Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner

ABSTRACT The transcription factor TFEB drives the expression of lysosomal, autophagic, and immune-responsive genes in response to LPS and phagocytosis. Interestingly, compounds that promote TFEB activity enhance bactericidal activity, while intracellular pathogens like Mycobacterium and Salmonella repress TFEB. However, Salmonella enterica sv. Typhimurium (S. Typhimurium) was reported to actively stimulate TFEB, implying a benefit to Salmonella. To better understand the relationship between S. Typhimurium and TFEB, we assessed if S. Typhimurium regulated TFEB in macrophages in a manner dependent on infection conditions. We observed that macrophages that engulfed late-logarithmic grown Salmonella accumulated nuclear TFEB, comparable to macrophages that engulfed Escherichia coli. In contrast, stationary-phase S. Typhimurium infection of macrophages actively delayed TFEB nuclear mobilization. The delay in TFEB nuclear mobilization was not observed in macrophages that engulfed heat-killed stationary-phase Salmonella, or Salmonella lacking functional SPI-1 and SPI-2 type 3 secretion systems. S. Typhimurium mutated in the master virulence regulator phoP or the secreted effector genes sifA, and sopD also showed TFEB nuclear translocation. Interestingly, while E. coli survived better in tfeb −/− macrophages, S. Typhimurium growth was similar in wild-type and tfeb −/− macrophages. Moreover, Salmonella survival was not readily affected by its growth phase in wild-type or knockout macrophages, though in HeLa cells late-log Salmonella benefitted from the loss of TFEB. Priming macrophages with phagocytosis enhanced the killing of Salmonella in wild-type, but not in tfeb− /− macrophages. Collectively, S. Typhimurium orchestrate TFEB in a manner dependent on infection conditions, while disturbing this context-dependent control of TFEB may be detrimental to Salmonella survival. IMPORTANCE Activation of the host transcription factor TFEB helps mammalian cells adapt to stresses such as starvation and infection by upregulating lysosome, autophagy, and immuno-protective gene expression. Thus, TFEB is generally thought to protect host cells. However, it may also be that pathogenic bacteria like Salmonella orchestrate TFEB in a spatio-temporal manner to harness its functions to grow intracellularly. Indeed, the relationship between Salmonella and TFEB is controversial since some studies showed that Salmonella actively promotes TFEB, while others have observed that Salmonella degrades TFEB and that compounds that promote TFEB restrict bacterial growth. Our work provides a path to resolve these apparent discordant observations since we showed that stationary-grown Salmonella actively delays TFEB after infection, while late-log Salmonella is permissive of TFEB activation. Nevertheless, the exact function of this manipulation remains unclear, but conditions that erase the conditional control of TFEB by Salmonella may be detrimental to the microbe.

1.If there are differences between stationary and late-log bacteria, which genes are differentially induced or suppressed between the two growth phases of bacteria?2. Inactivation of the SPI-1 and SPI-2 T3SS of S. Typhimurium abrogated the inhibition of TFEB-nuclear translocation.The same effect was also seen when the master regulator, phoP, or the secreted effectors sopD2 or sifA were deleted.What are the mechanisms by which these effectors modulate the TFEB nuclear translocation?Although the findings are interesting, the paper suffers from the lack of a mechanism.3. Interestingly, IgG-primed phagocytosis, but not resting macrophages, restricted intracellular Salmonella, however, the mechanisms are vague.
Reviewer #2 (Comments for the Author):

Summary
The study by Inpanathan et al 'Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time dependent manner' attempts to understand the effect of bacterial growth conditions on TFEB activation.The authors have clearly shown that infection with stationary phase Salmonella exhibits a delay in TFEB activation.The study also highlights the importance of Salmonella virulence factors in causing this delay.While it is an interesting finding that the metabolic status of Salmonella modulates TFEB activation, the study lacks in exploring the downstream consequence of this delayed TFEB activation.As TFEB is considered as the master regulator of lysosomes and autophagy, this study provides further understanding of the regulation of this important transcription factor.Major comments 1.The major question that the authors have not addressed is 'What is the consequence of delayed TFEB activation'?The readouts for most of the key experiments are only microscopic analysis of TFEB and not taking into consideration the downstream effect of TFEB activation.a) What happens to the expression of lysosomal and autophagy target genes after TFEB activation in stationary phase grown E. coli and Salmonella? b) There is immediate TFEB translocation during infection with log phase Salmonella.Does this correspond to increased autophagy capture compared to stationary phase Salmonella? 2. It is surprising that authors do not see any difference in replication of Salmonella in TFEB -/-macrophages.Is it cell type specific?As macrophages have multiple other mechanisms (antibacterial peptides/ROS) to prevent bacterial infection.It is therefore essential for authors to check the essentiality of TFEB in epithelial cells where lysosome-mediated killing and autophagy are important defence mechanisms against bacteria.If possible, intestinal epithelial cells that are relevant for Salmonella infection could be used.3. The authors should discuss in detail the physiological relevance of this study during in vivo infection.Especially, how the growth conditions of bacteria play a role during in vivo Salmonella infection and replication.
Minor comments 1. Kindly revise the manuscript for typographical errors.For example, in figure 2, 'TFEB' is labelled as 'TFEG'.2. It will be easy to understand if graph legends are consistent.In certain graphs such as figure 1B

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The paper "Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner" by Inpanathan et al. showed that the effect of Salmonella on TFEB was affected by the bacterial growth conditions and time of infection, whereby stationary, but not late-log bacteria, delayed TFEB activation.Some data are interesting; however, the paper suffers from the lack of detailed mechanisms.Here are the major comments: 1.If there are differences between stationary and late-log bacteria, which genes are differentially induced or suppressed between the two growth phases of bacteria?
2. Inactivation of the SPI-1 and SPI-2 T3SS of S. Typhimurium abrogated the inhibition of TFEB-nuclear translocation.The same effect was also seen when the master regulator, phoP, or the secreted effectors sopD2 or sifA were deleted.What are the mechanisms by which these effectors modulate the TFEB nuclear translocation?Although the findings are interesting, the paper suffers from the lack of a mechanism.

Editorial comments
"I have received the reviews; they agree that this study addresses an important question in field and provides insight into the role of TFEB during Salmonella infection.While Spectrum does not necessarily require mechanism, the reviewers raise valid questions regarding the downstream impact on Salmonella when TFEB activation is delayed, as well as how your findings fit into the broader context of infection.Reviewer #2 also suggested revisions to the graphs to improve readability." Rebuttal: We would like to thank you and to thank our peers for the constructive feedback of the original work.We acknowledge that we do not define a mechanism by which Salmonella manipulates TFEB but agree that understanding the functional consequences of TFEB modulation by Salmonella is important.As enumerated below, we have assessed several measures of downstream impact by i) measuring the expression of genes previously connected to TFEB by qRT-PCR; ii) quantifying the effect on autophagy and xenophagy, and iii) extending studies of Salmonella survival and TFEB activation to a non-macrophage cell line.Details of these experiments and findings are summarized below.

Reviewer 1 comments
1.The paper "Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner" by Inpanathan et al. showed that the effect of Salmonella on TFEB was affected by the bacterial growth conditions and time of infection, whereby stationary, but not late-log bacteria, delayed TFEB activation.Some data are interesting; however, the paper suffers from the lack of detailed mechanisms.Here are the major comments: 1.If there are differences between stationary and late-log bacteria, which genes are differentially induced or suppressed between the two growth phases of bacteria?
To determine if stationary and late-log Salmonella infection differed in the expression of host genes, we measured the expression of LC3, LAMP1, and cathepsin D by qRT-PCR in infected cells over 2, 5 and 20 h post-infection.These are canonical lysosomal and autophagy genes that can be controlled by TFEB under at least some specific conditions.We also tested if any such changes caused by infection with Salmonella at different growth phases was dependent on TFEB/TFE3 by using wild-type, tfeb -/-(SKO), and tfeb - /-tfe3 -/-(DKO).For disclosure, we did all these conditions simultaneously at least three independent times, but because of the number of possible combinations, we re-analysed the same data in different ways to address specific questions and to make analysis manageable.
i) First, we didn't observe changes in basal level of these genes in resting wild-type, SKO, and DKO macrophages (Fig. 8A).
ii) Second, we then tracked expression of LAMP1, cathepsin D, and LC3 mRNAs in wild-type and mutant macrophages infected with late-log vs. stationary-grown Salmonella over 2, 5, and 20 h of infection.a. Within the parameters we tested, we didn't observe any differences in LAMP1 mRNA expression among any condition (Fig. 8B, C), suggesting that Salmonella infection and TFEB expression do not change LAMP1 mRNA levels within 20 h post-infection.b.For cathepsin D, the pattern was more complex.We observed a relative increase in cathepsin D mRNA in wild-type macrophages infected for 20 h with stationary or late-log Salmonella (Fig 8D).Thus, Salmonella infection seems to modulate cathepsin D expression.Interestingly, SKO macrophages did not exhibit an increase in cathepsin D mRNA after infection with either Salmonella condition, but we cannot conclude that TFEB is required since basal levels of mRNA appeared to be higher overall (though not statistically significant relative to wild-type; Fig. 8D).Moreover, when we compare mRNA levels at 20 h infection between wild-type, SKO, and DKO macrophages, we did not observe a difference (Fig. 8E).Thus, Salmonella infection seems to promote cathepsin D expression, but this may not depend on growth phase or TFEB/TFE3.c.We then assessed expression of LC3 mRNA.Here, there was no difference in mRNA expression in wild-type macrophages infected with Salmonella (Fig. 8F).However, late-log Salmonella infection of both SKO and DKO macrophages led to a statistically significant increase in LC3 mRNA levels after 20 h of infection.This was not observed in mutant macrophages infected with stationary-grown Salmonella (Fig. 8F, G).These data suggest that Salmonella growth phase can affect expression of LC3 mRNA in macrophages in a way that is promoted by the absence of TFEB and/or TFE3, not dependent on itwhich was not as expected.This again suggests a complex interplay that likely involves other factors.d.While we present these data, we also acknowledge that there are challenges in the assays we chose given the heterogeneity of infection conditions.We propose that single-cell assays that examine gene expression activity may be better suited to determine differences caused by Salmonella growth phase and TFEB/TFE3 instead of population-based methods like qRT-PCR.Given that we only examined three models, transcriptomics may also be suitable in the long-term.These statements can be found starting in line 450.

Inactivation of the SPI-1 and SPI-2 T3SS of S. Typhimurium abrogated the inhibition of TFEB-nuclear translocation.
The same effect was also seen when the master regulator, phoP, or the secreted effectors sopD2 or sifA were deleted.What are the mechanisms by which these effectors modulate the TFEB nuclear translocation?Although the findings are interesting, the paper suffers from the lack of a mechanism.
Thank you for the feedback.We agree that revealing the mechanism by which these effectors modulate TFEB would be interesting.However, we are currently constrained in our resources to further build on these observations.Thus, we believe that it is beneficial to disseminate these observational findings to encourage the community to resolve said mechanisms.Our main goal with this work was to disseminate that Salmonella manipulates TFEB in a context-dependent manner, which may guide future work by better defining key parameters.We do discuss possible mechanisms by how these effectors might modulate TFEB in the discussion, between lines 429-439.
We have previously demonstrated that phagocytosis of IgG-beads engages TFEB, increases lysosome genes in a TFEB-dependent manner, and boosted lysosomal and bactericidal activity (Gray et al., 2016).In that work, we provided evidence that phagosome-lysosome fusion triggered Ca 2+ released via the TPRML1 lysosomal channel to activate TFEB.We now explicitly discuss this starting in lines 334, 381, and 472.
Reviewer 2 comments

The major question that the authors have not addressed is 'What is the consequence of delayed TFEB activation'? The readouts for most of the key experiments are only microscopic analysis of TFEB and not taking into consideration the downstream effect of TFEB activation. a) What happens to the expression of lysosomal and autophagy target genes after TFEB activation in stationary phase grown E. coli and Salmonella?
b) There is immediate TFEB translocation during infection with log phase Salmonella.Does this correspond to increased autophagy capture compared to stationary phase Salmonella?
A) Thank you for raising this issue, which was also asked by Reviewer 1.As such, please see our response above under Reviewer 1, point 1.

The authors should discuss in detail the physiological relevance of this study during in vivo infection. Especially, how the growth conditions of bacteria play a role during in vivo Salmonella infection and replication.
We have commented on the physiological relevance of growth phase and Salmonella infections in vivo in our discussion highlighted (Lines 455-467).Overall, there is limited literature on the impact of Salmonella growth-phase on in vivo infection; some evidence suggests stationary-phase Salmonella delays animal mortality, is less susceptible to clearance early infection and has reduced colonization in peripheral organs.Some of these effects however, seem to be temporal in nature as the differences between growthphases disappear with time, as we observe for repression of TFEB nuclear entry.
Fixed.We chose to display p values for survival data (current Figure 11 and 12) because the magnitude of the p value may be informative to the reader in cases where the threshold was close to p<0.05, but not under.
November 1, 2023 1st Revision -Editorial Decision Re: Spectrum04981-22R1 (Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner) Dear Dr. Roberto J. Botelho: As you can see from the reviewer's comments, there are still concerns from reviewer 2 regarding the lack of mechanism and impact on downstream bacterial infection.While these are valid criticisms, after careful evaluation, I agree that these are followup studies and the current manuscript fulfills Spectrum's requirements of high quality work that will be useful for the community.
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Reviewer #1 (Comments for the Author): My concerns have been cleared.

Reviewer #2 (Comments for the Author):
As I said before, the authors have a potential story here but are not able to explain their results in the context of Salmonella infection.I am particularly concerned that the TFEB data is inconclusive, and the authors do not provide a satisfactory/alternative explanation or hypothesis.I am sharing below the information that may be useful to the authors: Concerns: 1) RT-PCR results: Lamp1, LC3, Cathepsin D: high variation and no conclusive results.
-D both colour and symbol (circle/square/triangle) are different whereas in other graphs like 2B-D only colour is different.3. Maintain consistency in representing significance in graphs.Only figure 8 has 'p' values mentioned.

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