Lecture 19 and 20 - Type 6 Secretion Systems Flashcards
How are T6SS used by gram negative bacteria?
T6SS are used by gram negative bacteria to kill neighboring cells.
It is conserved in gram-negative pathogens.
Explain the journey of discovery of the Type 6 secretion system.
In 2000, the first T6SS gene - icmF was discovered.
In 2003, a group recognized that the T6SS was an entire gene cluster and was important, and recognized it was important for some sort of secretion.
In 2006, it was given the name T6SS.
What technique was first used to uncover the mechanism of T6SS.
Comparison to a known homologue.
Dotu/Icmf have variants in a T4bSS used by Legionella to deliver toxins into eukaryotic cells.
clpV gene also similar to an ATPase part of the degradasome complex in bacteria (ClpXP).
ClpXP acts as a protein pump that threads unfolded proteins through it.
This ClpV protein was fluorescently labelled, but was wrong.
What was the connection to phages?
A lot of genes in the operon are homologous to genes in contractile tail phages.
This created the conclusion that the T6SS may be an “inverted phage.” The T6SS ‘fires’ a needle out of the cell.
Again the details of the mechanism were seen to be wrong.
How was Cryo EM used to study T6SS?
Using Cryo-EM, a structure for the T6SS was uncovered.
A conical structure was seen in the periplasm and outer membrane.
Two types of tubules were seen in the cell.
An extended sheath (longer, thinner, and density filled), with an inner Hpc2.
A contracted sheath (shorter, thicker, and hollow), where the Hpc2 is pushed out of the cell.
Explain the details of the sheath in the T6SS.
The sheath is made up of VipA sheath proteins.
They were studied by using GFP as a fluourescent label.
Polymerizes from the membrane out in around 30s.
Contracts to 50% in less than 5ms
Disassembles in around 30s.
What did knockout gene studies and visualization of dynamic structures show?
The ClpV gene is not essential for T6SS activity.
ClpV does not co-localize with extended sheath
Whole contracted sheath gets quickly recognized by ClpV
ClpV spot marks T6SS that have just “fired”, and disassembles.
How is T6SS activity powered?
The T6SS sheath assembles in a “high-energy” state.
This energy is transformed into mechanical force when the sheath contracts .
ClpV then uses ATP to disassemble the contracted sheath.
This allows sheath subunits to reform in their high energy conformation.
Describe the delivery of the secrets substances.
Secreted substrates associate either directly or indirectly with the T6SS spike or the inner tube.
When the tube/spike is fired out of the cell, the cargo goes along with it.
The Hcp “tube” does NOT form a stable channel out of the cell.
The effectors/substrates are located downstream of the VgrG protein gene (the spike).
How were T6SS assembly dynamics studied?
T6SS assembly was simply made bigger.
They were treated by ampicillin, which blocks peptidoglycan, making cells very large and round.
How do we know which end T6SS assembled from?
From Fluorescence Recovery after Photobleaching (FRAP) studies, we can see that the distance of the photobleached area, is maintaining the same distance to the structure as the tube grows.
This is indicating the subunits are added to the distal end.
How does T6SS know when to stop growing?
The operon contains homologues TssA and TagA.
The N-termnus ends of both these protiens are similar, however the C-terminals differ.
The C-terminus of TssA contains VasJ, which allows for the addition of more subunits.
The C-terminus of TagA contains a hydrophobic patch, which binds to membranes.
Once the tube reaches the other end of the membrane, TagA is recruited and binds to the membrane, so the tube stops growing.
What happens with deletion of TagA?
The tube is not able to terminate extension, so it bends to allow more growth.
While T6SS activity can still occur, it cannot kill neighboring bacteria as well.
What is the Tit-for-Tat concept and its mechanism?
When attacked by a T6SS of a different bacterium, Pseudomonas retaliates.
Proteins located in the inner and outer membrane pass a signal onto a serine-threonine kinase, which phosphorylates a protein called Fha1, which enables the unlocking of the T6SS.
Retaliating bacteria react to any kind of T6SS system, conjugation, and chelating agents.
This is to do with membrane permeation.
How do conjugative elements fight back?
Conjugative elements can turn off T6SS if it can infiltrate Pseudomonas.
