PREreview of ISG15-modification of the Arp2/3 complex restricts pathogen spread

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

ISG15-modification of the Arp2/3 complex restricts pathogen spread Yifeng Zhang1, Brittany Ripley1, Wei Ouyang2,3, Miranda Sturtz1, Ellen Upton1, Emma Luhmann1, Madeleine Vessely1, Rocio Coloma4, Nathan Schwery1, Scott M. Anthony5, Adam 5, Thomas O. Moninger6, John T. Harty5, Aloysius Klingelhutz1, Emma Lundberg2,3,7, 5, Balaji Manicassamy1, Christopher Stipp8, Susana Guerra4, and Lilliana Radoshevich1* doi: https://doi.org/10.1101/2022.12.27.522022   We will adhere to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

SUMMARY: ISG15 is a Ubiquitin Like (UbL) protein that is linked to antimicrobial defense. Like all UbL proteins, ISG15 can be covalently linked to substrate proteins, a process known as ISGylation. ISGylation is intimately linked to interferon (IFN) responses; in fact, IFN responses drive transcriptional activation of the genes that encode the ISGylation machinery, including ISG15 itself. As ISG15 substrate proteins are identified, understanding of how ISGylation affects the fate of substrate proteins and relevance to antimicrobial defense must be addressed. Here, Zhang, Y., et al. build upon their previous comprehensive proteomics studies that identified ISGylated proteins in Listeria monocytogenes (Lm) infected animals (REF: Zhang Y., et al (2019) Nat. Commun. 10, 5383. Doi: 10.1038/s41467-019-13393-x). They employed a very clever approach that compared infected WT, ISG15 KO and USP18 C61A animals. In particular, the USP18 mutant animals displayed hyper-ISGylation due to the defective ISG15-specific USP18 deconjugase, which allowed the authors to distinguish between ubiquitin, ISG15 and the NEDD4 UbL modifications, all of which yield a di-glycine dipeptide on tryptic peptides during processing for mass spectrometry. Here, the authors report that hyper-ISGylation paradoxically increases Lm lethality in mice and increases pathogen spread in vivo and in vitro. They hypothesized that if hyper-ISGylation aids Lm cell to cell spread, which depends on the propulsive force of actin tails, then tail components may be substrates. They cross-referenced their list of candidate ISGylated proteins from Lm-infected mice with a list of host proteins known to be associated with Lm actin tails and discovered ISGylation of several proteins from the actin branching Arp2/3 complex, including Arp2 and Arp3 themselves. They demonstrated that ISG15 is present in Lm actin tails, and that hyper-ISGylation was associated with altered Arp3 localization on the Lm cell surface (rather than being restricted to the tail). Hyper-ISGylation was also associated with shorter tails and decreased Lm motility. Knowing that Arp3 is a substrate, they modelled ISGylation on 3 substrate lysines, which suggested the possibility of decreased Arp2/3 complex activity through steric hinderance. In the hyper-ISGylation cells, they noticed a phenomenon of groups of bacteria, which they called ‘bazookas’, invading neighboring cells. Careful time-lapse microscopy analysis suggested a failure of daughter bacteria to separate post-division in the hyper-ISGylation cells, which the authors conclude is the likely cause of the grouping phenomenon. They were able to link this phenomenon back to Arp2/3 complex ISGylation using USP18 C61A cells in which Arp3 was knocked out and complemented with a non-ISGylatable mutant Arp3 construct (with the 3 key lysines substituted); in these cells, hyper-ISGylation was no longer able to cause the bacterial segregation defect.

Finally, to link these studies to broader control of cell biology and development by ISGylation of Arp2/3 complex proteins, the authors determined that hyper-ISGylation causes the accumulation of thick cortical actin in fibroblasts, stronger attachment to a fibronectin matrix, slower cell migration, and general thickening of epithelial layers in mice.

OVERALL ASSESSMENT: Discovering new UbL modifications of substrate proteins and determining the effects of these modifications on protein fate are difficult tasks. In this manuscript, Zhang Y. et al., demonstrate that Arp2/3 complex proteins are ISGylated and that perturbations in ISGylation affect the actin tails that propel Lm. Conclusions are generally well supported by the data throughout, particularly with regard to the Lmmotility data. The authors employ advanced microscopy to demonstrate that increased global ISGylation causes defects in actin tails that support greater cell-to-cell spread of bacteria via a clustering effect. In particular, the role of Arp3 ISGylation in driving these defects is well supported through the study of mutant alleles of Arp3 that cannot be ISGylated. Overall, we considered this study to be an impressive contribution to the understanding of how UbLs can affect host-pathogen interactions. By contrast, we considered the final figure that focused on general effects of ISGylation on fibroblast actin cytoskeleton dynamics, cell shape, motility and epithelial barrier function, to be less well developed and somewhat disconnected from the strong host-pathogen interactions work that preceded it.

STRENGTHS: The novelty of this paper is a strength, and larger conclusions are supported by the data. The authors provide new mechanistic insight into how ISGylation affects host-pathogen interactions by modifying the Arp2/3 actin branching complex, which we considered a big advance considering our generally poor understanding of ISGylation to date. This also suggests that the proteomic dataset from their Nature Communications paper may contain more valuable ISGylated host proteins that could be the subject of future studies.

WEAKNESSES: We identified weaknesses that we would like to address:

1.     There was broad agreement that while the alterations in fibroblasts and epithelial barrier in the hyper-ISGylated state (Fig. 5) are fascinating, this aspect of the study is less well developed compared to the host-pathogen studies that preceded it. For example, while there were clearly changes in the fibroblast actin cytoskeleton, shape and motility that could be plausibly linked to ISGylation of the Arp2/3 complex and global defects in actin branching, only the motility phenotype was clearly linked to Arp2/3 complex, and this information was relegated to the extended data. Even if additional confirmatory experiments were performed to shore up this aspect of the manuscript, it would remain conceptually peripheral to the main take-home message. We generally agreed that the manuscript would be stronger without Figure 5.

2.     There were some statements in the manuscript that were not well supported and should be reconsidered. This includes the following:

a.     Title: “ISG15-modification of the Arp2/3 complex restricts pathogen spread” - this is confusing as the main message is that aberrant hyper-ISGylation of Arp2/3 results in actin tail alterations (shorter, thicker) and failed bacterial segregation post-division, which nevertheless increases spread due to the formation of bazookas. In this light, ‘restriction’ doesn’t seem like the right description of the change. It is certainly true that the ISG15 KO cells display an enhanced bacterial spread phenotype, but we didn’t think this was the main take-home message. Also, the use of ‘”pathogen” is meant to include VACV, but there was not very much virus infection data in the manuscript to support this broader scope of the title.

b.     The flow of information in the abstract does not match the order of figures. It doesn’t have to, but if the general effects of hyper-ISGylation on cortical actin and lamella need to be in the 3^rd sentence of the abstract, then maybe the data in Figure 5 belongs in the first figure.

c.      The final sentence of the abstract is an impact statement that goes beyond the scope of the study. “Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.”  The authors should consider revising this statement to keep focus on the main findings of the study, as the therapeutic options remain uncertain, and the main point of this compelling study is deepening understanding of host-pathogen interactions, not cancer.

3.     The experiments involving the Arp3 KO cells complemented with the non-ISGylatable mutant Arp3 allele provide essential mechanistic information linking phenotypes to the Arp2/3 complex and steering the reader away from potential indirect effects of ISGylation on cell physiology. As such, we think that the data in Extended Data Figure 4 should be retrieved and put in the main dataset.  

4.     We know from Extended Data 3b/c that the ActA-R148S mutant Lm bacteria that could not stably recruit Arp3 lost its enhanced cell-to-cell spread phenotype in hyper-ISGylation cells. This clearly indicates that ISGylation of Arp2/3 complex components AND their recruitment to bacteria is required for this phenotype. However, there is much more to learn from the ActA-R148S mutant Lm bacteria. For example, how do we expect these mutant bacteria to perform in vivo, in WT or USP18C61A/C61A mice? Do the mutant bacteria still replicate and spread efficiently in the liver, and does the hyper-ISGylated state have any effect?  

5.     We found the intravital microscopy data in Figure 1 challenging; there appear to be changes, but they are difficult for the non-expert to interpret properly. We think that retrieving the 2-photon microscopy images from the Extended Data could aid interpretation.

6.     Infectious doses, timings, and cell types for infection assays varied throughout the manuscript, but these changes were usually not explained or justified in the text (sometimes this information was found in the figure legends). For example, some imaging was performed at 6 hpi and some was performed at 24 hpi, and this was not clearly justified. We think that providing this kind of information in the text would enhance readability.

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.  Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2

● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.]

Figure 1:

·       The intravital microscopy in Figs.1b/1c were hard for non-experts to interpret. It might help the reader if the two-photon microscopy images were retrieved from the extended data and paired with the IVM images as these are much easier to interpret. Also, why was a different CFU dose used in Fig. 1a compared to Fig. 1b/c?

·       1F – is a heatmap the best way to present this data? Some readers are quite adept at interpreting heatmaps at a glance, but others could use some more exposition.

·       1j-1l: Why were A549 cells used for plaque assays but U2OS cells for immunofluorescence microscopy? Please justify the use of different cell types.

       Figure 2:

·       2I - Figure label says 12 hpi but figure legend says 24 hpi,

·       2K – This method of data presentation was viewed as unhelpful, as the plots on the right had individual data points that obscured the lines that aid interpretation. This turned into a discussion of the use of these plots throughout the manuscript. We think that these side-by-side plots could be replaced by single violin plots that provide the reader with better information at a glance.

Figure 5:

●     Here, the authors use isg15^+/+ usp18^+/+, usp18^C61A/C61A, and isg15^-/- interchangeably with WT, USP C61A, and ISG15 KO. We think that harmonizing labels throughout would help the reader. Furthermore, the superscripts in the former terms are difficult to read in the PDF even with high magnification.

●     Are specific analyses performed using methods that are consistent with answering the specific question?

·        Generally, this was viewed as acceptable throughout.

●     Is there appropriate technical expertise in the collection and analysis of data presented?

·        Generally, this was viewed as acceptable throughout. 

●     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

●      Most noticeably in Figures 2 f-h, the authors compare means with bar plots. Given that in these panels they also provide the data points overlaid with the bars, we think they mean to draw attention to the distribution of the data as well as the differences between test groups. These plots would instead be more succinctly presented as violin plots. The bars, on the other hand, should not be used for mean comparison, as they do not convey the distribution of the data as well as the mean. As detailed by Chenxin Li in this GitHub entry, there are other, better options. We think that instead of two graphs per panel, a single violin plot or at minimum a box plot is a better representation of this data.   

●      There are other instances of these plots (like Fig 1E) with data overlaying the bars. We think this one could also become a violin plot to neatly display the comparison. For some other plots like in Fig 4E the bars are not legible under the data points. This panel would benefit from being just bars with SEM or CI bars. Perhaps if the authors wish to show each individual data point, they may consider supplementing this piece with FigShare or another figure data repository.

●     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

●      OK

2. Quality: Completeness (1–3 scale) SCORE = 2

●     Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

●     Title: “ISG15-modification of the Arp2/3 complex restricts pathogen spread” - this is confusing as the main message is that aberrant hyper-ISGylation of Arp2/3 results in actin tail alterations (shorter, thicker) and failed bacterial segregation post-division, which nevertheless increases spread due to the formation of bazookas. In this light, ‘restriction’ doesn’t seem like the right description of the change. It is certainly true that the ISG15 KO cells display an enhanced bacterial spread phenotype, but we didn’t think this was the main take-home message.

●     Conclusions could have been better supported by making better use of the ActA Lm strain that could not stably recruit Arp3. How does this strain perform in vivo in WT or hyper-ISGylation mice, in terms of spread and invasion in the liver? Similarly, the mutant Arp3 construct that cannot be ISGylated could have been better leveraged to confirm and extend abstract-level conclusions.    

●     We discussed the following statement: “More importantly, our data indicate that ISGylation of mediators of actin-based motility acts a critical host defense strategy for both bacterial and viral pathogens, when properly regulated”, This statement needs more support, as there are only Lm and VACV included in the paper, the VACV studies were underdeveloped, and the definition of “properly regulated ISGylation” was not well described.

●     Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

●     We did not identify a fatal flaw in the study, but we were most concerned about the fibroblast/epithelial layer studies presented in Figure 5. For example, while there were clearly changes in the fibroblast actin cytoskeleton, shape and motility that could be plausibly linked to ISGylation of the Arp2/3 complex and global defects in actin branching, only the motility phenotype was clearly linked to Arp2/3 complex, and this information was relegated to the extended data.

3. Quality: Reproducibility (1–3 scale) SCORE = 1.5

●     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

●      Fig2 – there was some discussion about whether more biological replicates for Figs2A-C were required to help understand the high variation in Arp3 localization on Lm in the hyper-ISGylation cells. There was some concern about the statistical tests performed that yielded strong statistical significance to remarkably similar values for tail thickness in Fig. 2G.

●      As mentioned above, in several instances the reader would be better served by data presented as violin plots rather than combo box/scatter plots with different Y-axis scales.

●     Is there sufficient raw data presented to assess rigor of the analysis?

●      We viewed this as generally quite good throughout the study. We appreciated that the authors uploaded code to github, and raw data for re-analysis to imjoy.io. However, their link points to the code editor, not to the github repository directly; we suggest changing it to https://github.com/radoshevichlab/YZhang

●      We also appreciated that, although they don't upload all their data, they pledge to upload to Zenodo (this will also be the home for our preprint peer review document).

●     Are methods for experimentation and analysis adequately outlined to permit reproducibility?

●      The methods were generally clear, although, as mentioned above, we think that some rationale for variation in MOI, sampling time, etc. would help the reader. Also, there was one error with regard to Figs 5C/D, in which the description of the experiment in the main text and the figure legend do not agree. Is this a trypsin-resistance assay or a PBS/EDTA(Versene)-resistance assay? The interpretation is the same, but obviously it must be clearly described for the reader for the sake of reproducibility.

●     If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?

●      N/A

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 2

●     Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

●     OK

●     Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

●     OK

●     Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant basis for decisions.

·     There were some statements in the manuscript that were not well supported and should be reconsidered. This includes the following:

§  Title: “ISG15-modification of the Arp2/3 complex restricts pathogen spread” - this is confusing as the main message is that aberrant hyper-ISGylation of Arp2/3 results in actin tail alterations (shorter, thicker) and failed bacterial segregation post-division, which nevertheless increases spread due to the formation of bazookas. In this light, ‘restriction’ doesn’t seem like the right description of the change. It is certainly true that the ISG15 KO cells display an enhanced bacterial spread phenotype, but we didn’t think this was the main take-home message. Also, the use of ‘”pathogen” is meant to include VACV, but there was not very much virus infection data in the manuscript to support this broader scope of the title.

§  The flow of information in the abstract does not match the order of figures. It doesn’t have to, but if the general effects of hyper-ISGylation on cortical actin and lamella need to be in the 3^rd sentence of the abstract, then maybe the data in Figure 5 belongs in the first figure.

§  The final sentence of the abstract is an impact statement that goes beyond the scope of the study. “Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.”  The authors should consider revising this statement to keep focus on the main findings of the study, as the therapeutic options remain uncertain, and the main point of this compelling study is deepening understanding of host-pathogen interactions, not cancer.

·     This is perhaps a small point, but we discussed whether ‘bazooka’ is actually a good description for the ISGylation-mediated bacterial clustering and spread phenomenon, since bazookas propel a single missile at a target. Maybe we need a different term to describe the phenomenon. Some rockets have multiple modules/boosters that cluster together, which provides imagery closer to the true phenomenon. Cluster bombs? We also discussed the parallels of this idea to the way that enveloped viruses (or non-enveloped viruses wrapped in host membranes) can sometimes operate as a cluster.

·     In this paper, the ISGylation in CA cells was described as “unchecked ISGylation”, “enhanced ISGylation”, and “dysregulated ISGylation”. Could these descriptors be harmonized throughout for clarity?

·     We discussed the following statement: “More importantly, our data indicate that ISGylation of mediators of actin-based motility acts a critical host defense strategy for both bacterial and viral pathogens, when properly regulated”, This statement needs more support, as there are only Lm and VACV included in the paper, the VACV studies were underdeveloped, and the definition of “properly regulated ISGylation” was not well described.

·     The github repository should have requirements.txt file that specifies versions of modules used.

·     Some typos: Figure2I, on the panel it says 12hpi but says 24hpi in the legend. There is no figure 1k on the panel.

MORE SUBJECTIVE CRITERIA (IMPACT)

1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.5

A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.]

●     How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

●      The novelty of this paper is a strength, and larger conclusions are supported by the data. The authors provide new mechanistic insight into how ISGylation affects host-pathogen interactions by modifying the Arp2/3 actin branching complex, which we considered a big advance considering our generally poor understanding of ISGylation to date. We think that keeping focus on the bacterial infection aspect of the study while helping the reader understand the extensive knowledge gaps in the UbL/ISG15 field, will maximize impact.

Impact: Extensibility (1–4 or N/A scale) SCORE = 2.5

●     Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

●     This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

●     Yes, it has been extended to viral (VACV) infection and there is also some data about ISG15-mediated regulation of epithelial barriers and fibroblast function in vivo. However, we worried that these aspects of the study were underdeveloped and detracted from the robust main focus.

Competing interests

The author declares that they have no competing interests.