Topic 4: Concepts of pathogenicity Flashcards
what does opportunistic mean
commensal but can cause disease in the right circumstances (e.g. Strep A or Serratia)
what does zoonotic mean
animal reservoir, pathogen or commensal can be transmitted to cause disease in humans
what do virulence factors do
allow colonisation and infection of the host -> are responsible for the symptoms of the disease (cause damage)
examples of virulence factors
adhesins to stick to host tissues
nutrient acquisition
motility and chemotaxis
damage - toxins and proteases
etiological agent of disease
casual link
pathogen definition
an organism that by its action causes harm to the host
commensal definition
a commensal organism benefits from the interaction with the host and the host neither benefits nor is harmed
how do you determine that a microorganism is the causal agent of a particular disease
- microorganism (MO) found in abundance in all organisms suffering from the disease but not in healthy organisms
- MO must be isolated from the diseased organism and grown in pure culture
- MO should cause disease when introduced into a healthy host
- MO most be re-isolated from the diseased experimental host and be identical to original
exceptions to kochs postulates
- MO secretes something that causes diseases eg. botulism, bac doesn’t need to be there to see disease state
- organism cannot be grown in pure culture, nutritional requirements eg. chlamydia/ growth in lab –> loss of virulence
no animal model is available (most common problem)
what is the definition of a strict pathogen
organisms highly adapted to pathogenic lifestyle
cannot survive outside the host eg. due to nutritional requirements, reduction in genome
examples of strict pathogens
helicobacter pylori
Neisseria gonorrhoeae
shigella dysentery
mycobacterium tuberculosis
chlamydia trachomatis
examples of opportunistic pathogens
Pseudomonas aeruginosa – wound/burn infections, lung infections in cystic fibrosis
Clostridioides difficile – inflammatory infections of the large intestine but only after antibiotic treatment
Staphylococcus epidermidis – skin and wound infections
Staphylococcus aureus – skin and wound infections, endocarditis, osteomyelitis, etc.
what is a virulence factor
any product of the bacterium that contributes to pathogenicity
what makes a bacterium virulent
invasiveness
damage
invasiveness of a pathogenic bacteria
ability of a bacterium to get into a particular niche, survive and proliferate there
- transmissibility
- adherence +colonisation
- evasion of host defences + competition
- nutrient acquisition
damage of pathogenic bacterium
damages the host using toxins and digestive enzymes
cell lines
immortalised or primary cultured cells
example of a pathogen that works well in animal models
pseudomonas aeruginosa diseases such as cycstic fibrosis pneumonia
example of a pathogen that needs a surrogate pathogen in animal models
salmonella enterica (typhoid) only causes a mild disease in rodents
also whooping cough
cell line pros
simpler cheaper scalable + less ethical issues
cell line cons
only model a single cell type
don’t mimic disease
immortalised so different to original tissue
growth conditions needed can affect bacterial behaviour
organoids
Multiple cell types and 3D architecture
organoids pros
similar to cell lines
Less ethical issues
Simpler, cheaper, less variability, easily scalable
organoids cons
similar to cell lines
Will not mimic disease – although can model aspects and more than monoculture
Immortalised cells (if used) often display difference to original tissue
Specialised growth conditions can affect bacterial behaviour
what can be studied using a cell line
Study adherence of bacteria to a particular cell type
Study invasion
Study toxicity
Study changes in cell biology
How do we use models to study virulence factors?
Immunological tools
Biochemical tools
Genetic tools
Immunological tools
Antibodies found in convalescent plasma tell us what the host immune system sees
Antibodies raised experimentally against a particular protein can help us to understand what that protein does, e.g. antibody to adhesin prevents colonisation in animals or adhesion to cells
Biochemical tools
Purified protein on its own causes a phenotype in animals or cells, e.g. toxins
Genetic tools
Gene over-expression, knock-outs, etc. can be used to understand the function of the encoded protein
gain of function - see if it enhances a phenotype or introduces a new phenotype
loss of function - see if the pathogen behaves differently without the encoded protein
Koch’s Postulates for the molecular era
1) The phenotype or property 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.
motility and chemotaxis
move towards attractants (nutrients) and away from repellents (threats)
directional movement using flagella or tumble
type IV pili twitching motility
adherence
early steps in initiating an infection
surfaces under flow such as lungs, urinary tract, GI tract
simple adhesins or multiprotein fimbriae/pili/flagella
binds to some kind of receptor (protein or carbohydrate) on the host side
describe the structure of non-fimbrial adhesins
proteins (beta barrel) on the outer membrane (gram -ve) or cell wall (gram +ve)
what is the problem with iron - nutrient acquisition
there is very little free iron in the body, it is bound by lactoferrin, transferrin, ferritin and haem
how do pathogens solve the problem with iron - nutrient acquisition
scavenge what free iron they can (siderophores - bind Fe with high affinity and import it into the cell)
free it from lactoferrin and transferrin
liberate more from host cells (damage/haemolysis)
damage
combines with colonisation to make pathogens virulent
- balance between the 2, a bacteria which is not a good pathogen may good at damaging
toxins - exo and endo
exotoxins
soluble proteins
secreted or released by cell lysis
most are heat labile
can form pores or modify a host target
endotoxin
aka lipopolysaccharide, major component of the outer membrane of gram -ve
highly potent toxin when released –> cell death in the host, induces cytokine release
what is the difference between AB toxins and non-AB toxins
AB = multi subunit
B (Binds receptor) and A (Active)
non = single polypeptide
Clostridium botulinum - exotoxin
most potent toxin known
leads to - flaccid paralysis
AB type
encoded as a single polypeptide - proteolytic cleavage but held together by disulphide bonds
exploits synaptic reuptake machinery. stops neurotransmission
Vibrio cholera - exotoxin
1:5 AB toxin increases cAMP levels in the cell, increasing ion and water efflux
structure of endotoxin
LPS - lipopolysaccharide
O region - repeating 4-5 sugar units
core polysaccharide - linking region
lipid A - C14 fatty acids linked to two N-acetyl glucosamines – TOXIC BIT – solubilised by the res
example of endotoxin
Neisseria meningitidis sepsis
mechanism of action of endotoxin
TLR4 activated results in massive cytokine release
activates compliment and coagulation cascade
blood clotting sequesters blood to periphery –> hypertension –> death
what is septic shock
a potentially fatal overreaction of the immune system
example of an AB toxin + example which is not 1:1
botulinum toxin
vibrio cholera - 1:5
What are the a host’s defences to a disease?
Physical barriers, Complement, Macrophages, Antimicrobial peptides, Adaptive immunity, etc.
What are the Koch’s Postulates?
There are 4 criteria of identify etiological agents of a disease:
-The disease must be found in all organisms suffering from the disease but not healthy organisms
-The microorganism must be isolated from the host and grown in vitro, the culture microorganism should cause disease when introduced to healthy organism, the microorganism must be re-isolated and identified as identical to the original causative agent.
Why is botulism an exception to Koch’s postulates?
Botulism leads to the secretion of Botulinum toxin (the most toxic protein on the planet), when it grows on food sources the organism may die but the toxin remains, therefore will have symptoms associated. This contradicts koch because the symptoms are associated with the toxin rather than the organism itself.
What is true about the adverse effects of the immune system?
Sometimes the immune system’s response is more harmful than the effect of the pathogen itself (e.g. toxic shock or allergic reaction)
Colonisation:
Infecting bacteria willl occupy one or more niches within the host during the pathogenesis process.
To colonies they need to: get to the target niche, keep themselves there, feed, and survive attack by immune system.
What is pathogenesis?
The mechanism of the disease
What is pathogenicity?
The ability of a pathogen to cause disease
What is virulence?
The degree of pathogenicity.
When is the comparison of lethal dose 50 or infectious dose 50 apporpriate?
When comparing closely related strains of a disease causing pathogen.
What are virulence factors:
Any product of a bacterium that contributes to its pathogenicity.
What is the primary method of studying pathogenesis?
Phenotypic readout - Measuring of a property that contributes to virulence. A method is then used to study the contribution of individual genes to that phenotype. (e.g. gene knockouts + overexpression)
Phenotypic read outs:
Relies on the ability of a pathogen to cause a disease in the model species or surrogate phenotypic assay.
Animal models allow us to identify and study virulence factors and compare strains or a wt to a mutant.
What are the 3 R’s of research?
Replacement, Reduction, and Refinement.
What are the factors the contribute to the choice of animal model used on a pathogenesis investigation?
Ethics, practicality, and cost
What is an ideal animal model in the study of pathogenesis?
The ideal animal model will exactly reproduce the human disease when infected via the same route. (not really possible)
What are practicality and cost factors which might be considered during a pathogenesis study?
- Chosen species disease replication
- Animal handling, ease of breeding, housing and feeding (small rodents preferred)
-Availability of tools (e.g. antibodies, mutant animal lines etc.)
What are alternatives to animal testing?
Cell line testing and Organoids.
What are the two mutagenesis methods:
Targeted and random
Targeted Mutagenesis:
Multiple changes can be made:
Specific genes can be disrupted (via insertion) or deleted
More subtle changes can be done, altering the DNA sequence to change the structure or function of proteins
Primarily done through either Homologous recombination or CRISPR-based methods.
Random Mutagenesis:
Chemical mutagen or radiation introduces random changes.
or
Transposons are inserted randomly into the genome.
(Both of these methods are followed by DNA sequencing and comparing to the reference genome)
What are Fimbriae/Pili?
Rod-like structures on the bacterial cell surface that interact with a host cell receptors via an adhesin subunit at their tip.
What are the major pili types in E.coli?
Type I and P -> they recognise mannose-containing glycoproteins and glycolipids
Type P being critical in the adhesion of E.coli to the Urethral epithelia in UTI’s.
What is an example of a non-fimbrial adhesin?
Hek from E.coli K1 - an outer membrane beta-barrel protein that interacts with host heparinated-proteoglycans
Fe acquisition by siderophores:
The siderophore is secreted by the bacterium, sequesters Fe, transporting it back to the pathogen, and upon recognition of an cell surface receptor releases the Fe into the cytoplasm.
Haemolysis: Process
Haemolysins are pore forming toxins which are secreted and oligomerise and insert into host cell membranes -> leaking the cell contents and causing eventual lysis.
What is the primary target of haemolysis?
Erythrocytes (RBCs)
What are the two groups of exotoxins?
- Pore-forming Toxins (physically disrupt membranes e.g. haemolysins)
- Enzymatic Toxins (have a wide range of activities and targets)
What is the mechanism by which Botulinum toxin operates?
Botulinum toxin binds to polysialoganglioside (PSG) receptor and Syt or SV2 proteins in the presynaptic membrane -> Then endocytosed by neurotransmitter pathway -> vesicle acidification leads to membrane insertion and translocation of the light chain into the cytoplasm by reduction of S-S (disulphide bridges) -> light chain cuts VAMP, SNAP25, or syntaxin (depending on botulinum subtype), preventing neurotransmitter release into the synapse.
