Introduction

Question 1

What is commensalism

Answer 1

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

Question 2

What is mutualism

Answer 2

Mutualism: a symbiotic relationship where both organisms benefit

E.g. Largest source of B12 comes from the microbiota.

Question 3

What virulence factors allow the infection to become established

Answer 3

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)

Question 4

What virulence factors cause damage

Answer 4

Exotoxins and endotoxins etc.

Question 5

Koch’s Postulates

Answer 5

In 1876 Robert Koch put forward 4 criteria that must be met in order to identify the etiological agent of a disease

1. The microorganism must be found in abundance in all organisms suffering from the disease but not in healthy organisms/ controls.
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

Koch’s Postulates for the molecular era- updated version

Answer 6

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”

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 genes associated with the suspected virulence traits 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 7

How do we study pathogenesis?

Answer 7

Genetic manipulation –> some readout of virulence (animal or surrogate)

Reductionist biology: let’s identify virulence factors

Question 8

Tetanus toxin

Answer 8

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

Question 9

OmpA

Answer 9

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.

Question 10

What is reverse genetics

Answer 10

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

Question 11

What does forward genetics do

Answer 11

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

Question 12

How do we make a knockout

Answer 12

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)

Question 13

Techniques

Answer 13

Lambda Red, Group II introns (TargeTron), Homologous recombination (engineer a peice of DNA), Phage transduction, CRISPR…

Question 14

How do we complement?

Answer 14

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

Question 15

Where abouts on the genome can you change the expression and what do we need

Answer 15

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.

Question 16

What do we do in forward genetics

Answer 16

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

Question 17

Random mutagenesis

Answer 17

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

Question 18

TraDIS – Transposon Directed Insertion site Sequencing

Answer 18

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

Question 19

TraDIS - conditional essentiality

Answer 19

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)

Question 20

TraDIS in C. difficile

Answer 20

Made 77,000 transposon mutants – an insertion every 54bp

404 essential genes – out of approx. 4,000

1. 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

Question 21

Animal models

Answer 21

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

Question 22

Alternative models

Answer 22

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)