Tuesday, February 28, 2017

The Wolbachia "holy grail"

The once-obscure alpha-proteobacterium Wolbachia pipientis, was catapulted into medical relevance by the discovery that it inhibits the replication of RNA viruses.  However, it was first discovered for its odd quirk of manipulating insect reproduction. That's right, Wolbachia were called "reproductive parasites" and for a long time, no fitness benefit was attributed to them.  These intracellular masters do things like kill male offspring, feminize male offspring, induce parthenogenesis, and the most common phenotype, sperm-egg incompatibility (also known by its more complicated name: cytoplasmic incompatibility or CI) (Figure 1).  

Figure 1. Modified from Werren et al., 2008.  Wolbachia cause four distinct reproductive phenotypes in a range of arthropod orders (top). 

The mechanisms behind CI - meaning how does Wolbachia induce it - have been a mystery for some time.  When you make a cross between infected males and uninfected Nasonia females, unviable embryos result from a mistiming of nuclear envelope breakdown for the male and female pronuclei, suggesting some cell biology behind the failed crosses. With regards to the Wolbachia genes facilitating it, however, there are so many papers on potential models that explain the data - from lock/key to mod/rescue - in fact, when you search google scholar for these terms, you get 92 and 281 papers, respectively.  Folks have mined the existing Wolbachia genomes for potential candidate loci in many different analyses, without any substantial evidence for loci underpinning the interaction.  I, for one, had always been skeptical of these types of approaches because - is Wolbachia evolution so static as to only use one homolog or one mechanism for such a widespread phenomenon. 

So, back in June of 2016, when the Wolbachia meetings took place in Australia, two research groups presented data on two loci - WD0631 and WD0632.  It is with great anticipation that the Wolbachia community awaited the recent manuscript from both John Beckmann and the Bordenstein lab, which I review openly here.

The paper starts out with the premise of a bioinformatics search to identify CI candidate loci. They use the following criteria: present in all CI-inducing strains, and absent or diverged in non-CI strains, expressed in the gonads of infected insects (especially in the C. pipiens ovaries).  Amazingly, this list led them to only two loci - WD0631 and WD0632 (the wMel homologs).  These two loci are associated with Phage WO, the lambda of the Wolbachia world, and are actually be part of the "Eukaryotic Association Module" which is packaged into phage particles. This means that these loci are actually phage loci, not strictly Wolbachia loci.

Now, what other characteristics would we expect from CI-inducing proteins? Well, they would be highly expressed in young male flies (as these are the males for which the CI phenotype is most penetrant).  Are these two loci differentially expressed in young males? Yes, see Figure 2 below:

Figure 2. Expression (qPCR, normalized to Wolbachia groEL) for the two CI loci (WD0631 and WD0632) is upregulated in young male flies, as is the expression of other phage associated genes (WD0508, WD0640 and WD0625). A non-phage associated gene (WD0034) is not upregulated in the same way.

Yes, WD0631 and WD0632 seem to be more expressed in young males but interestingly, so are other loci here.  The uniting factor? These are phage associated...curiouser and curiouser.

What else would we expect of these loci if their protein production was actually behind CI? Well, we would expect to find that you could recapitulate CI by expressing the proteins in transgenic flies.   LePage et al do this (Figure 3b below)! Check out the cross between uninfected (unfilled) males expressing WD0631 and WD0632 and infected females AND the rescue between infected females and those same males.

Figure 3. Expression of CI genes in the reproductive tract of male flies recapitulates CI and is rescued by Wolbachia infected females - CI measured by counting hatched embryos.

And in the most convincing figure of this set (in my opinion) they show a dosage dependence of sorts by exacerbating CI in Wolbachia infected males by expressing the transgenes either alone or together (Figure 3c above).  What's interesting is that with one trangene alone, they could not get a phenotype (Figure 3A), suggesting that these two proteins work synergistically in some fashion.

To add the cherry on top, LePage et al look at embryonic defects in these transgenic crosses and find that, just as in "real" CI, when you express WD0631 and WD0632, you see embryonic defects such as chromatin bridging and regional mitotic failure. Importantly, these defects go away when you mate transgenic males expressing the CI loci to infected females! Bingo! They have satisfied all of the requirements necessary to show that these proteins are indeed involved in CI. 

But how do these proteins induce CI? How are they transferred to the host (by phage???!!!) and what do they do in the host cytoplasm? Do they function together? Are there other loci that underpin CI or other reproductive manipulations? Some of these points may be, in part, addressed by John Beckmann's mechanistic work.  I am sure there will be more interesting work to come from this group!