Department of Biochemistry University of Oxford Department of Biochemistry
University of Oxford
South Parks Road
Oxford OX1 3QU

Tel: +44 (0)1865 613200
Fax: +44 (0)1865 613201
Anaphase bridges in fission yeast cells
Whitby lab
Lactose permease represented using bending cylinders in Bendix software
Caroline Dahl, Sansom lab
Epithelial cells in C. elegans showing a seam cell that failed to undergo cytokinesis
Serena Ding, Woollard lab
Collage of Drosophila third instar larva optic lobe
Lu Yang, Davis lab
First year Biochemistry students at a practical class
Image showing the global movement of lipids in a model planar membrane
Matthieu Chavent, Sansom lab
Bootstrap Slider

Dynamic behaviour of bacterial injectisome component visualised in new study

Schematic of the bacterial Type III secretion injectisome which injects effector proteins directly into the host cell. The C-ring cytosolic component is shown in red

Schematic of the bacterial Type III secretion injectisome which injects effector proteins directly into the host cell. The C-ring cytosolic component is shown in red

Research in the department has shed light on a machinery that causes virulence in a group of pathogenic bacteria.

The work from Professor Judy Armitage's lab, led by Dr Andreas Diepold, reveals previously unobserved features of the injectisome, an essential virulence factor that is responsible for the transmission of bacterial proteins into host cells.

Published in PLoS Biology with collaborators from the Department of Physics in Oxford and the Biozentrum in Basel, the findings suggest the possibility of a novel target for the development of anti-virulence drugs (1).

Many Gram-negative bacteria such as Shigella, Salmonella and Yersinia use the Type III secretion system to help them to thrive in their host. The machinery, also called injectisome, uses a protruding needle to inject a range of effector molecules into host cells, allowing the bacteria to proliferate without being eliminated by the host immune system.

The bacteria only synthesise the injectisome when they are in the body. Once inside, the injectisome remains tightly sealed until it makes contact with the host cell, the trigger for exporting the effectors.

As Dr Diepold explains, researchers have started to build up a picture of the injectisome machinery. 'The part within the membrane has been structurally quite well characterised and is made up of a series of membrane-bound rings. The needles, which can be purified, have also been studied.'

What is not known is how the machinery works. For example, how are the effectors selected and how is the energy transduced for delivery? All of this is thought to take place via the cytosolic part of the machinery.

Cellular distribution of motile (blue) and stationary (red) labelled YscQ molecules

Cellular distribution of motile (blue) and stationary (red) labelled YscQ molecules in bacteria (Click to enlarge)

So Dr Diepold and colleagues decided to look at this region in more detail and in particular the C-ring, an essential and conserved cytosolic component.

To study its behaviour, they labelled the C-ring subunit (YscQ) with a fluorescent tag and tracked it in live Yersinia enterocolitica bacteria, a human pathogen. Using super-resolution microscopy in the departmental Micron facility together with facilities in the Department of Physics, they found that the protein is present in both a free-moving cytosolic state and a stable injectisome-bound state.

'We found that the C-ring is not a stable component of the injectisome but exchanges with a pool of C-ring subunits in the cytosol,' explains Dr Diepold. 'When we measured the exchange, we saw that the exchange is faster when the system is active and exporting factors.'

This is the first time that super-resolution analysis has been carried out on the injectisome, although the exchange has been observed in a related system - the equivalent protein in the bacteria flagellar motor, which is homologous to the injectisome, shows similar behaviour.

Micrograph showing representative images before and after photobleaching of a single fluorescent spot (marked by red circle) of labelled YscQ. The spot shows fluorescence recovery over time

Micrograph showing representative images before and after photobleaching of a single fluorescent spot (marked by red circle) of labelled YscQ. The spot shows fluorescence recovery over time

The group wanted to know whether other components of the injectisome show a similar dynamic exchange. 'We tested other proteins within the membrane part of the injectisome and found that they are stable,' comments Dr Diepold. 'Now we're interested to know whether the dynamic behaviour is specific to the C-ring or a common feature of the cytosolic machinery.'

Schematic of the injectisome showing the dynamic exchange of the C-ring subunit YscQ

Schematic of the injectisome showing the dynamic exchange of the C-ring subunit YscQ

As well as pinning down this dynamic C-ring behaviour, the researchers also studied the stoichiometry of the C-ring and how its subunits assemble into the cytosolic component of the injectisome.

They found that the C-ring comprises approximately 22 YscQ subunits per injectisome and confirmed the importance of a recently discovered shorter variant of YscQ, which forms through use of an internal translation initiation site. The smaller protein was found to be required for proper localisation of full-length YscQ and assembly of the cytosolic complex.

Dr Diepold says that the discovery that the C-ring exhibits dynamic exchange and that the rate of exchange correlates with function, points to a role for the C-ring in regulating the injectisome and integrating external cues coming into the system.

'We're trying to find out what factors could bind to the C-ring and in that way influence activity of the system. Maybe these could be signal proteins that are membrane-bound. We know that the system does respond to some external cues such as pH and oxygenation.'

He adds that any protein identified would be a good target for anti-virulence therapeutics. 'The therapy would be relatively generic and the bacteria would be less likely to develop resistance against it since we would be tackling virulence without putting pressure on processes that are essential for the survival of the bacteria outside the host.'

Reference

  1. Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome. Diepold A, Kudryashev M, Delalez NJ, Berry RM and Armitage JP. PLoS Biol 13(1): e1002039. doi:10.1371/journal.pbio. 1002039

Search

 

Related Information

Share This