Bacterial Virulence Flashcards
give us some examples of gram positive rods?
- Bacillus anthracis
- corynebacterium diphtheriae
- clostridium botulinum
- clostridium tetani
- clostridium perfringens
give us some examples of gram positive cocci ?
- staphyloccus aureus
- staphyloccus epidermidis
- streptococcus pyogenes
give some examples of rods gram negative bacterial pathogens?
- Salmonella enterica
- Shigella dysenteriae
- Escherichia coli
- Yersinia pestis
give some examples of cocci gram negative bacterial pathogens?
- Neisseria gonorrhoeae
- Neisseria meningitidis
give some examples of coccobacilli gram negative bacterial pathogens?
Bordetella pertussis
give some examples of curved rods ( spiral) gram negative bacterial pathogens?
Vibrio cholerae
Helicobacter pylori
Treponema pallidum
Borrelia burgdoferi
what are virulence factors?
products or structural components that allow an organism to enter and alter host function to cause disease
examples of virulence factors?
Adhesins - stick to host cells
Toxins – secreted into the environment or into host cells
Invasins – promote phagocytosis into host cells
Protein secretion systems - secrete toxins and enzymes to subvert host defences.
Iron uptake systems - scavenging essential iron in the low iron environment of the host.
Etc….. e.g. polysaccharide capsule to evade immune recognition.
What is Molecular Kochs postulates (steps) ?
- Gene (or its product) should be found only in strains of bacteria that cause the disease
- Gene should be isolated by cloning
- Disruption of genes in virulent strain should reduce virulence
- Gene is expressed by bacterium during infectious process in animal or human
Techniques of virulence factors ?
- cloning
- transposon mutagenesis
- Transcriptional fusions
- In vivo expression Technology (IVET )
- signature tagged mutagenesis
What do you know about cloning?
Shot-gun cloning of genomic DNA from a pathogen into a plasmid vector
Transform the plasmid library into E. coli and look for genes that make E. coli virulent
Can be more selective now genome sequences are available and can amplify candidate gene/s from genome, clone and express in non-pathogen to see if it confers virulence
old fashioned !
Take the genomic DNA from a pathogen. Digest it with a restriction enzyme clone into plasmid so have enormous library of different plasmids with different inserts in
Would probably not do it this way these days. Ethics of it – are making a new pathogen, could be viewed as bio-terrorism!
What is transposon mutagenesis?
Transposon mutagenesis of the pathogen
Take the individual transposon mutants and test them for loss of virulence
Take your pathogen and expose it to a transposon. I hope you all know what a transposon is – it’s a piece of DNA that gets inserted usually at random into chromosomal DNA.
You can tell when this has happened because the transposon has an antibiotic resistance gene on it so the bacterium becomes resistant to that antibiotic. Need to get lots of mutants, for example E. coli has 4000 genes so need to get at minimum least 4000 mutants to have a chance that you have an insertion in each gene.
Then take all of the mutants and do infection tests with them. This is very time consuming.
What is transcriptional fusions?
Make random or targeted promoter fusions
In the genome
Or on a plasmid (transformed into your pathogen)
Infect the host with the pathogen
Look for strains that express the
gene in the host environment
This is a more indirect method. Here you are looking for genes that are specifically switched on when a pathogen is infecting the host. The way this is done is to fuse a reporter gene to all of the promoters in a genome. The most common reporter that people use these days is GFP, green fluorescent protein.
This can be done in 2 ways by cloning the promoters onto a plasmid in front of promoterless GFP
Or use a transposon to deliver promoter-less GFP at random into the genome of your pathogen
Then take your pathogen that has been transformed with all of the promoter fusions on plasmids, or that has GFP at random in the chromosome and infect the host. Look for strains where they glow green on infection.
However this is indirect – it only tells you that genes are switched on in the host, it doesn’t tell you that they are virulence factors, could be other reasons for them to be switched on (eg aerobic – anaerobic switch)
What is In vivo expression technology (IVET) ?
Very powerful screening technique as it offers positive selection:
Approach: genes which are important for the survival of the pathogen in the host will be activated during the infection.
Use a strain of Salmonella typhimurium which is deficient in purine biosynthesis (purA- mutant). This strain grows fine in the lab but NOT in the host because there are no free purines in the mouse.
Prepare a transposon that contains a promoter-less purA gene and insert the transposon randomly into chromosome of S. typhimurium
IVET is a very powerful and relatively new tool for looking at promoters that are expressed in the host.
The reason it is so powerful is that it is a positive selection. How it works:
Place all of the promoters in front of a gene that can be used as a positive selection. In this example I have used the purA gene for purine biosynthesis. This gene is not essential for Salmonella to survive in the lab because we can add free purines to the growth media so the mutant will survive.
However there are no free purines in the mouse so only those strains where the transposon with the promoter-less PurA has jumped in front of a promoter that is active in the mouse will survive. This means that any promoters not switched on in the host will result in those bacteria dying out and only the purA expressing ones survive, so you have enriched for promoters that are active in the host.
Use an engineered transposon that has on it the purA gene without a promoter. Then allow this transposon to jump randomly into the genome of your pathogen of choice. It will hop into genes and the expression of purA on the transposom will then be regulated by the promoter of the gene you’ve jumped into.
What do we know about In vivo expression technology ?
In the original library, the purA gene is inserted into different regions of the S.
typhimurium chromosome under the control of different promoters. Because purines
are required for the growth of S. typhimurium in the mouse, only cells expressing purA
will survive after several days of infection. Only those cells where purA is behind a
promoter that is active when the bacteria infect the mouse will survive. These will
include the cells where purA was inserted behind a constitutive promoter or behind a
promoter that is induced only during infection.
How can you find out which promoters were only active in the host?
This is how we would do such an experiment, take all the millions of different bacteria from the transposon mutagenesis with the purA transposon. Pooled them all together and inject into the mouse. After 3 days the mouse is sacrificed and the bacteria are harvested.
All of these bacteria should contain purA genes in front of promoters that are active in the host.
BUT: Can be two circumstances. They may contain promoters that are only switched on in the host. OR they may have a constitutive promoter that is switched on all the time and not just in the host because it is required for the production of something that is essential (eg the subunits of RNA polymerase). How can you distinguish these?
