L19 & 20 : Type 6 Secretion System Flashcards
How is T6SS used by gram-neg bacteria?
T6SS used to kill neighbouring cells
Explain journey of T6SS discovery?
2000 - first identified T6SS as icmF and found it reduces conjugation frequency
2003 - recognition of entire gene operon cluster as important and found to be widely conserved in gram-neg bacteria
2004 - compared WT and mutant strains (bacteria causing edwardsiellosis) and analysed secreted components, mass spec and identification of relevant gene loci, confirmed as secretion system
2006 - genetic screening and mutagenesis to produce avirulent V.cholerae (not resistant to amoeba, revealed system and given name T6SS
Is T6SS conserved?
Conserved in gram-neg pathogens
~25% have some version of this SS
IcmF conserved in almost all T6SSs
How was the mechanism of T6SS uncovered?
Comparison to other known systems/homologues
DotU/icmF have variants in a T4bSS used by Legionella to deliver toxins into eukaryotic cells - thought to form some transmembrane complex
Triple ATPase clpV also similar to ATPase within protease clpXP where clpX unfolds and threads protein through into degradation complex - thought to be pumping proteins out through SS
Fused to GTP and monitored location fluorescently, producing first incorrect T6SS model
What is the connection between T6SS and phages?
Several T6SS genes have strong structural homology to T4 bacteriophage
VipA/VipB - sheath
gp25 - baseplate
Hcp - tail tube
VgrG - tail spike
PAAR - tail tip
Led to proposal that T6SS may be ‘inverted phage’, firing a needle out of the cell through sheath contraction
How was cryo-EM used to study T6SS?
Allowed visualisation of T6SS structure within cells
Up to 5 tubules seen in WT cells, none in various T6SS mutants
Conical structure seen in periplasm and outer membrane
T6SS sheath (up to ~700 nm long)
What are the two types of tubules visible inside cells?
Extended sheath:
Longer, thinner, inner tube filled with density
Contracted sheath:
Shorter, fatter, follow
What are general T6SS sheath dynamics?
Sheath proteins (VipA) can be labeled with GFP
Polymerises from membrane out in ~30s
Contracts to ~50% in less then 5ms
Disassembles in ~30s
Whole cycle restarts at apparently randomised location
How is protein secretion powered differently to bacteriophages?
Initially thought T6SS would function similarly to bacteriophafes, with distinct steps of membane penetration and cargo delivery
- Secreted ‘effectors’ also structural componetns
- Unlike phage, hcp tube not stable without sheath
- Presumed substrate ‘pump’ ClpV not essential
What is the function of ClpV in T6SS and how was this discovered?
Analysed ClpV and VipA together temporally
ClpV doesn’t colocalise with extended sheath, instead quickly recognises contracted sheath
ClpV actively unfolds contracted sheath and recycles sheath components
Describe assembly and disassembly of sheath?
Sheath is assembled in high energy state
Energy transformed to mechanical force when sheath contracts
Inner tube and spikes ‘fired’ out of cell
ClpV then uses ATP to disassembly contracted sheaths and allow subunits to reform in high energy conformation
How are secreted substrates delivered?
Secreted substrates associate directly or indirectly with T6SS spike (VgrG/PAAR) / inner tube (hcp)
When spike/tube fired out of cell, cargo goes with it
Hcp tube does not form stable channel out of cell
How and why was the T6SS assembly made bigger?
Ampicillin blocks peptidoglycan assembly (cell wall), makes cells very large and round
Note : must maintain proper osmotic conditions to prevent lysing
T6SS assemblies will span width of these larger scale cells, so easier to visualise and investigate dynamics
Which end does T6SS assemble from and how was this shown?
Fluorescence recovery after photobleaching (FRAP) uses strong beam of light to bleach fluorophores and wait for recovery
Can observe that distance of photobleached area is maintaining same distance to structure as tube grows
Concluded that T6SS subunits add to the distal end of the structure during assembly
What is the function of proteins tssA and tagA?
Operon contains homologues tssA and tagA
Similar N-terminal ends but different C-terminal ends
tssA CTD: contains VasJ allowing recruitment of additional subunits to assemble
tagA CTD: contains hydrophobic patch which binds to and associates with membranes
How does T6SS know to stop growing and cap sheath?
Initially, tssA associated with cap facilitating acquisition of more subunitsl
Once tube reaches other end of membrane, tagA is recruited and doesn’t allow further extension
What happens when tagA is deleted?
Tube not able to terminate extension so bends to allow more growth
No longer perpendicular to cell surface
Doesn’t inhibit T6SS activity but may reduce ‘aim of firing’
What is the tit-for-tat retaliation mechanism?
P. aeruginosa can sense attack of different bacterium, assembly and construct T6SS at location of initial attack and retaliate directly at the aggressor
Selective to aggressor and not ‘neutral bystanders’
Retaliating bacteria respond to any kind of membrane penetration
- T6SS
- Conjugation
- Chelating agent
What is the general pathway for sensing aggressor attack?
Proteins in inner/outer membrane pass signal onto serine-threonine kinase
Causing phosphorylation of protein Fah1
Enabling activation of T6SS
What effect does T6SS have within co-cultured bacteria?
P. aeruginosa protects E. coli from V. cholerae attack
Also prevents conjugative element spread with conjugative donors being selectively eliminated (~60-fold fewer transconjugants)
Conjugant elements can turn off T6SS if it can penetrate Pseudomonas
