Response to Dr. Newton

This is a response to a blog post about my paper written by Dr. Irene Newton:
Wolbachia the Holy Grail – Now go away or I will taunt you a second time
I will reprint her words in Red Quotations in order to address the issues she raises.

“Beckmann et al start out with a different premise from that of Le Page et al. in that John had already identified two candidates, wPa_0282 and wPa_0283, from another study, which focused on proteins found in Wolbachia-modified mosquito sperm. They state that these proteins must have been “secreted into germline cells by Wolbachia.” but to be honest, that is not clear from the data. Yes, the proteins may have gotten to the germline but we don’t yet know the mechanism.”

Specifically testing secretion via Type IV system is a waste of my time. Here is why:
1. We already know the proteins get out of Wolbachia because they were detected in a tissue where Wolbachia do not exist. How did it get there? I don’t really care. The mechanistic point is that the protein gets out of Wolbachia and into the sperm. It doesn’t matter to me how it gets there, though the most likely explanation IMO is secretion.

2. CidA binds CidB. Where you find one protein you will likely find the other. CidB is a deubiquitylating enzyme (DUB). The Ulp1 DUB domain is not an internal bacterial enzyme. They are eukaryotic modulators. Why does a Bacteria have a Eukaryotic enzyme? Bacteria do not have Ubiquitin Proteasome Systems. Bacterial DUBs are only found in pathogenic gram negative bacteria where the enzymes are secreted as host effectors. All bacterial DUBS identified thus far have already been shown to be secreted. This long list includes but is not limited to Ssel (Salmonella), SdeA (legionella), ElaD (E.coli), ChlaOTU and ChlaDUB1/2 (Chlamydia), XopD (Xanthomonas)… All these DUBs have already been shown to be secreted via Type III or IV secretion systems in the literature. Why bother testing another bacterial DUB for secretion? We already know it gets out of the cell.

3. In our manuscript we never explicitly say it is “secreted.” Here are the exact quotes a) “In a previous proteomic study we had therefore searched for Wolbachia (wPip strain) proteins associated with Wolbachia-modified mosquito sperm and identified the protein WPA0282”
b) “Wolbachia bacteria have a type IV secretion system that could translocate the CidA and CidB proteins into the host cytoplasm.” It is difficult for me to imagine why Dr. Newton has problems with these words.

“Beckmann et al take the stance that this orientation, and conservation of orientation, must mean these genes are co-transcribed, in an operon. I am not so sure that there is very strong evidence of these being in an operon – “that said, I am not sure that matters much here, except for pushing the idea that this is a toxin-antitoxin system.”

I am in complete agreement here. Whether it is an operon or not will not prove or disprove the TA hypothesis. It’s entirely semantic and of no functional consequence… The only way to disprove the TA hypothesis is to find the rescue factor (that’s your job if you don’t like the TA hypothesis). My job is to prove that A can rescue B in the fly and TA won’t be validated until that happens. Whoever gets their experiment to work wins the hypothesis battle. Go!

“Operons” should be assessed in a case by case basis. The case for the operon construction of CidA/BwPip and CinA/BwPip is made in the 2013 paper by reverse transcriptase amplifying a polycistronic mRNA molecule that spans the intergenic gap between both A and B proteins. Both “operons” are “operons” according to this data. How else does one prove operon functionality, and why is it so important to you?

“Based on the analysis of these three homologs the statement is made that: “As Wolbachia strains evolve within different host species, they accumulate mutations in their corresponding CI systems and become bidirectionally incompatible.” and also “The evidence to date is most consistent with a duplication and divergence from a common CinB-like ancestral operon, although the converse cannot be ruled out, namely, that an ancestral CidB-like operon picked up a nuclease domain and later lost the DUB domain.” These are testable hypotheses, sure, but certainly not shown by the reference cited nor really by the data provided in the manuscript, although in the Supplementary Discussion, they do describe some of the variants and homologous loci in other Wolbachia.”

Feel free to test these hypotheses now that I have identified and verified the CI genes. I will be testing them too! Nature Letters are extremely limited space formats. We can only include so much work.

“In this paper, they show that cognate cidA-cidB proteins interact in vitro and that cinB will not interact with CidA variants (wMel or wPip), but this is as far as the pattern goes with regards to this dataset. Again, it will be cool to explore this further with the different variants out there.”

Yup, already done. But do you realize how much cloning work is involved with making an A and B protein for every operon in existence from major model systems? Quite a bit, I know because I did it and have them. Hope to get data out soon.

“So, next comes the real meat of the paper, identifying DUB activity for the CidB protein. One thing that is awesome is that they have made catalytically inactive mutants by altering the residues known to be important for the DUBs and the nuclease. The beginning of this work is done in yeast, expressing the proteins and looking for growth defects (as we’ve done with Wolbachia effector WalE1). One thing I would’ve liked to have seen in their yeast work is a graphic of constructs. Are they really expressing these as an operon in yeast as well? That wasn’t super clear to me from the manuscript, but maybe I missed it.”

The co-expression of the genes occurs not as an operon but on two separate vectors. The A proteins are under Gal1 promoter in a 2-micron high copy Leu vector (pRS425gal1). B protein toxins are in a low copy CEN Ura vector pRS416gal1. I’m not sure what you gain by me drawing it out. These are standard well characterized yeast vectors with standard promoters and terminators.

“Also, co-expression of CidA, the upstream gene, rescued the phenotype to some extent (the yeast cells still look plenty unhappy and probably if you compared the CidB+CidA at 37C yeast to the Vector 37C yeast you’d see a significant growth defect).”

I am not exactly sure what you mean by this, but all comparisons are made to the far-right uninduced control panel in which all the plasmid genes are turned off in the presence of glucose at 37C. There you see absolutely no growth defect, so all toxicity and rescue are entirely due to the heterologous plasmid protein expression.

“They had a harder time identifying the exact substrate in yeast, as the profile for Ub-conjugated proteins upon CidB expression did not dramatically change.”
This is an incorrect understanding of what that supplemental figure 5c represents and means. Some DUBs are very promiscuous and will cleave all ubiquitylated substrates within a cell. Others are very specific and target individual substrates for ubiquitin removal. A promiscuous DUB expressed within a yeast cell will drastically reduce global ubiquitylation. Figure 5c shows that global ubiquitylation levels are unchanged upon expression of CidB. This means that the DUB is not promiscuous and has a very specific target, in a very specific spot, at a very specific time. Identifying the biochemical substrate was not the goal here.

“That said, I am sure they are taking advantage of the yeast deletion library to identify candidates for both synthetic rescue and lethality. Regardless, they do identify one substrate for CidB (UbVME), which they use in their in vitro assays (see below).”

The substrate is Ubiquitin. UbVME is just Ubiquitin chemically synthesized to the VME chemical group. There are two tiers of “substrate” in ubiquitin biochemistry. The first tier is the Ubiquitin-like moety: does the enzyme cleave Nedd8, SUMO, Ubiquitin, ISG-15, URM1, ect.. ? The second tier is what protein does the DUB cleave Ubiquitin off of? So what protein in the cell has ubiquitin tags attached that this protein removes. We are addressing these mechanistic questions.

“Riffing on the idea that this is a toxin-antitoxin system, they go on to try and explore if CidA inhibits CidB activity in vitro (see panel (a) above).” It does not. So, how do we explain that CidA expression seems to rescue CidB expression in yeast?

It doesn’t bother us that CidA doesn’t inhibit CidB DUB catalytic activity specifically and it actually makes sense with CI evolution and biology. Think about it this way: If, as we discussed above, the systems are evolved from an ancestral nuclease operon. That means whatever the ancestral rescue factor was, it was able to rescue effects of the nuclease. How then would that rescue factor change into a rescue factor for a vastly different chemical enzyme like a DUB? The only way it can make sense is if rescue happens in a way independent of enzyme activity. We propose the likely rescue methodology in the paper.

“Are these plasmids kept at the same copy number?”
Yes, 2-micron plasmids are maintained at about 60 copies per cell with the standard 2-micron origin. CEN plasmids are maintained at 1-3 copies per cell with the CEN origin.

“They suspect that perhaps CidA rescues CidB toxicity by changing localization of CidB. Why not look at localization in yeast? See if that changes with CidA expression?”
Yup, will publish that soon hopefully.
“Moving on to the final part of the study – showing that expression causes CI in flies. They go about making their constructs in a different way from Le Page et al. First, they codon optimize their constructs (this is not mentioned in Le Page et al so I am guessing they did not). Second, like the black night that never dies (“It’s just a flesh wound!”) the hypothesis that these loci are in an operon persists, so they do this interesting trick with T2A to make sure that these syntenic genes would be expressed and translated as two different proteins. Why the obsession with it being an operon.”
Are you really criticizing us for the key insight that made everything work? Why do you think LePage et al. thought to put them together… operon…? The T2a peptide is really cool whether you like the TA hypothesis or not. Cool science is cool science and good construct design is good construct design.

“Also, they do not show rescue, either with a Wolbachia infection (because they don’t have wPip in Drosophila melanogaster) or with cidA or cidA-cidB expression (which suggests that cidA is not really a rescue factor).”
We are cautious not to overinterpret negative data in systems that are extremely experimentally complex where no one has performed the appropriate controls to make conclusions based off the negative data. Reasons are provided in the paper.

“They show in supplementary figure 2 that E. coli Lon protease can cleave CidA, but Wolbachia Lon does not seem to. No matter, if we believe these are acting in the eukaryotic cell, then wouldn’t a eukaryotic protease be involved?”
The Lon stuff was just a way to improve recombinant protein expression without getting cleavage. We are not arguing that Wolbachia Lon cleaves A proteins or that Lon is relevant to the TA hypothesis and we argue against that in the manuscript. But we did test it just in case.

These points support the TA hypothesis:
A) the two proteins bind in cognate manners (our paper).
B) The operons exhibit basic TA functionality in yeast (our paper).
C) Genomics of colleagues at the wolb2016 meeting on a very unique genome supports TA operon hypothesis. *this point has been censored retroactively to protect unpublished results.*

If the TA hypothesis is wrong that means there is a yet unidentified rescue factor. A third gene. My question to you is why did comparative genomics from Me, Bordenstein, and Klasson all fail to find it? All comparative genomics points internally to the operon.

It is perfectly fine to doubt the TA hypothesis we are working to provide data to prove it. I hope you will be working equally as hard to find and prove rescue from another protein. The data will win in the end.