Lecture 5 - Identification of Virulence Determinants Flashcards
Koch's postulates 1) 2) 3) 4)
1) The pathogen must be present at the site of infection
2) The pathogen must be isolated from lesions, can be grown in a pure culture
3) Pathogens from pure culture must cause disease when inoculated into a susceptible host
4) Pathogen must be able to be cultured from lesions from experimentally-infected host
Issues with Koch’s postulates
1) Some infections are asymptomatic
2) Some bacteria can’t be cultured
3) A good animal model can’t always be found
4) Some infections cause systemic effects
Example of a disease which causes systemic effects
Staphylococcal scalded skin syndrome
Bacterial infection is in nose
Skin all over the body peels off, despite bacteria not being present in skin
Example of bacteria that can’t be grown in culture
Treponema pallidum
Mycobacteria leperae
Falkow’s different approach to Koch’s postulates
Focus on pathogenic genes, not organism
Falkrow’s postulates
1) Measure virulence of organism
2) Isolate, mutate gene of interest. Measure virulence
3) Restore mutated gene to wild type. Measure virulence
Order of wild type, mutant and restored wild type virulence determining genes in terms of virulence
1) Wild type (most virulent
2) Restored wilt type
3) Mutant
Why is the restored wild type gene normally less virulent than the wild type?
Gene might have inserted into a less-advantageous part of the genome.
Wild type has evolved to be in the optimal location
Ways to measure virulence
1)
2)
3)
1) Inoculate a number of mice with pathogen. Observe mortality rates
2) Inoculate mice with pathogen tagged with dye. Scan mice, see where, when bacteria colonise
3) Tag several different pathogens with dyes/resistance plasmids/genetic signatures. See which pathogen is most successful, lest successful
How to find virulence gene 1) 2) 3) 4)
EG: for toxin-producing gene
1) Introduce transposon containing dye tag or resistance gene.
2) Assay bacteria, see which are resistant
3) See which of the resistant bacteria produce toxin.
4) The bacteria which don’t produce toxin had transposon insert into toxin-producing gene, or into an area which disrupts toxin-producing gene
What is a transposon?
DNA with self-replicative capacity, jumps to different parts of genome by non-homologous translocation
Transposase gene
Inverted repeats
Structure of a transposon
Core transposase gene, flanked by inverted repeats.
Other genes can be spliced into core
Compound transposon
Resistance gene flanked by insertion sequences.
Insertion sequences are flanked by inverted repeats
Complex transposon
Resistance and transposase genes flanked by inverted repeats
Use of transposons to determine protein synthesis
1)
2)
3)
1) Include a gfp gene in transposon
2) If transposon inserts into the end of a gene, the gene might produce protein tagged with gfp
3) Can see where, how much protein is expressed.