron in humans

Bacteria must:
- Scavenge what they can – Bind free iron with very high affinity
- Steal what they can – Capture host storage proteins
- Damage the host to liberate more – Toxins

Question 33

What are siderophores? (hint - nutrient acquisition)

Answer 33

Compound synthesised and secreted by bacteria that has high affinity for free iron (scavenge)
This is then imported back to bacteria

Question 34

How is Transferrin and Lactoferrin acquired? (7 steps)

Answer 34

1. Fe-Tf binds TbpA + TbpB
2. TbpA and TbpB extract iron from Fe-Tf
3. Iron binds plug domain
4. TonB initiates transport by pulling plug
5. Localised denaturation of plug results in iron release
6. Iron is captured by FbpA
7. TbpB facilitates Apo-Tf release from surface

Question 35

What is haemolysis?
- What facilitates this?
- Effective for release of what?

Answer 35

Secreted pore forming toxins that oligomerise and insert into host cell membranes –> Leakage of cell contents and eventual lysis

Effective for releasing haemoglobins from RBC

Question 36

Virulence relies on damage. How is damage caused by a pathogen? (hint - 2 types of toxins; Gives traits))

Answer 36

Exotoxins
- Soluble proteins
- Heat sensitive
- Made by all classes of bacteria

Endotoxins
- A.K.A lipopolysaccharide (LPS)
- Major component of gram -ve outer membrane

Question 37

What are the 2 groups of exotoxins?
Single polypeptide or multi-subunit (AB)?

Answer 37

Pore-forming toxins that physically disrupt membranes (e.g. haemolysins)
Enzymes that modify host target

Can be both single polypeptide or multi-subunit (AB type)

Question 38

Botulinum toxin is an AB type exotoxin. What does it do? (hint - presynaptic)

Answer 38

Binds polysialoganglioside (PSG) receptor and proteins in the presynaptic membrane
Exploits normal neurotransmitter reuptake

Vesicle acidification leads to membrane insertion and translocation into cytoplasm
Cuts certain proteins (e.g. Syntaxin) preventing neurotransmitter release into synapse

Question 39

What does the gram -ve pathogen Vibrio cholera produce? (hint - encoded by ctxA and ctxB)
What do they do? (hint - uptake and efflux

Answer 39

Produces a 1:5 AB toxin (CtxAB) and CtxA toxin

CtxAB binds ganglioside GM1, stimulating uptake transport to ER

CtxA ribosylates (activating) G protein-coupled receptor
This upregulates adenylate cyclase, increasing cAMP levels and stimulating ion and water efflux

Question 40

What does endotoxin/LPS do when released and how? (hint - cytokine)

Answer 40

Causes cell death in host

LPS activates TLR4 leading to massive cytokine release, activating a complement and coagulation cascade
This causes blood clotting leading to organ failure and hypotension, which kills