You say "effector", I say "artifact" - how do we know a protein is truly secreted?

We've been thinking a lot lately about how our favorite endosymbiont Wolbachia manipulates host biology. Because Wolbachia are not genetically tractable - or even culturable outside of host cells - it's pretty difficult to figure out the mechanisms used by the bacterium. However, we can start with some assumption about our expectation: Wolbachia likely secrete proteins into the host cell and change cell biology to facilitate infection. This is not ground breaking or novel in any way - invading intracellular microbes must deal with the host during infection - and many do this via secretion of proteins, called "effectors." We know that these effectors often contain eukaryotic domains and homologies because they interface with the eukaryotic cell - a completely different domain of life. But how do we identify the proteins secreted by Wolbachia, or any symbiont?

There have been some pretty clever work arounds. For example, John Beckmann and Anne Fallon used mass spectrometry to identify Wolbachia proteins present in the spermathecae of female mosquitos. The assumption here is that Wolbachia does not infect sperm, so proteins identified may be involved in manipulating reproduction. Others have started with a screen of genomic libraries, expressed in a cell culture line, to find toxic Wolbachia proteins. We have taken a bioinformatics approach to start, identifying candidate secreted proteins based on their eukaryotic homologies. We can then use a growth screen in a eukaryotic model (yeast) to identify interactions between the Wolbachia protein and eukaryotic cell machinery (an approach also used successfully to identify the mechanism behind the CI-inducing loci). Finally, in collaboration with Pete Christie, we generated a surrogate secretion system for Wolbachia effectors. All of these approaches provide evidence for the identification or characterization Wolbachia's effectors and I have no doubt that as these methods become more widespread, we will see an increase in the number of labs using them and effectors identified. I think we are at the brink here for the generation of a new field within Wolbachia and symbiosis

But it is worthwhile to think about our expectations for secreted effectors and our standards, within the community, for what we consider to be evidence of Wolbachia-host interaction. I will focus specifically on proteins secreted by Wolbachia.

1) Wolbachia must express the protein.
2) The native proteins must be found external to the microbe itself (so in the cytosol, or on the vacuole surface, or in uninfected tissue).
3) Of course, the gold standard, which will be difficult for Wolbachia, is to show direct interaction between secretion machinery and the effector.

My point is, heterologous expression of a Wolbachia protein and induced phenotypes is not evidence that it is secreted. Indeed, Wolbachia transketolase and pyruvate phosphate dikinase kill Drosophila S2 cells upon expression. Does this mean they are secreted by Wolbachia to manipulate the host? Surely you don't think that. The truth is, that although toxicity in yeast has been used by many as a primary screen for bacterial effectors (although see false positives here), we don't really know what happens when you express random Wolbachia proteins in the fly. Maybe bupkis. But it's also important to consider that bacteria modify their proteins in ways that would not be replicated upon heterologous expression in an insect system. Indeed, you might expect other modifications to these proteins that wouldn't be present in the native, Wolbachia-expressed protein. Therefore, it's important to interpret this kind of work with its caveats in mind.

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