Does Wolbachia really grow inside Saccharomyces?

Obligate intracellularity is a bit like unculturability – dogma that was popular decades ago but is now being questioned given advances in our technology and methodologies for cultivation. Who wouldn’t be inspired by the recent ability to cultivate Coxiella or Hamiltonella? Remember the 99.9% uncultivable microbes? Many studies (here and here, for example) have revealed that it was our methods - the media conditions - that limited our ability to culture many microbes. Yet, for my favorite bacterium, Wolbachia pipientis, obligate intracellularity is like a badge of honor (or shame!); I cannot tell you how many times I’ve written obligate intracellular in grant proposals and manuscripts to describe the bug. Wolbachia, you see, is part of the ancient intracellular Rickettsiales clade - including Ehrlichia, Anaplasma, Rickettsia, and none of these microbes have been cultivated outside of host cells. They have reduced genomes and generally infect arthropods - although sometimes the pathogenic species cause trouble in vertebrates. 

Therefore, I was quite surprised by a recent publication in MicrobiologyOpen with the following bold result: Wolbachia strain wAlbB grows inside Saccharomyces cerevisiae. It is an astonishing claim that Wolbachia can make its way inside the yeast cell (past the cell wall) much less grow inside. It seems that all the authors had to do was spin a Wolbachia preparation (from infected Aa23 cells) onto yeast cells (strain W303) and wait while the magic occurred. The only special ingredients they added to their media was ferric citrate and FBS (1%). I have asked the authors to send me their strain, and it may be on the way but the authors feared the Wolbachia infection would be lost in the mail. Therefore, I've tried to replicate their inoculation here.

First, let me point out the methodological differences between what I did and what Uribe-Alvarez et al. published.

1) I isolated Wolbachia from JW18 cells, not from Aa23 cells - so I am working with wMel while they were likely working with wAlbB. This could potentially be a huge difference.

2) I did not have access to "expired human erythrocytes" but as the authors claimed ferric citrate and FBS did just as well in supplementing YPD, that is what I used.

3) I used the anti-FtsZ antibody in addition to anti-Wsp to identify replicating Wolbachia in addition to the stability of the Wsp protein.

4) I incubated my Wolbachia and Saccharomyces cocultures in broth over a period of 7 days, under elevated CO2, sampling the cocultures every few days to identify the fraction of Wolbachia that was active under three conditions: Yeast alone (Y), Wolbachia alone (W), and the coculture (B).

Figure 1. Wolbachia does not replicate in coculture with Saccharomyces cerevisiae. Three conditions (yeast alone (Y), Wolbachia alone (W), and both together (B)) were incubated at 25C, with elevated CO2, in YPD supplemented with ferric citrate and FBS as per Uribe-Alvarez et al., 2018. Control = source strain for wMel, JW18 cell lines.

Wolbachia is not actively replicating in any of these conditions as you can see from the anti-FtsZ blot. The wsp protein is membrane associated and based on these results, quite stable. In our lab we have seen wsp stick around for a month in media - not sure why it is protease resistant but the truth is that it does not make the best probe for live, dividing Wolbachia. I therefore would take the western blots in the paper with a big heaping spoonful of salt - wsp does not tell us Wolbachia are actively replicating.

But, you say, there is a lot of other evidence provided in the published paper, including qPCR of wsp and fluorescence in situ hybridization against the bacterial 16S. The images from the paper are indeed convincing - it really does look like Wolbachia is inside the yeast cells. However, in order to get the FISH probe in to the yeast, the authors would've had to make spheroplasts - something that is clearly not evident in their images - and not listed in their methods.  The authors also provide qPCR evidence of Wolbachia persistence -- but this could just as easily be extracellular DNA persisting.

You can see that I am not convinced. The claim that Wolbachia infects Saccharomyces is unbelievable and unlikely -- but that does not make it untrue. I do hope to get my Wolbachia infected yeast strain in the mail, as promised by the authors. At that point, we'll see if the infection is indeed "lost in the mail."