Bacterial Virulence Flashcards
Describe some Gram positive bacterial pathogens.
Rods: Bacillus anthracis (95% fatal even with treatment. Bacteria produce spores which make it persistent. Used as a bioterrorism agent, and illegal to work on in UK).
Corynebacterium diphtheriae (causes diptheria).
Clostridium botulinum.
Clostridium tetani.
Clostridium perfringens.
(all of the Clostridium ones are anareobic growing and produce spores. Also produce toxins.)
Cocci: Staphylococcus aureus (commensal organism on skin. Multi drug resistant). Staphylococcus epidermidis (causes spots). Staphylococcus pyogenes (causes tonsilitis).
Describe some Gram negative bacterial pathogens.
Rods: Salmonella enterica. Shigella dysenteriae. Escherichia coli. (these three cause food poisoning). Yersinia pestis (causes black death. carried by deer. Can be killed by penicillin).
Coccobacilli: Bordetella pertussis (causes whooping cough).
Cocci:
Neisseria gonorrhoeae.
Neisseria meningitidis.
Curved rods (spiral): Vibrio cholerae. Helicobacter pylori (causes stomach ulcers). Treponema pallidum (causes syphilis). Borrelia burgdoferi (causes Lyme disease).
What is the definition of virulence factors?
Products or structural components (usually proteins) that allow an organism to enter and alter host function to cause disease.
Describe some virulence factors.
Adhesins- on surface of bacteria. Sticks to host cells.
Toxins- secreted into the environment or into host cells.
Invasins- on surface of bacteria, a bit like adhesins. Promote sticking to host cells, then phagocytosis into host cells.
Protein secretion systems- secrete toxins and enzymes to subvert host defences. Some are dedicated for pathogenesis.
Iron uptake systems- scavenging essential iron in the low iron environment of host (Release iron from host when it isn’t redily available eg. haemoglobin).
Polysaccharide capsule- featureless, so can evade immune recognition for longer.
What are Molecular Koch’s Postulates and what do they do?
Address how a particular gene contributes to virulence.
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.
(Koch discovered Mycobacterium tuberculosis)
How is cloning used to identify virulence factors?
Make new strains of pathogenic bacteria- insert pathogen gene into non pathogenic bacteria. Very old fashioned method.
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.
How is transposon mutagenesis used to identify virulence factors?
Transposon mutagenesis of pathogen.
Take individual transposon mutants and test them for loss of virulence.
How is transcriptional fusions used to identify virulence factors?
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 really really uses Koch’s postulates.
Things can be switched on depending on host environment. Method is bit indirect.
How is In Vivo Expression Technology (IVET) used to identify virulence factors?
This is a newer, Very powerful screening technique as it offers positive selection.
Find promoters switched on in the host.
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.
(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.)
Which ones were on in the mouse and which ones are on all the time?
Plate post mouse cells onto rich media: all the cells that survived during infection will form colonies.
Replicate each colony onto two new plates- purine media and no-purine media.
Ones only in mouse won’t grow on no-purine media. Promoter only effective under infection conditions.
Isolate DNA form cells where purA is controlled by the promoter induced in the mouse, sequence flanking regions and identify the promoter and the genes it controls.
How is signature tagged mutagenesis used to identify virulence factors?
New method.
A very powerful technique- uses a negative screening approach. A high throughput technique can screen many millions of mutants at the same time. Invented by David Holden (imperial College London)- designed to identify bacterial mutants that disappear in the population during infection.
High throughput transposon library, infect mice, which ones have died in the mice?
Signature tag is like a barcode. Different transposon mutates a different bacteria.
Only one copy of the transposon is inserted into the chromosome in each bacterial cell. In different cells, transposons insert into difefrent sites in the chromosome. Each transposon carries a different barcode sequence.
Grid out the mutant library (approx. 20,000 colonies), replicate onto filters. Pool the mutants together and inject into mouse. Recover surviving bacteria after 3 days. PCR the tags from all the survivors from the starting pool and hybridise the original filter.
Bacterial clones that contained transposon insertions in the genes required for growth and survival in the mouse will be missing in the recovered pool. PCR identifies the missing ones. Tags that have no hybridisations are the virulent ones.
This techniques can be applied to many species of bacteria, and has a very high throughput.
How is TRADIS used in identifying virulence factors?
Transposon Directed Insertion Site Sequencing- most recent iteration of STM.
New method that is most up to date. Uses next generation sequencing. Can be done in a week or two, and can find very small virulence factors.
Make a large transposon library of your pathogen of interest.
Determine all of the insertion sites using next generation sequencing.
Grow under selective conditions (e.g. in the presence of bile) and sequence the survivors.
Those that do not survive are in genes essential for survival under those growth conditions.
What are the genetic elements that encode pathogenicity factors?
Bacteriophage- diptheria toxin, cholera toxin, Shiga toxin, botulinum toxin.
Plasmids- Shigella flexerni, Salmonella enterica, Yersinia species, Clostridium tetani, Enterococcus faaecalis.
(Can move around between organisms.)
Transposons- heat stable toxin of ETEC.
Can spot transposons (inverted repeats).
Pathogenicity Islands (PAIs)- can be up to 200kb in size.Virulence related genes situated within continuous segments of the chromosome. Found in both Gram positive and negative bacterial genomes.
What are some common features of PAIs?
Crry one or more genes encoding virulence factors.
Present in pathogenic organisms but absent from genomes of non-pathogenic organisms of the same or closely related species.
Occupy relatively large genomic regions (>10-200kb).
Often have different G+C content and codon usage.
(GC content- E.coli has 50% AT and 50% GC, other organisms have different ratios.
The use of PAIs in virulence identification is just as valid as any of the experimental ways.)
Often flanked by Direct Repeat sequences- related to the way they are integrated.
Often associated with/integrated near tRNA genes.
Often carry cryptic or functional genes encoding mobility factors (have to move around. Once integrated, mobility factors become degenerate- become a permanent part of the chromosome).
Represent unstable DNA regions.
May represent integrated plasminds, conjugative transposons, or bacteriophage.
Where are PAIs found?
Found in almost very organism, even non-pathogenic ones- they can pick up PAIs from environment.
Particular species include: Enterobacteriaceae (E.coli, S.enterica, Yersinia species), V.cholerae, P.syringae, Leigonella pneumophila, Listeria species, S.aureus.
What are some virulence associated genes on PAIs?
Adhesins Secretion systems Toxins Iron uptake systems O antigen synthesis- relates to capsule and surface.
