p62 limits Salmonella Typhimurium in macrophages through its role in cell signalling
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The intracellular autophagy receptor p62 (also known as Sequestosome-1) plays a dual role in autophagic flux and downstream Toll-like receptor signalling and has been implicated in modulating immune responses. However, its specific function in controlling intracellular bacterial survival, particularly in macrophages, remains less well characterized. Salmonella enterica serovar Typhimurium ( S . Tm) is a major global pathogen and a leading cause of gastroenteritis-associated morbidity. We have previously demonstrated that host restriction of S . Tm in macrophages involves the GTPase Rab32 and the BLOC-3 complex. In the present study, we identify a novel interaction between p62 and Rab32. Notably, p62 restricts Salmonella survival independently of the Rab32/BLOC-3 pathway. Indeed, p62-knockdown in macrophages resulted in significantly increased intracellular bacterial survival, an effect that did not correlate with altered recruitment of canonical autophagy-related proteins, as assessed by fluorescence microscopy. Through real-time polymerase chain reaction (RT-qPCR) and infection assays, we further show that p62-depleted macrophages exhibit a dampened pro-inflammatory response, which corresponds with the increased bacterial burden. These findings provide new mechanistic insight into the role of p62 in modulating the macrophage inflammatory response during Salmonella infection, highlighting its contribution to host defence beyond its canonical functions in autophagy.
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The work presented is clear and the arguments well formed. This is a study that would be of interest to the field and community.
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Comments to Author
Summary: Underwood et al demonstrate the importance of the autophagy adaptor p62 in restricting intracellular Salmonella during in vitro infection models, and provide evidence to suggest this p62-mediated restriction occurs in a manner that is distinct from the canonical role of p62. While an initial link between p62 and the host GTPase Rab32 is made, the authors describe how restriction of Salmonella by p62 seems to occur independently of the established Rab32/BLOC-3 pathway. Notably, the authors describe how p62 may contribute to the host inflammatory response that cause restriction of intracellular bacteria, which provides new insights into non-canonical roles of p62. Overall, the claims of the manuscript seem well-supported by the data, which includes a range of experimental techniques. The …
Comments to Author
Summary: Underwood et al demonstrate the importance of the autophagy adaptor p62 in restricting intracellular Salmonella during in vitro infection models, and provide evidence to suggest this p62-mediated restriction occurs in a manner that is distinct from the canonical role of p62. While an initial link between p62 and the host GTPase Rab32 is made, the authors describe how restriction of Salmonella by p62 seems to occur independently of the established Rab32/BLOC-3 pathway. Notably, the authors describe how p62 may contribute to the host inflammatory response that cause restriction of intracellular bacteria, which provides new insights into non-canonical roles of p62. Overall, the claims of the manuscript seem well-supported by the data, which includes a range of experimental techniques. The Discussion in particular is a balanced and thoughtful perspective on the possible biological relevance of these findings. I have only minor suggestions below that might improve readability and accessibility. Minor comments: 1) Throughout the manuscript, the authors refer to S.Tm ΔgtgEΔsopD2 (or SB2527) as S.Tm ΔΔ. While I appreciate this is done for the sake of brevity, I wonder if simply referring to this strain as S.Tm ΔgtgEΔsopD2 might improve readability, and bring the manuscript in line with other publications that use this nomenclature (e.g. PMID: 26867180). The abbreviated version can also cause confusion when the authors later describe the '2−ΔΔCT Method' for RT-qPCR analysis. 2) Line 50: it might be useful to specify that the authors refer to the SPI-2 T3SS. This is already implied due to intravacuolar nature of these bacteria, but the reader may not understand whether this refers to the SPI-1 or SPI-2 T3SS. 3) Methods: a small editing concern - in some sentences a degrees symbol is correctly used (e.g. 4°C), whereas in other sentences another circle symbol is used (particularly in the description of RT-qPCR methods). 4) Fig 1E: The dark red circles seem to describe 'iBMDM Scrm S.Tm WT' while the pink triangles describe 'iBMDM WT S.Tm ΔΔ' - should the pink triangles not be labelled 'iBMDM Scrm S.Tm ΔΔ'? My reading is that these groups serve as controls to the corresponding p62 KD infected cells, though perhaps I have misread.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
In this study, Underwood and colleagues investigate the potential interaction between p62 and Rab32 and their role in restricting Salmonella pathogenesis. The authors showed that, although p62 and Rab32 can be co-immunoprecipitated, they are rarely observed together at the SCV (approximately 3% of SCVs), suggesting that they interact at distinct intracellular sites, as they discuss. Furthermore, p62 localizes to a subpopulation of intracellular Salmonellae and restricts bacterial proliferation independently of the effectors SopD2 and GtgE, indicating that p62 acts independently of Rab32. Silencing p62 did not affect LC3 or Rubicon recruitment to the SCV, suggesting that p62 is not required for the recruitment of these autophagy-related proteins in macrophages. Finally, p62 silencing in macrophages …
Comments to Author
In this study, Underwood and colleagues investigate the potential interaction between p62 and Rab32 and their role in restricting Salmonella pathogenesis. The authors showed that, although p62 and Rab32 can be co-immunoprecipitated, they are rarely observed together at the SCV (approximately 3% of SCVs), suggesting that they interact at distinct intracellular sites, as they discuss. Furthermore, p62 localizes to a subpopulation of intracellular Salmonellae and restricts bacterial proliferation independently of the effectors SopD2 and GtgE, indicating that p62 acts independently of Rab32. Silencing p62 did not affect LC3 or Rubicon recruitment to the SCV, suggesting that p62 is not required for the recruitment of these autophagy-related proteins in macrophages. Finally, p62 silencing in macrophages reduced the pro-inflammatory response. These findings bring novel insights on alternative involvement of p62 in host defense mechanisms. Overall, the text and figures are clearly presented, the study is logically developed, and the results are appropriately discussed. Nevertheless, the following points should be addressed to improve the manuscript: 1. Fig1A - Please specify the number of replicates and provide quantification. 2. Lines 235-236 - Add the reference corresponding to "as shown previously." 3. Lines 239-240 - Remove the statement "suggesting a possible interaction on the SCV," as the data do not provide strong evidence for this hypothesis. 4. Fig1B - Please add a scale bar and outline the cell shape, particularly for cells where the cytosolic signal is not detectable. 5. Silencing - The authors state that KD levels were determined by WB and qPCR. Given the number of negative data associated with p62 KD, the validation of the KD should be shown. 6. Fig2A - The lack of significance may reflect excessive data dispersion rather than biological independence. Could the authors perform a paired t-test to account for experimental variation? 7. Section title "2" - The data indicate that the recruitment of autophagy-related proteins to the SCV is independent of p62, rather than the reverse. 8. Fig3A-B, caption, and lines 282-286 - There are inconsistency between text, figure and caption with either the x-axis or the timing (5 hpi and 24 hpi). Please review and correct for consistency. It is currently difficult to interpret these data. 9. Lines 297-301: "Supernatants from infected wild-type macrophages significantly enhanced bacterial killing in both wild-type and p62-depleted cells, compared to control media from uninfected cells. In contrast, supernatants from infected p62-depleted macrophages failed to promote bacterial clearance." These statements are not supported by the data presented in Fig 3D. Please include the appropriate statistical tests (matching the comparisons described in the text) and revise the statements accordingly. 10. Fig3E-F, please label the conditions at the top of the WB.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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