Introduction Flashcards
What is commensalism
We describe non-pathogenic members of the normal flora as “commensals”- oversimplification
Commensalism: an association between two organisms in which one benefits and the other derives neither benefit nor harm.
Commensals can sometimes cause disease given the right circumstances
What is mutualism
Mutualism: a symbiotic relationship where both organisms benefit
E.g. Largest source of B12 comes from the microbiota.
What virulence factors allow the infection to become established
adhesions (proteinaceous surface structures that allow to adhere onto a surface or body site)
invasins (allows pathogens to penetrate deeper into the tissue)
nutrient acquisition (ion scavenging)
motility and chemotaxis (can swim around and deters so can move towards nutrition or away from danger)
What virulence factors cause damage
Exotoxins and endotoxins etc.
Koch’s Postulates
In 1876 Robert Koch put forward 4 criteria that must be met in order to identify the etiological agent of a disease
- The microorganism must be found in abundance in all organisms suffering from the disease but not in healthy organisms/ controls.
- The microorganism must be isolated from a diseased organism and grown in pure culture.
- The cultured microorganism should cause disease when introduced into a healthy organism.
- The microorganism must be re-isolated from the diseased experimental host and identified as being identical to the original causative agent.
Koch’s Postulates for the molecular era- updated version
In 1988: “a form of molecular Koch’s postulates is needed when examining the potential role of genes and their products in the pathogenesis of infection and disease”
- The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species.
- Specific inactivation of the genes associated with the suspected virulence traits should lead to a measurable loss in pathogenicity or virulence
- Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity.
How do we study pathogenesis?
Genetic manipulation –> some readout of virulence (animal or surrogate)
Reductionist biology: let’s identify virulence factors
Tetanus toxin
Tetanus (gram positive) spore forming anaerobe, most important, produces clostridial neurotoxin that induces rigid paralysis, leading to death and prevents the muscle from relaxing. Its the second most potent toxin known
OmpA
Dominant E. coli outer membrane protein, essential for evasion of macrophage killing and invasion of the blood brain barrier. Can cause meningitis (from neonatal e.coli). It’s lethal in 50% of cases
All e.coli strains have ompA - protein sequences are identical between the lab strains.
What is reverse genetics
Reverse genetics seek to assign a function to a particular gene/ sequence. You start with a hypothesis and it uses directed mutagenesis.
You try to figure out the function of a gene by starting with a hypothesis on what it does
Its normally backed up by another sort of data e.g biochemical assays to demonstate an enzyme activity
What does forward genetics do
Seeks to identify the genetic basis of a particular phenotype
It does not require prior knowledge.
It uses random mutagenesis.
Its a experimental approach designed to screen for phenotype
How do we make a knockout
Two main options:
Insertional or deletion
Deletions are the gold standard.
Insertions are quicker and easier but can have downstream effects (stand alone, then this disruption can work well but can affect genes downstream)
Techniques
Lambda Red, Group II introns (TargeTron), Homologous recombination (engineer a peice of DNA), Phage transduction, CRISPR…
How do we complement?
Again two main options: plasmid (clone gene into plasmid) or insertion at an distal locus
Cloning into a plasmid is the easiest to do. Cloning into a recombination vector is harder
Plasmids are present on more than one copy so you get more of the gene copy expressed if you’ve got increased plasmid copy, sometimes overexpression has negative effects
Where abouts on the genome can you change the expression and what do we need
Plasmid: Selectable markers (antibiotic resistance set of some sort), Origins, Transformation/Conjugation (get DNA into species- impossible for a lot of species), Promoters, etc.
Insertion: A suitable insertion site, Homologous recombination, Unstable/conditional plasmid, Counter selection, etc.
What do we do in forward genetics
No genetic basis of that phenotype- phenotype to gene
Incubate the bacteria with different types of human uncultured cells- then you have screen. Screen - high throughput.
Can be extremely labour intensive – technology helping
Requires a relatively straightforward and scalable phenotypic screen- need to do lots in parallel
Relies on random mutagenesis so can be challenging to identify the responsible mutation
Random mutagenesis
Originally done with chemical mutagens or radiation
Now we largely rely on Transposons
Random insertion into genome
Screen library
But how many mutants to screen?
In corn- transposons changes the colours - she discovered them - can insert themselves into any DNA
TraDIS – Transposon Directed Insertion site Sequencing
Take transposon (encodes antibiotic resistance) and introduce into bacteria- the transposon will jump into the genome of that bacteria completely randomly at different points in the chromosome- can end up with enormous collections of mutants.
Within your library you’ve got an insertion every 4-5 genomes
Each has a different mutation
If that transposon has gone into an important survival gene- mutant immedietaly dies (10% of bacterial genome is essential)- they’re not present in library
Take remaining 90% extract genomic DNA- mixture of molecules each with a transposon in a different place
Then do illuminia sequencing and PCR amplification- amplify the junction - get a million different junctions then we sequence- tells us the insertion side of every one in the library
Also see genes with no reads mapping
TraDIS - conditional essentiality
Can take transposon library and apply some selective pressure to it- selective pressure could look like bile salt (emulsifying agent - helps digests fats- they’re also stressful for bacteria because they break down their membranes)
TraDIS in C. difficile
Made 77,000 transposon mutants – an insertion every 54bp
404 essential genes – out of approx. 4,000
- difficile is a spore former
Sporulate the library, kill all the vegetative cells and sequence again. Only mutants capable of sporulating will be present – 798 sporulation genes- double the essential number- highlights how complex this process is
Animal models
Rodents are the most commonly used – why?
Inbred lines – reduces variation
Small, cheap, reproduce quickly
Lots of available tools, e.g. antibodies, mutant lines
Mice, rats, rabbits, hamsters, etc.
But: License restrictions, Ethical issues, Mice aren’t humans
Alternative models
Fish and non-vertebrate models becoming more popular
Lots of tools in development, particularly for Zebrafish
Light touch regulation
Can use large groups sizes for statistical robustness
But: Further away from humans
More primitive immune systems
Cultured cell lines
Can use human cells
Normally immortalised so easy to culture and scalable
No ethical issues*- whats the origin of those cultured cells?
But: Usually cancer cells so have many genetic changes (e.g. alterations in surface protein expression)
