Lecture 5 - Identification of Virulence Determinants

Question 1

Koch's postulates
1)
2)
3)
4)

Answer 1

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

Question 2

Issues with Koch’s postulates

Answer 2

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

Question 3

Example of a disease which causes systemic effects

Answer 3

Staphylococcal scalded skin syndrome
Bacterial infection is in nose
Skin all over the body peels off, despite bacteria not being present in skin

Question 4

Example of bacteria that can’t be grown in culture

Answer 4

Treponema pallidum

Mycobacteria leperae

Question 5

Falkow’s different approach to Koch’s postulates

Answer 5

Focus on pathogenic genes, not organism

Question 6

Falkrow’s postulates

Answer 6

1) Measure virulence of organism
2) Isolate, mutate gene of interest. Measure virulence
3) Restore mutated gene to wild type. Measure virulence

Question 7

Order of wild type, mutant and restored wild type virulence determining genes in terms of virulence

Answer 7

1) Wild type (most virulent
2) Restored wilt type
3) Mutant

Question 8

Why is the restored wild type gene normally less virulent than the wild type?

Answer 8

Gene might have inserted into a less-advantageous part of the genome.

Wild type has evolved to be in the optimal location

Question 9

Ways to measure virulence
1)
2)
3)

Answer 9

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

Question 10

How to find virulence gene
1)
2)
3)
4)

Answer 10

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

Question 11

What is a transposon?

Answer 11

DNA with self-replicative capacity, jumps to different parts of genome by non-homologous translocation
Transposase gene
Inverted repeats

Question 12

Structure of a transposon

Answer 12

Core transposase gene, flanked by inverted repeats.

Other genes can be spliced into core

Question 13

Compound transposon

Answer 13

Resistance gene flanked by insertion sequences.

Insertion sequences are flanked by inverted repeats

Question 14

Complex transposon

Answer 14

Resistance and transposase genes flanked by inverted repeats

Question 15

Use of transposons to determine protein synthesis
1)
2)
3)

Answer 15

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.

Question 16

Principle of competition assays

Answer 16

1) Introduce wild type and mutant bacteria into host organism
2) See which one outcompetes the other

Question 17

Signature-tagged mutagenesis principle

Answer 17

1) Have many different mutants of pathogen, tagged with different dyes
2) Inoculate into host organism, see which pathogens outcompete others, which die off
3) Mutants in which transposon has inserted into a vital gene will not survive, won’t be present on assay

Question 18

Ways to tag pathogens for signature-tagged mutagenesis

Answer 18

1) Using dye
2) Using resistance gene
3) Using DNA signature - PCR can be used to see how much of a DNA-signature-tagged organism is present