VL11: Genetic identification of virulence factors Flashcards
What can bioinformatics be used for? (4)
- checl genome for virulence-like genes
- check genome for genes that are universally conserved in human pathogens
- compare protein coding sequences between closely realted pathogenic and non-pathogenic species
- Check for horizontally transfgerred DNA (HGT) (codon usage bias, GC content, dinulcleotide pattern, identify candidates for PAI and virulence genes)
How can bioinformatics be used to identify candidate genes for virulence phenotypes?
compare genes present in virulent strain to harmless strain
What is a brute force screening?
knock-out iindividual genes and check effect in animal model and in vitro
How can genes be inactivated?
transposon ¨hop¨ and insertion (random genes are inactivated)
.linear transformation (lambda red recombineering) deactivates interest genes
How does transposon mutagenesis work and what are its advantages and disadvantages?
recombination between short dna sequences at the ends of atransposon and a random sequence in the host dna, catliyed by transposase
- stable knock-out mutations
- random isertion
- negative data- no insertions found in essential genes
- no direct information on virulence, individual mutants or the entire pool must be tested in animal model
How does lambda red recombineering work? advantages+ disadvantages
- pcr amplify a drug resistant cassette using oligos -which also have homology to the specific target gene or genes
- induce the lambda red genes in the recipient bacteria
- transform in the pcr product
- select transformant- the target gene will be replaced
- assay the phenotype is the gene essential for viulence?
very specific can knock out or replace any gene regon
very efficient
must know what you eant to knowck out
no direct information on virulence- follow up witn animal model tests
method only developed for a few bacterial species
name 3 methods for virulence gene screening
IVET in vivo expression technology
DFI differential fluorescence induction
STM signature-taggged mutagenesis
How does IVET work?
a) IVET= In Vitro Expression Technology.
-Goal: identify bacterial genes that are specifically induced during an infection.
-Premise: most virulence genes will be transcriptionally induced during an infection.
Salmonella requires an active purA gene to survive in a mouse.
In IVET, random pieces of DNA are cloned in front of a promoterless purA gene, creating a library of
Salmonella mutants.
These grow OK in vitro (because purA expression is not required in vitro), but they will only survive in the mouse if the piece of cloned DNA contains a promoter sequence that is active in the mouse and drives transcription of purA.
The lacZ gene is also placed immediately downstream of the promoterless purA and its expression also depends on the presence of a promoter in front of purA.
lacZ activity (visualized as blue colony colour on agar containing X-gal) is used as a control to identify all of the bacteria that express purA-lacZ in vitro (these promoters are not of interest)
the interest with IVET is to identify those bacterial promoters that express the purA specifically in the mouse (and not in vitro).
After passaging the bacterial population through a mouse, surviving bacteria are collected and examined to determine if they make blue colonies on X-gal agar (not interesting). The remaining bacteria are regarded as interesting – capable of surviving in the mouse (meaning they expressed purA) but not blue on X-gal (meaning the expression is specifically induced in the mouse).
DNA sequencing is then used to determine which sequence is present in front of purA.
These sequences are mapped to the chromosome sequence to identify the genes they regulate, and the assumption (or at least, hope) is that they are promoter sequences for genes or operons that are important during some stage of infection.
What is DFI?
Trap gene promoters that are activated inside macrophages
Use GFP as the signal of activity
9 steps of how to do DFI
- Clone random fragments of Salmonella chromosome into a
promoterless gfp plasmid - Introduce the plasmids into Salmonella
- Infect macrophages with Salmonella harboring gfp fusion plasmids
- Sort GFP-active Salmonella with FACS
- Lyse macrophage, isolate bacteria, sort bacteria with FACS
- Discard Green bacteria (constitutive promoters), keep GFPinactive
bacteria (macrophage-activated promoters) - Re-infect Salmonella into macrophage, confirm that GFP
specifically activated by macrophage - Isolate plasmid, sequence promoter, identify gene by Blast
- Have identified potential virulence-specific gene
What is STM? How does it work?
generate a collection of recombinant transposons, each containing a unique tag sequence that are flanked bu arms that allow for pcr amplification
create libbrary of mutants
bring them into pathogen -> put the ones that are viable in vitro in vivo and see if they can survive there too. if not the knocked out gene was important for virulence -> identify gene with after pcr amplification
What are the advantages of STM?
Major advantages of STM
1. Directly identifies genes involved in virulence rather than
indirectly by in vivo expression.
2. Like DFI, it does not depend on selection and can be used to
identify genes that are expressed transiently or at low levels.
3. STM is easy to adapt to many host-pathogen systems and,
unlike IVET methods, it is not dependent on preferential
transcription in host tissues. Rather, it identifies functions
required for survival within the host.
How does TraDIS work?
Transposon-directed insertion site sequencing (TraDIS)
- TraDIS helps to determine the location of transposons -DNA from bacteria population is broken (sheared) into ca 300 bps long fragments
- DNA fragments are ligated to linkers
- > PCR reaction with one primer specific for the linker sequence and the second primer specific for a sequence close to one end of the transposon.
- This PCR reaction selects specifically for fragments carrying DNA containing one of the junctions between the transposon and the part of the chromosome where it is inserted.
- Sequencing of PR fragments
- compare to known chromosomal DNA sequence and identifiy exactly where every transposon is inserted (and the depth of sequence at each gene indicates how frequently a transposon was inserted at that location).
- Do the same with DNA recovered after infection of the animal model,
- compare genes
- the genes that are present in vitro but absent in vivo are potential virulence genes
(The absence of certain transposon insertion sites post infection, and differences in depth of sequence coverage post infection, indicate which genes were important at some stage during the infection. )
Thus, genomes in which transposons inactivate genes
that are important for infection will be absent (or have a lower read depth) from the post-infection library.
How can virulence genes be validates?
Test: Oral and intraperitoneal infection
Test: Immunocompromised and
immunocompetent mice
Describe a model of how Shigella evolved
SHI-1 and SHI-2 are the Shigella pathogenicity islands that make it different from e.coli
