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Pathogenesis Lecture 16: identification of Virulence Factors- Bacterial approaches
Terms in this set (35)
What advances in molecular biology was considered the "2nd wave revolution" with the identification of virulence factors?
When virulence factors are purified in vivo, what can it be used to study?
-Recombinant protein expression/purification
-Vaccine and/or drug development
What is expressed when measuring virulence phenotype?
Bacterial toxins, secreted enzymes, LPS
What are the assumptions of the traditional biochemical approach of studying virulence factors that should be taken into account?
-Some VF are expressed in vivo but not in vitro
-Some toxins don't result in host cell damage bc it wasn't introduced normally
How is a virulence factor expressed in an avirulent strain?
1. Insert DNA fragments with gene of interest into cloning plasmid to obtain library of genome fragments
2. Introduce cloned plasmids into bacterial cells
3. Select clones carrying plasmid DNA
4. Test the library of clones for gene of interest
5. Grow bacterial clones with plasmid DNA of interest
6. Isolate plasmid DNA and sequence
How was virulence factor expression studied in Listeria monocytogenes?
1. Isolate LLO gene on plasmid and introduce (transform) to B. subtilis
2. pH 6.5 produces LLO, escape from endosome, studied and sequenced
2. pH 4.5 , degradation pathway
What are the limitations for using molecular biology to study expression of virulence factor?
-Best applied to close species
-Can only analyze small segments of genome (~30kb)
What are transposons? What are its 2 methods?
segments of DNA that can relocate b/w genomic sites that encode enzymes required for transposition. They often carry antibiotic resistance or tractable genes.
Cut-and-paste & Copy-and-paste
How are the properties of transposons that satisfy it as a good genetic tool?
-Trackable via reporter gene/antibiotic resistance
-Disrupted gene of interest can be identified by PCR
How is transposon mutagenesis used as a genetic tool?
1. Transposon introduced into chromosome
2. Plate on medium that detects for resistance
3. Collect colonies which has transposons on different sites
4. Screen colonies for ability to invade tissue culture cells & find colony that no longer invades
4. Clone genes interrupted by transposon or sequence clones to identify disrupted gene
What are the potential limitations of transposon mutagenesis?
-Polar effects causing transcriptional termination
- Can only be used to identify nonessential genes
Where can a transposon go to induce a polar effect on downstream gene expression?
Operon and regulon
Where is the gene transposed that may cause a pleiotropic effect in a gene that has a regulon?
What is used to measure virulence gene regulation?
What is the difference between transcriptional fusion and translational fusion?
Transcriptional fusion measures where/when a virulence gene promoter is active
Translational fusion measures where/when a virulence gene is translated
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