245 2013 lecture 3

8/13/2019 245 2013 lecture 3

http://slidepdf.com/reader/full/245-2013-lecture-3 1/18

8/13/2019 245 2013 lecture 3


8/13/2019 245 2013 lecture 3


Generation of mutants in putative VF genes

Random: chemicalradiationtransposons

transposons/transposable elements :- designation “Tn X ”- G-ves e.g. Tn 5 - G+ves e.g. Tn916 and others- mobile genetic elements (2-20 kb)- cannot autonomously replicate- genes: transposition

resistance to antibiotics/toxicmetals

inverted repeats

transposease gene antibiotic R gene

General transposon structure

8/13/2019 245 2013 lecture 3


Advantages of transposons:

- only mutants selected

- single mutations (check by PCR/

Southern blotting)

- non-“leaky” (“knockout” or “null”)

- marked (“tagged”) mutation: easy toclone

Disadvantages of transposons:

- polar (phenotype may not be due totagged gene)

- may not be completely random

- can’t obtain conditional lethal

mutations (only way of isolatingmutations in essential genes)

8/13/2019 245 2013 lecture 3


Transposon mutagenesis

8/13/2019 245 2013 lecture 3


Use of genome sequences to identify virulencegenes.

• In the last 10 years, bacterial genome sequencinghas become routine

• Hundreds of bacterial pathogens, including

multiple strains of the same species, have been orare being sequenced

• Use sequence homologies to identify putativevirulence factors

• Amplify gene(s) by PCR, clone into vector,mutate gene by insertional inactivation with anantibiotic resistance marker

• Select mutant bacteria by allelic exchangemutagenesis

• Screen for loss of virulence in an appropriateanimal/cell model

Strategy to identify virulence factors:

8/13/2019 245 2013 lecture 3


1. Use genome sequence of bacterium “X” to design primersto PCR amplify and clone the WT gene plus flankingsequence into a E. coli plasmid vector that will notreplicate in bacterium “X”.

2. Use restriction enzymes to cut out/delete most of the gene.3. Replace with a gene encoding resistance to an antibiotic that

bacterium “X” is normally sensitive to.4. Transform plasmid into bacterium “X” and select for

antibiotic resistance.

antibiotic resistancecassette

flankingsequence

flankingsequence

your favourite gene

Cloned DNA fragment

Chromosome

Use ofAntibiotic resistance “cassettes”for making mutants by allelic exchange

To test the effect of gene deletion on phenotype – reverse genetics

Homologous recombination

Now test the mutant phenotype in an appropriateanimal or other model…

8/13/2019 245 2013 lecture 3


Use of Animal Models

Identification of VF’s requires the use of a “modelsystem” where the effects of the VF are apparent

Commonest approach is to compare a wild-typestrain with a mutant defective in a putative VF gene

Best system for this is an appropriate animal model

Considerations in the choice of animal model:

(i) pathogen must be able to infect by sameroute:

e.g. Pseudomonas aeruginosa :

keratitis ! scratched cornea model

burn wound infections ! burned mouseCystic fibrosis pneumonia ! CF mouse

8/13/2019 245 2013 lecture 3


However, this is NOT always possible:

Alternatives:

(a) compromised animals

- infant (immature immune system)- gnotobiotic (“germ-free”) no

resident flora

- immunocompromised :1. irradiated

2. genetic defect

genetic defects:

- nude mice (no functional T cells)

- SCID mice (no functional B/T cells)

[severe combined immunodeficiency]

- transgenic : IFN " knockout(No activated macrophages)

(b) unnatural route of infection

- intraperitoneal injection

Drawback: cannot assess ability to colonise orinvade

8/13/2019 245 2013 lecture 3


Nude mouse – FOXN1 mutationcauses absent thymus gland

SCID mouse – NOD mutationCauses lack of both T and B cells

8/13/2019 245 2013 lecture 3


(ii) Pathogen should cause similar symptoms:

Again, not always possible because pathogen may:

- be human-specific

- cause different disease symptoms in the model

Alternative: use related pathogen, e.g:

1. Whooping cough Bordetella pertussis ! primates exhibit

similar symptoms(expensive and unethical?)

! cataarh in rabbits

B. bronchiseptica ! pigs, dogs (“kennel cough”)

2. Typhoid FeverSalmonella enterica serovar Typhi

! less serious in rodents

S. enterica serovar Typhimurium! lethal systemic infection in rodents

(gastroenteritis in humans)

# pathogenic mechanisms probably similar

8/13/2019 245 2013 lecture 3


(iii) Types of animal to be used

• rodents desirable

• cheap to maintain, require limited space,short generation time

use to measure:

! ID50

! LD 50

! histopathological changes ( i.e. signs of inflammation)

! bacteraemia

! effect of specific mutations

• Can use sufficient number to bestatistically significant

8/13/2019 245 2013 lecture 3


Real example – each open square is thecolonisation level of Campylobacter jejuni (wild-type or a mutant in the cj0415 gene ) in an individualanimal . Note the variation! Hence must use statisticsto test significance

8/13/2019 245 2013 lecture 3


Cell Lines - tissue culture

Advantage:

(i) simpler system:

- single cell type

- grown under reproducible conditions

- cheap and easy to maintain

- avoids animal suffering/ethicalissues

Disadvantages:

(i) Often tumour-derived

- immortalised, rapidly dividing

- many genetic changes

- loss of traits, i.e. cell surface receptors

(ii) loss of shape/polarity

(iii) composition of tissue culture medium

(bile salts, mucus etc)

8/13/2019 245 2013 lecture 3


Quantitation of individual steps in

microbial pathogenesis using tissue culture:

Adherence assay Invasion assay

cell monolayer

add known number of bacteria

wash monolayer gently add antibiotic

microscopic examination wash & lyse

monolayer

count adherent count released bacteria bacteria

Can compare wild-type with mutants…

8/13/2019 245 2013 lecture 3


Toxicity assay

purified toxin or crude extract

cell line or animal model

cell morphology LD-50

Adherent bacteria visualised by fluorescent staining

8/13/2019 245 2013 lecture 3


Organ Cultures

- tissue explants- maintained for short periods- surmounts disadvantages (i) and (ii)for tissue-cultures:

(genetically wild-type, originalcell shape)

- multiple cell types present

Haemophilusinfluenzaeattached toadenoid cellsin organ culture

8/13/2019 245 2013 lecture 3


Summary

• Need suitable methods (preferable genetic) to

identify putative VF genes

• Global mutagenesis by e.g. transposons ortargeted allelic exchange mutagenesis provides aset of mutants for any given pathogen that can be

tested in a host model.

• Best host models are whole animal models,ideally rodents where large numbers can beinfected to give statistically significant results for

comparisons between wild-type and mutantstrains

• Alternatives are cell-cultures for adhesion/invasion or organ cultures, which are cheaper and

have fewer ethical issues.

245 2013 lecture 3

Documents

Transcript of 245 2013 lecture 3