Transcriptional Regulation of Culex pipiens Mosquitoes by Wolbachia influences Cytoplasmic Incompatibility

An intriguing title for an article published in PLoS Pathogens on Halloween.  Needless to say, many of us in the Wolbachia community anxiously await the discovery of the mechanism behind Cytoplasmic Incompatibility.  For those of you who aren't Wolbachia-philes, the short of it is that this bacterium has figured out a nifty way to spread through insect populations. First, it's transmitted via the germ line -- so that means eggs come pre-loaded with Wolbachia from infected mothers.  Second, Wolbachia affect reproduction in a variety of ways but the most common is that infected females can mate with uninfected or infected males.  However, if you are an uninfected female -- if you don't carry the bacterium -- you cannot mate with infected males.  This drops the fecundity of uninfected females and allows Wolbachia, and the host carrying it, to spread.  There are also some interesting incompatibilities that result when hosts are infected with two different Wolbachia -- sometimes the crosses are compatible and sometimes they are not.  This has led the Wolbachia research community to come up with all sorts of complex, mathematical models and explanations for the observed data.

So, what is the molecular mechanism behind these incompatible crosses? How does Wolbachia prevent embryos from hatching when an infected male mates with an uninfected female?  Lots of elegant work has been published by both the Sullivan and Frydman labs suggesting that Wolbachia both associated with cytoskeletal elements (microtubules) and alter the cell cycle progression.  This cell cycle defect has been correlated with cytoplasmic incompatibility.  So, it makes sense to focus on the cell cycle when you're going after CI.

The Pinto et al. paper in PLoS Pathogens this last month takes a candidate gene approach to the CI phenotype.  They start with the Drosophila melanogaster gene grauzone (grau).  This gene is pretty darn important to the fly -- mutants are sterile and lay eggs with aberrant chromosomal segregations which arrest in development at metaphase II.  Pinto et al.  figure out that this gene is over-expressed in Wolbachia-infected mosquitos -- by almost 2 fold at some time points -- compared to uninfected mosquitos (see Figure 1 below from the paper, A,B are females and males, respectively while C,D are their reproductive organs).

Figure 1. Transcription analysis of CPIJ005623 in the Culex pipiens complex.

I think it's neat to find host genes that are differentially expressed when a bacterium invades.  Transcriptomics has been done before in Wolbachia infected cell lines but many of the candidates identified were immunity genes, seemingly irrelevant to the CI phenotype.   Even more interesting is the fact that this gene is important to female reproduction and specifically, meiosis.

Pinto et al. then ask whether by suppressing the grau homolog in mosquitos, they could replicate the CI phenotype.  I guess the idea is that this factor could be the "rescue" factor or the "key" that is produced by the female when crossed with an infected male.  An alternative hypothesis would be that this factor is upregulated in infected insects as a result of Wolbachia increasing the mitotic activity of the germ line cells, a result reported by the Frydman lab a few years ago.  Anyway, they go with hypothesis #1 and use RNAi knockdowns (KD) of this gene to then observe what happens if we cross KDed infected females with infected males?  Interestingly, they observed an increase in the percentage of unhatched embryos, compared to a LacZ control (see Figure 2D below):

Figure 2. Knockdown analysis of CPIJ005623 in C. molestus Italy females.

However, they ddidn't include some important experiments here -- how do we know that this grau knockdown is specific to the CI phentoype? Wouldn't it have been important to cross these KDed infected females with an uninfected male?  What if the observed increase in unhatched embryos is similar?  Since it seems that Wolbachia actually increase the expression of grau, wouldn't you want to take an uninfected female, overexpress grau, and cross her to an infected male?

Moving on, next, they do some interesting crosses. As it turns out, the Wobachia they are working on exists as many different strains in mosquitos, each exhibiting an interesting incompatibilities.  For example, two of their Wolbachia strains, wPel and wItaly, cannot be crossed to each other -- that is, infected females or males from either background do not produce viable embryos (no eggs hatch).  Pinto et al. knockdown grau in wPel infected males and crossed them to wItaly infected females, they also performed a KD in wPel and in wItaly and crossed these two KDed lines.  The results: no difference - no viable embryos.  BUT, they do observe a statistically significant change in the number of embryos reaching stage II and III (which, I forgot to mention, was the opposite phenotype observed for infected KDed females crossed with infected males - Compare Figure 4C below to Figure 2D above).

Figure 4. Knockdown analysis of CPIJ005623 in C. pipiens males.

So it seems like Wolbachia CI-like effects can be modulated by host grau expression (although clearly this isn't the entire "rescue" or the "key" story).

The final part of this paper is a bit disappointing.  As has been done time and time again, the researchers attempt to identify genomic differences in the Wolbachia strains infecting these mosquitos to determine what may be the factor that is changing expression of grau.  Disappointingly, although they identify some regions present in some strains and absent from others, they don't go far enough to establishing mechanism.  In specific, they point out that a transcriptional regulator, which they call wtrM, is present in wPipMol but absent in wPipPel.  What is their evidence that this transcriptional regulator is altering grau expression? Sadly, none. They show it is expressed in ovaries -- note: this is not that surprising since this is where Wolbachia actually hang out.  They go out on a limb and say that wtrM is actually secreted by Wolbachia and modulates host gene expression, so it presumably makes its way to the nucleus and actually binds to host DNA.  No evidence is presented for this presumed activity -- no chromatin immunoprecipitation, no DNA footprinting, not even nuclear localization when expressed in their mosquitos.

wtrM, it turns out, is a pretty well conserved XRE transcriptional regulator  -- that is, a Xenobiotic Response Element.  It's found within the Rickettsiales -- there are even homologs in Anaplasma, Ehrlichia, and Bartonella.  What do XRE's do, you ask? Well, they are known to respond to environmental stimuli in many systems but are probably most famous for their involvement in phage response. It is therefore not so surprising that Pinto et al. find this gene associated with the Wolbachia prophage.  That's not to say that the prophage isn't interesting -- it sure darn is! -- but the connection between this specific XRE element and grau is tenuous, at best.