Lecture 2 Autotransporter secretion tool and target Flashcards
What pathway is used for biogenesis of IMP?
SRP pathway (signal recognition particle). SRP homologous with eukaryotic SRP.
FtsQ is used as a model protein in crosslinking studies to study interactions of nascent membrane proteins. Why? What sort of protein is this?
It is a simple IMP. Central role of FtsQ in divisome (large complex of membrane proteins, assembles in middle of the cell right before division) assembly.
•Interactions with many divisome proteins (role in assembly);
Closest interactor: FtsB. So, Structural analysis of soluble FtsQ-FtsB subcomplex: tool for development of inhibitors of this crucial interaction. Would make a good antibiotic.
T5SS is also known as? What kind of secretion system is it?
also known as Autotransport.
•most widespread secretion system to deliver virulence factors (eg. IgA protease, pertactin, Haemoglobin protease or Hbp). Hbp = model protein.
How does Autotransporter secretion go?
- Translocation over the IM by the Sec machinery
- Chaperones keep the protein protected + organize targeting to the BAM
- BAM complex helps in folding and inserts OMP: helps to assemble beta-domain into the membrane and at the same time helps to translocate the passenger domain across the OM.
- Autotransporter cleaves itself mostly from the beta-domain: released into the host.
Autotransporter = domain in OMP. Use of the Autotransporter Hbp for recombinant protein secretion and surface display: advantages?
- Ubiquitous, relatively simple process
- Efficient/specific
- Transfer between Gram-negative bacteria possible
- extent of display can be manipulated
How can the extent of display be manipulated?
(mutating the cleaving of the beta-domain: inducing cleaving for secretion of proteins: biotech or preventing cleaving for display of proteins)
What can be a toepassing for the display of antigens?
Display of antigens for live vaccine development
> Fuse antigens/-fragments from pathogenic bacteria to expose them at the surface of harmless bacteria. Harmless salmonella strain in which the Hbp + antigen is expressed.
> Surface display enhances humoral and CD4 and CD8 T cell responses.
How to check if antigens are expressed?
> Immuno-gold labeling: Label antibodies to and see if they attached.
Why is it difficult to get live vaccines accepted?
Still regulatory issues:
Can it revert to a virulent strain?
Can it spread in the environment and mutate? -> Difficult to get a vaccine accepted.
How can Outer Membrane Vesicles (OMV) be used in vaccines? How are they ‘gekweekt’?
- Nanoparticles that shed from the outer membrane
- Safe, but still intrinsic adjuvant activity (immunogenic molecules, OMP, LPS), so don’t need adjuvant (stimulating particle for immune response)
- Present in licensed vaccines (Bexsero, GSK); cost-effective production
- Antigen display on OMVs:
- Hypervesiculating strain: S. typhimurium (ΔaroA ΔtolRA)
- Efficient expression of Hbp-H56 at surface of OMVs:
What are the advantages of using omv (‘platform’?)
High density display of multiple sizeable antigens for optimal immune responses
- Intrinsic adjuvent ativity
- Fast development & cost-efficient production
- Safe attenuated strain, non-replicating derived OMVs
- Compatible with mucosal and parental delivery routes
Disadvantages of OMV (platform) ?
Intrinsic adjuvant activity sometimes too strong
Limited capacity for introduction of (multiple) complex antigens
How to increase platform capacity?
–> Coupling of antigens after translocation of the carrier to the cell surface (not hampered by the limited translocation)
How to connect the proteins?
Protein ligation: Spy-technology
Streptococcal adhesin CnaB: divide into SpyCatcher + SpyTag. Add SpyTag to protein A and SpyCatcher to protein B. Now you can covalently attach A + B.
Autotransport is also a target for antibiotics, as it is used for secretion/surface display of many virulence factors. Name some examples of these virulence factors.
- Adhesion to cells
- Uptake of iron
- Immuno-evasion
- Host cell destruction
- Biofilm formation
Why would you inhibit virulence factors and not necessarily inhibit growth?
That is good for e.g. microbiome
With the help of what assay can you find out whether a compound can inhibit the translocation across the OM? (BAM complex)
When inhibiting this translocation, proteins will aggregate in the periplasm. This will cause stress which will lead to the expression of proteases and chaperones. Stress promoter: fused to a GFP. So, when there is stress, the cells turn green.
How did we perform this assay (HTS assay based on sigma E stress) in the lab?
- Start growth in normal culture up to steady-state growth
- Transfer to multiwell plate
- Add compounds: continue growth in multi-well plate reader
- Measure fluorescence and OD in time
Autotransport + ? use Bam?
OMP biogenesis
BamA = ?
+ 4 lipoproteins BamB-E
Beta-barrel.
- Key role in Autotransporter secretion, hence in virulence
- Key role in OMP biogenesis, hence in OM functioning
- BamA is surface exposed
- Structures known
Rcs system is used for?
- responds cell-envelope integrity stress (o.a. proper insertion in OM).
(RcsF (sensor located in the OM) senses defects in:
•Bam complex
•LPS
•Peptidoglycan
•Lipoprotein trafficking
Kinetics of response is related to target. Stress is induced and there is an upregulation of RprA promoter.)
What are fluoroquinolones ?
-> chemical compounds that inhibit DNA gyrase (act in the cytosol)
3 domains of T5SS?
passenger domain, beta-domain, protease domain
