The key principles Flashcards
What is a pathogen?
A microorganism that causes disease.
What is disease?
Disease: any change in a host to an unhealthy abnormal state in which part or all of the host’s body is not capable of carrying out its normal function
What is pathogenicity?
The ability of a pathogen to cause disease in the tested host.
Pathogenicity is an absolute term:
Either a pathogen
Or a non-pathogen
Define a primary pathogen
Able to establish an infection in a normal host
Define an opportunistic pathogen
Requires a compromised host to establish an infection
Define virulence
The relative potential of a microorganism to cause disease
Quantifies strains within a species
Virulence is a continuous variable
Dependent not only on the microorganism but also on the host
How is virulence measured?
- Severity of symptoms
- Infectious dose
- Fatalities
Typically measured by calculating the median lethal dose (LD50), the dose that kills 50% of the population
Challenge with multiple doses & record fatalities.
Survival curves - challenge with a set dose & record fatalities, calculating mean survival time
What are the infection models?
- Animals
- Invertebrate
- Plant models
- Cell culture models
Define virulence factors
“A microbial product, usually a protein or carbohydrate, that contributes to virulence or pathogenicity”
“Enable the microorganism to carry out specific functions whose effect on the host can cause host cell or tissue damage.
In order to cause disease a pathogen has to?
Colonise host tissues \+ Grow within host tissues \+ Avoid host defence mechanisms \+ Cause damage to the host
Examples of virulence factors
- Secreted products (incl. toxins)
- Adhesion pili
- Capsule
- LPS
- Endotoxins
- Adhesins
- Flagella
- Secretion systems
- Iron acquisition proteins
- Surface proteins
Define virulence & molecular Koch’s postulates
The original Koch’s postulates, formulated in 1884, are the criteria used to establish a causative relationship between a microbe and a disease.
The “molecular Koch’s postulates” follow the same principle to establish a causative relationship between a gene and virulence
Molecular Koch’s postulates:
All strains causing disease have the gene encoding the putative virulence factor
Deletion of this gene leads to attenuated virulence or avirulence
Re-introduction of the gene restores virulence.
How do we identify putative virulence factors?
Bioinformatics (in silico) approach: Homology searches for known virulence factors Comparative genomics Pathogen versus non-pathogen Pathogen-specific proteins Can’t identify novel virulence factors
Expression-based approach:
Characterize microbial gene expression under relevant conditions
e.g. Characterize gene expression within a relevant infection model and compare to that observed during standard growth
Typically now performed via sequencing (“RNA-seq”)
Genes identified by either approach will require experimental validation.
Genome-wide mutagenesis:
Define Transposon mutagenesis
Transposons (Tn) are mobile genetic elements that integrate randomly into the genome.
Transposon mutagenesis can rapidly create a library of thousands of individual mutants harbouring the transposon in different genes.
This library of mutants can then be screened for phenotypes of interest.
Sequencing outwards from the transposon (into the genome sequence) enables the identification of the gene into which the transposon inserted.
Transposon-directed insertion site sequencing (TraDIS)
Combines transposon mutagenesis with high-throughput sequencing.
Provides a very powerful tool for identifying genes required under conditions of interest, including during infection.
A library of random transposon mutants is generated. That library is cultured and mixed together. Sequencing of that “input pool” is performed to identify those genes in which the transposon has inserted into. This input pool is then put through whatever experimental condition is of interest. That could be an infection model, but equally it could simply be a culture condition. After that model/condition, the transposon mutants are recovered (the “output pool”) and the mutants are sequenced again. The sequences obtained from the output pool are compared to those from the input pool. If we no longer have mutants corresponding to a particular gene, then that gene must be required for the experimental growth condition being studied.
