Author response:
Public Reviews:
Reviewer #1 (Public review):
This a comprehensive study that sheds light on how Wag31 functions and localises in mycobacterial cells. A clear link to interactions with CL is shown using a combination of microscopy in combination with fusion fluorescent constructs, and lipid specific dyes. Furthermore, studies using mutant versions of Wag31 shed light on the functionalities of each domain in the protein. My concerns/suggestions for the manuscript are minor:
(1) Ln 130. A better clarification/discussion is required here. It is clear that both depletion and overexpression have an effect on levels of various lipids, but subsequent descriptions show that they affect different classes of lipids.
We thank the reviewer for the comments. We will improve Ln130 in the manuscript. The lipid classes that get impacted by the depletion of Wag31 vs overexpression are different. Wag31 is an adaptor protein that interacts with proteins of the ACCase complex (Meniche et al., 2014; Xu et al., 2014) that synthesize fatty acid precursors and regulate their activity (Habibi Arejan et al., 2022).
The varied response to lipid homeostasis could be attributed to a change in the stoichiometry of these interactions with Wag31. While Wag31 depletion would prevent such interactions from occurring and might affect lipid synthesis that directly depends on Wag31-protein partner interactions, its overexpression would lead to promiscuous interactions and a change in the stoichiometry of native interactions, ultimately modulating lipid synthesis pathways.
(2) The pulldown assays results are interesting, but links are tentative.
The interactome of Wag31 was identified through the immunoprecipitation of Flag-tagged Wag31 complemented at an integrative locus in Wag31 mutant background to avoid overexpression artifacts. We used Msm::gfp expressing an integrative copy (at L5 locus) of FLAG-GFP as a control to subtract non-specific interactions. The experiment was performed in biological triplicates, and interactors that appeared in all replicates were selected for further analysis. Although we identified more than 100 interactors of Wag31, we analyzed only the top 25 hits, with a PSM cut-off ≥18 and unique peptides≥5. Additionally, two of Wag31's established interactors, AccD5 and Rne, were among the top five hits, thus validating our data.
Though we agree that the interactions can either be direct or through a third partner, the fact that we obtained known interactors of Wag31 makes us believe these interactions are genuine. Moreover, we performed pulldown experiments for validation by mixing E. coli lysates expressing His-Wag31 full-length or truncated protein with M. smegmatis lysates expressing FLAG-tagged interacting proteins. The wash conditions used were quite stringent for these pull-down assays—the wash buffer contained 1% Triton X100, eliminating all non-specific and indirect interactions. However, we agree that we cannot conclusively state that the interactions are direct without purifying the proteins and performing the experiment. We will describe this caveat in the revised manuscript.
(3) The authors may perhaps like to rephrase claims of effects lipid homeostasis, as my understanding is that lipid localisation rather than catabolism/breakdown is affected.
In this manuscript, we are trying to convey that Wag31 is a spatiotemporal regulator of lipid metabolism. It is a peripheral protein that is hooked to the membrane via Cardiolipin and forms a scaffold at the poles, which helps localize several enzymes involved in lipid metabolism.
Homeostasis is the process by which an organism maintains a steady-state of balance and stability in response to changes. Depletion of Wag31 not only results in delocalisation of lipids in intracellular lipid inclusions but also leads to changes in the levels of various lipid classes. Advancement in the field of spatial biology underscores the importance of native localization of various biological molecules crucial for maintaining a steady-cell of the cell. Hence, we have used the word “homeostasis” to describe both the changes observed in lipid metabolism.
Reviewer #2 (Public review):
Summary
Kapoor et. al. investigated the role of the mycobacterial protein Wag31 in lipid and peptidoglycan synthesis and sought to delineate the role of the N- and C- terminal domains of Wag31. They demonstrated that modulating Wag31 levels influences lipid homeostasis in M. smegmatis and cardiolipin (CL) localisation in cells. Wag31 was found to preferentially bind CL-containing liposomes, and deleting the N-terminus of the protein significantly decreased this interaction. Novel interactions between Wag31 and proteins involved in lipid metabolism and cell wall synthesis were identified, suggesting that Wag31 recruits proteins to the intracellular membrane domain by direct interaction.
Strengths:
(1) The importance of Wag31 in maintaining lipid homeostasis is supported by several lines of evidence.
(2) The interaction between Wag31 and cardiolipin, and the role of the N-terminus in this interaction was convincingly demonstrated.
Weaknesses:
(1) MS experiments provide some evidence for novel protein-protein interactions. However, the pull-down experiments lack a valid negative control.
We thank the reviewer for the comments. We will include a valid negative control in the experiment. We would choose ~2 mycobacterial proteins that are not a part of our interactome study and perform a similar pull-down experiment with them and a positive control (known interactor of Wag31).
(2) The role of the N-terminus in the protein-protein interaction has not been ruled out.
Previously, we attempted to express the N-terminal (1-60 aa) and the C-terminal (60-212 aa) proteins in various mycobacterial shuttle vectors to perform MS/MS experiments. Despite numerous efforts, neither was expressed with the N/C-terminal FLAG tag nor without any tag in episomal or integrative vectors due to the instability of the protein. Eventually, we successfully expressed the C-terminal Wag31 with an N and C-terminal hexa-His tag. However, this expression was not sufficient or stable enough for us to perform Ni affinity pull-down experiments for mass spectrometry. The N-terminal of Wag31 could not be expressed in M. smegmatis even with N and C-terminal Hexa-His tags.
To rule out the role of the N-terminal in mediating protein-protein interactions, we plan to attempt to express N-terminal of Wag31with N and C-terminal hexa-His tag in E. coli. If this clone successfully expresses in E. coli, we will perform pull-down experiments as described in Figure 7.
Reviewer #3 (Public review):
Summary:
This manuscript describes the characterization of mycobacterial cytoskeleton protein Wag31, examining its role in orchestrating protein-lipid and protein-protein interactions essential for mycobacterial survival. The most significant finding is that Wag31, which directs polar elongation and maintains the intracellular membrane domain, was revealed to have membrane tethering capabilities.
Strengths:
The authors provided a detailed analysis of Wag31 domain architecture, revealing distinct functional roles: the N-terminal domain facilitates lipid binding and membrane tethering, while the C-terminal domain mediates protein-protein interactions. Overall, this study offers a robust and new understanding of Wag31 function.
Weaknesses:
The following major concerns should be addressed.
• Authors use 10-N-Nonyl-acridine orange (NAO) as a marker for cardiolipin localization. However, given that NAO is known to bind to various anionic phospholipids, how do the authors know that what they are seeing is specifically visualizing cardiolipin and not a different anionic phospholipid? For example, phosphatidylinositol is another abundant anionic phospholipid in mycobacterial plasma membrane.
We thank the reviewer for the comments. Despite its promiscuous binding to other anionic phospholipids, 10-N-Nonyl-acridine orange is widely used to stain Cardiolipin and determine its localisation in bacterial cells and mitochondria of eukaryotes (Garcia Fernandez et al., 2004; Mileykovskaya & Dowhan, 2000; Renner & Weibel, 2011). This is because it has a stronger affinity for Cardiolipin than other anionic phospholipids with the affinity constant being 2 × 106 M−1 for Cardiolipin association and 7 × 104 M−1 for that of phosphatidylserine and phosphatidylinositol association (Petit et al., 1992). Additionally, there is not yet another stain available for detecting Cardiolipin. Our protein-lipid binding assays suggest that Wag31 preferentially binds to Cardiolipin over other anionic phospholipids (Fig. 4b), hence it is likely that the majority of redistribution of NAO fluorescence that we observe might be contributed by Cardiolipin mislocalization due to altered Wag31 levels, with smaller degree of NAO redistribution intensity coming indirectly from other anionic phospholipids displaced from the membrane due to the loss of membrane integrity and cell shape changes due to Wag31.
• Authors' data show that the N-terminal region of Wag31 is important for membrane tethering. The authors' data also show that the N-terminal region is important for sustaining mycobacterial morphology. However, the authors' statement in Line 256 "These results highlight the importance of tethering for sustaining mycobacterial morphology and survival" requires additional proof. It remains possible that the N-terminal region has another unknown activity, and this yet-unknown activity rather than the membrane tethering activity drives the morphological maintenance. Similarly, the N-terminal region is important for lipid homeostasis, but the statement in Line 270, "the maintenance of lipid homeostasis by Wag31 is a consequence of its tethering activity" requires additional proof. The authors should tone down these overstatements or provide additional data to support their claims.
We agree with the reviewer that there exists a possibility for another function of the N-terminal that may contribute to sustaining mycobacterial physiology and survival. We would revise our statements in the paper to accurately reflect the data. Results shown suggest that the tethering activity of the N-terminal region may contribute to mycobacterial morphology and survival. However, additional functions of this region can’t be ruled out. Similarly, the maintenance of lipid homeostasis by Wag31 may be associated with its tethering activity, although other mechanisms could also contribute to this process.
• Authors suggest that Wag31 acts as a scaffold for the IMD (Fig. 8). However, Meniche et. al. has shown that MurG as well as GlfT2, two well-characterized IMD proteins, do not colocalize with Wag31 (DivIVA) (https://doi.org/10.1073/pnas.1402158111). IMD proteins are always slightly subpolar while Wag31 is located to the tip of the cell. Therefore, the authors' biochemical data cannot be easily reconciled with microscopic observations in the literature. This raises a question regarding the validity of protein-protein interaction shown in Figure 7. Since this pull-down assay was conducted by mixing E. coli lysate expressing Wag31 and Msm lysate expression Wag31 interactors like MurG, it is possible that the interactions are not direct. Authors should interpret their data more cautiously. If authors cannot provide additional data and sufficient justifications, they should avoid proposing a confusing model like Figure 8 that contradicts published observations.
In the literature, MurG and GlfT2 have been shown to have polar localization (Freeman et al., 2023; Hayashi et al., 2016; Kado et al., 2023), and two groups have shown slightly sub-polar localization of MurG (García-Heredia et al., 2021; Meniche et al., 2014). Additionally, (Freeman et al., 2023) they showed SepIVA to be a spatio-temporal regulator of MurG. MS/MS analysis of Wag31 immunoprecipitation data yielded both MurG and SepIVA to be interactors of Wag31 (Fig. 3). Given Wag31 also displays polar localisation, it likely associates with the polar MurG. However, since a sub-polar localization of MurG has also been reported, it is possible that they do not interact directly, and another protein mediates their interaction. We will modify the model proposed in Fig. 8 based on the above.
We agree that for validation of interaction, we performed pulldown experiments by mixing E. coli lysates expressing His-Wag31 full-length or truncated protein with M. smegmatis lysates expressing FLAG-tagged interacting proteins. The wash conditions used were quite stringent for these pull-down assays—the wash buffer containing 1% Triton X100, which eliminates all non-specific and indirect interactions. However, we agree that we cannot conclusively state that the interactions are direct without purifying the proteins and performing the experiment. We will describe this caveat in the revised manuscript and propose a model reflecting our results.
References:
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