The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-to-cell spread of Listeria
Tina Rajabian, Balramakrishna Gavicherla, Martin Heisig, Stefanie Müller-Altrock, Werner Goebel, Scott D. Gray-Owen and Keith Ireton. Nature Cell Biology 11:1212-1218. 20 September 2009
This paper addresses the role of InlC in Listeria monocytogenes infection of a cell. They investigate the role of this protein in cytosolic replication of the bacteria and in the formation of actin comet tails and conclude that it plays no role in either of these two functions. They further investigate actin tails in the cytosol versus ones that are formed at the plasma membrane with the use of ezrin, a protein existing only in actin tails that protrude out of the plasma membrane. 
Figure 1: InlC is needed for efficient spreading and protrusion formation in a polarized cell line.
In the remainder of the paper the continue to look at the role of inlC in protrusion formation and manage to identify TUBA, as a cellular factor that interacts with inlC.
Figure 2: InlC interacts with the mammalian adaptor protein Tuba.
They further look at the region of TUBA, an SH3 domain that interacts with inlC and go on to show this interaction with pull-downs of GST-tagged proteins and subsequent Westerns. 
Figure 3 - Tuba and N-WASP control protrusion formation.
They find out that this specific SH3 region had been found to interact with N-WASP, a cellular nucleator of actin. They go on to show that as inlC concentrations are increased, the amount of N-WASP that can be pulled down with GST-tagged TUBA decreases, leading them to conclude that inlC works by displacing N-WASP from its interaction with TUBA. 
Figure 5: Model for InlC-mediated cell-to-cell spread of Listeria.
They continue to look at the morphology of apical junctions, performing microscopy and measuring the curvature of the junctions, concluding that inlC helps to perturb these junctions. This is useful for Listeria because the bacterium is able to protrude out of the membrane more efficiently in the presence of inlC and spread to neighboring cells.
Figure 4: InlC, Tuba and N-WASP control the morphology of apical junctions.
This is important in finding out how Listeria is able to infect cells and spread its infection and it demonstrates an interesting use of a bacterial protein to help spread an infection. The paper had very thorough experiments that covered what they were trying to show. My one complaint would be the blot that they show for N-WASP knockdown in Figure 3 doesn’t seem to have knocked down that much of the protein. If the remainder of the characterizations were down on such a small knockdown some of their conclusions may not be as accurate.
7 comments:
Are there any relevant figures in the article that you could post to clarify your summary?
Also, I am having difficulty piecing together all of the different experiments together and understanding how they help support the authors' conclusions. Again, perhaps some figures could help clarify how their methodology lead to their conclusions.
Well, I don't think it'll let me edit the post or add images in a comment, but if you start with their Fig.5, you can see the role of the membrane rigidity in cell to cell spread of Listeria.
If you go backwards from there, in Fig.4 they show that when they knockdown TUBA or N-WASP the membranes of the cells are affected. Similarly since inlC is believed to be a bacterial protein that interacts with the cellular protein TUBA, when cells are infected with the wild type Listeria, you see a change in the membranes, whereas when the Listeria are lacking the inlC gene or when they have the K173A mutation, the membranes are unaffected. Previous figures basically show that inlC plays an important role in infection and interacts with cellular TUBA. Hope that helps, and sorry I wasn't able to upload the figures.
Do you know if TUBA is actually necessary in the actin nucleation process? Also what cells were being used in studying the infection of listeria?
They present a model in which they believe TUBA acts to activate N-WASP, leading to cortical tension. So they believe TUBA is one of the many activators of N-WASP, in this case leading to cortical tension. InlC of Listeria binds to the same domain of TUBA that would normally activate N-WASP, competing with the N-WASP reaction.
They used human enterocyte cell line Caco-2 BBE1 (ATTC; CRL-2102).
Fascinating paper!
Actin comet tails seem to be a feature found in many actin forming eukaryotes. The ability of lnlC to inhibit comet tail formation many be very useful as a "part" to control the structural dynamics of a cell. One model organism of great interest is yeast, which forms actin. I was wondering, is there any evidence that lnlC is specific to mammalian cells?
Furthermore, is there any information concerning homologues of lnlC in viruses for plants or yeast? If those exist, is there any evidence that the homologues behave the same way in yeast and plants?
So InlC is the Listeria (bacterial protein) that functions to interfere with regular cellular function.
I'm not aware of whether other bacteria have a homologue of this protein, or whether any viruses do. I would guess that viruses wouldn't have this protein. It serves a minor function (seeing as Listeria can still infect cells, just at a lower efficiency) and viruses are generally more limited in the number of proteins that they can encode
Can you explain more about ezrin and the actin tails that protrude out of the plasma membrane? It seems like this has a function in listeria spreading from cell to cell, but how is it able to cross the plasma membrane, and with an actin tail, no less.
If it perturbs the apical junctions, then does that mean that the role of Inlc is mainly for cell to cell spreading?
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