Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems

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   Sep 15, 2025

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   Manuscript number: RC-2025-03098

   Corresponding author: Pedro Escoll

   1. General Statements

   Our study investigates the interplay between the metabolism of host cells and the intracellular replication of Salmonella enterica serovar Typhimurium (ST). Type III Secretion Systems (T3SSs) are considered essential for ST to replicate within macrophages. However, we found that restricting macrophages to different bioenergetic contexts, such as supplementing them with glycerol, modulates bacterial replication and remarkably, enables a T3SS-deficient ST mutant (ΔprgH/ΔssaV) to replicate intracellularly. This T3SS-independent replication occurs within the Salmonell…

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   Manuscript number: RC-2025-03098

   Corresponding author: Pedro Escoll

   1. General Statements

   Our study investigates the interplay between the metabolism of host cells and the intracellular replication of Salmonella enterica serovar Typhimurium (ST). Type III Secretion Systems (T3SSs) are considered essential for ST to replicate within macrophages. However, we found that restricting macrophages to different bioenergetic contexts, such as supplementing them with glycerol, modulates bacterial replication and remarkably, enables a T3SS-deficient ST mutant (ΔprgH/ΔssaV) to replicate intracellularly. This T3SS-independent replication occurs within the Salmonella-containing vacuole (SCV) and is driven by the capacity of the host cell to provide these preferred nutrients, rather than by the host glycolytic activity itself.

   2. Description of the planned revisions

   __Reviewer #1 (Evidence, reproducibility and clarity): __

   Summary:

   In this manuscript, the authors investigate how host cell metabolic heterogeneity influences the intracellular replication of Salmonella enterica serovar Typhimurium. They use live-cell imaging of infected human primary macrophages to reveal that bacterial replication does not occur uniformly across infected cells. They demonstrate that supplementation with specific carbon sources-used by Salmonella during infection-promotes bacterial replication and increases the proportion of macrophages supporting intracellular growth. These effects are seen even in the absence of functional Type III Secretion Systems (T3SS), using a ΔprgH/ΔssaV double mutant. The authors further suggest that this replication enhancement is not strictly dependent on host glycolytic activity but rather on the host cell's ability to import nutrients. Their findings imply that intracellular Salmonella can exploit host cell metabolism to grow, even without its canonical virulence secretion systems, under nutrient-favorable conditions.

   Major Concern:

   While the topic is potentially interesting, the novelty is not fully clear. The concept that nutrient availability impacts intracellular Salmonella replication, largely via T3SS2 function, has been addressed previously (e.g., Liss et al., 2017). The finding that added exogenous carbon sources can enhance bacterial growth is thus not unexpected. The key claim-that Salmonella can replicate intracellularly even in the absence of T3SS function-would be significantly strengthened by demonstrating whether this is specific to Salmonella, or whether similar effects are seen with non-intracellular organisms such as E. coli K-12. If the phenomenon is unique to Salmonella, this would suggest a pathogen-specific mechanism beyond general metabolic support.

   As acknowledged by the Reviewer, the novelty and key claim of our work is that Salmonella can replicate intracellularly even in the absence of T3SS. To experimentally sustain that claim, we showed evidence that providing macrophages with the preferred carbon sources used by *Salmonella *during infection, such as glycerol, bypass the requirement of both T3SS by Salmonella to grow, intravacuolarly, inside macrophages.

   With respect to the article mentioned by the Reviewer (Liss et al. 2017, ref 36 in the manuscript), there are three important novel insights provided by our work:* i)* we show that Salmonella can replicate intracellularly in the SCV even in the absence of T3SS if certain carbon sources are provided;* ii) *we show the preference of Salmonella for certain carbon sources intracellularly such as glycerol and galactose (but not preferentially glucose); and iii) we have extended our observations to primary human macrophages in addition to RAW cells.

   We are not convinced that the experiment suggested by the Reviewer to use E. coli K12 (ECK12) is necessary to support our findings for Salmonella, but we propose to add the requested experiment. Briefly, we will infect hMDMs and RAW macrophages with ST-WT-GFP, ST-ΔprgHΔssaV or ECK12-WT-GFP, while culturing macrophages on different carbon sources (glucose, glycerol, galactose, fructose). Then we will monitor intracellular bacterial growth. By comparing bacterial growth of ST double mutant with ECK12-WT-GFP under favorable carbon sources such as glycerol, the results will be definitive to answer whether this phenomenon is unique to Salmonella or not.

   Specific Comments:

   Figure 1H: The effect shown here is not compelling due to inconsistent y-axis scaling. Panels 1B, 1C, and 1D should use a unified axis range with 1H to allow direct visual comparison of growth dynamics.

   Thank you, we will change it as suggested.

   Figures 1B, 1C, 1G, 1H: The current presentation of individual growth traces makes it difficult to appreciate the population-level trend. A smoothed average line overlaid on these plots could better represent the average dynamics of replicative vs. non-replicative infections. Or alternatively the total fraction of cells that proliferate summarized as a segmented bar plot (possibly binned per time point).

   We will plot the results as suggested, the total fraction of infected cells harboring bacteria that proliferate as a segmented bar plot, binned per time point.

   Figure 2G: This panel would benefit from including a comparable condition with the SPI-1/SPI-2 double mutant to aid interpretation. Additionally, the authors should explore whether this nutrient-supported replication is seen in non-phagocytic cells such as HeLa or Caco-2, which would help delineate whether the observed phenomenon is macrophage-specific.

   The graph asked by Reviewer is Figure S1D. As we are representing ST growth in macrophages supporting Salmonella replication, some of the conditions, such as lactate, cannot be shown in the infection conditions using the double mutant because there are no cells supporting the replication of the double mutant, so there are no cells to plot.

   As suggested, we are also going to perform the same experiments in HeLa cells to investigate whether the observed phenomenon is macrophage specific.

   Line 117: The sentence stating that the double mutant can undergo "exponential intracellular growth even in the absence of T3SS-dependent secretion" is an overstatement. The data suggest only a modest improvement in growth, restricted to a minority of infected cells. This claim should be revised accordingly, as should similar overstatements in the discussion (e.g., lines 203-204).

   We will remove the term 'exponential' and revise the sentence at line 117 and those in the discussion. Line 203-204 will be: 'we demonstrated that providing macrophages with preferred nutrients allows a subpopulation of ST to replicate intracellularly without the need for a functional T3SS'.

   Line 162: The authors should clarify that glycerol had the strongest effect in primary macrophages, while multiple alternative carbon sources had notable effects primarily in RAW cells.

   We will add this clarification in the text.

   Lines 198-201: This relates to the major concern. The authors should assess whether the observed growth enhancement is unique to Salmonella by testing other bacteria not known for intracellular replication. This would clarify whether the effect is due to general nutrient-driven host cell permissivity or a pathogen-specific adaptation.

   As outlined above, we will perform the suggested experiment with E. coli K12 to answer whether this phenomenon is unique to Salmonella or not.

   RAW 264.7 Observations: The modest intracellular growth of SPI-1/SPI-2 double mutants in RAW cells is consistent with prior observations in the field. The idea that nutrient availability explains this is noteworthy. The authors might consider whether differences in standard culture media (e.g., glucose concentration) influence these outcomes. This could have broader implications for reproducibility in infection models.

   Thank you for the suggestion, we will include a paragraph discussing whether differences in standard culture media might influence bacterial replication. Indeed, to answer also a question from Reviewer #2, we will include a new supplementary Figure where we have already compared "no Glucose" (0 mM), "low Glucose" (2 mM) and standard culture media Glucose levels (10 mM). Our results show that differences in Glucose levels in the culture media influence Salmonella intracellular growth in hMDMs and RAW macrophages (see Figure below).

   Reviewer #1 (Significance):

   This manuscript highlights how host cell metabolism and nutrient availability can influence intracellular Salmonella replication. While the findings are intriguing, the current framing overstates their novelty and impact. Key revisions-such as comparative experiments with non-pathogenic bacteria and non-phagocytic cells, consistent figure scaling, and more measured language-would improve the clarity and significance of the work. If the authors can show Salmonella-specific mechanisms at play, the study could offer important insights into host-pathogen metabolic interactions.

   We believe that performing all experiments suggested by the Reviewers, as well as the requested changes in the text to avoid overstatements, will improve the manuscript and will offer readers new insights and details to better understand the metabolic interactions happening between host and pathogens and how they can shape bacterial virulence.

   Reviewer #2 (Evidence, reproducibility and clarity):

   Summary: In their study titled "Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems", Dr. Garcia-Rodriguez et al. describe the influence of the host cell metabolism on the intracellular proliferation potential of Salmonella during infection. The authors investigate whether the supplementation of the media with different carbon sources has an impact on the intracellular lifestyle of Salmonella. By using single cell tracking in live-cell microscopy, including the use of different reporter strains, they describe that glycerol benefits Salmonella's ability to grow within its vacuolar niche, in part, interestingly, in a Type-3-Secretion System independent manner.

   They furthermore highlight the dependence on host background for this observation by showing that effects differ between cells of varying metabolic activity. Throughout their study, they use cutting-edge methodologies, as well as Salmonella strains that could be of versatile use in other investigations. This work, while limited to in vitro models for now, has implications for the better understanding of how pathogens and their host are intertwined. This, in turn, has significance for the development of new anti-infective strategies further down the line. I therefore believe that it should be disseminated to the research community. The following comments summarize ideas how the quality of the study could be improved:

   Major comments:

   Salmonella, especially when cultured to activate the SPI-1 T3SS, introduce rapid cell death in their host - most commonly through activation of the NLRC4 inflammasome and downstream pyroptotic signaling. The authors don't describe the effect of the infection in differently supplemented media on host cell death, yet it would be important to elucidate whether this cellular response is also altered.

   We have performed these experiments and tracked host cell death by measuring Annexin-V levels in single cells, during infection in the conditions using the different supplements. We will include these results in the revised version of the manuscript and main text. Please see the Figure below showing that the different carbon sources did not affect macrophages cell death significantly (future Figure S1E and S1F)

   The aspect of partially T3SS-independent growth enhancement by glycerol (and depending on the host background glucose) is most curious. The authors quantify this by determining the percentage of cells containing proliferating Salmonella and by tracking individual cells over the time course of the infection. I am missing a general statement on whether the initial infection rate (i.e. timepoint 0) is comparable across conditions and mutants, and whether possible discrepancies in the infection rate could have downstream effects on the statements and claims made in the manuscript. This is, to my mind, also important for the quantification of cytosolic and vacuolar bacteria. There, the authors always speak in "percent of infected cells", so it is relevant whether the number of infected cells varies among conditions (see e.g. Figure 3).

   We thank the reviewer for this comment. The initial infection rate at t=0 significantly differs between WT and mutants in RAW 264.7 macrophages, and carbon source supplementation has no effect. However, as we only analyze infected cells, this does not affect the final results. In any case, we are going to add the graphs of % of infected cells at t=0 as supplementary Figures S1G-K.

   The authors use a concentration of 10mM for all supplemented alternative carbon sources. It would be useful to discuss the rationale behind this approach, including whether all chemicals have the same ability to be taken up by the cell. A concentration series (at least for some of the tested compounds) may be beneficial to bolster the conclusions that the authors make.

   We use 10 mM as this is the concentration of Glucose in standard culture media. By using 10 mM for all the different carbon sources, we can thus compare them keeping concentration constant (10 mM). Indeed, to answer also Reviewer #1, we will include in the manuscript a paragraph discussing whether differences in standard culture media might influence bacterial replication. As this Reviewer suggested, we will include a new supplementary Figure comparing no Glucose (0 mM), low Glucose (2 mM) and standard culture media Glucose levels (10 mM), showing that the concentration of glucose has a gradual effect in supporting the replication of the T3SS-deficient strain in RAW macrophages (see Figure below).

   I think it would strengthen the study, if the authors used host cell mutants in certain metabolite transporters, or alternatively Salmonella mutants that are deficient in uptake or metabolism of some of the compounds used in this study. This point is alluded to in the discussion, and I believe if the authors could show that in certain host mutant backgrounds the impact of supplementation with alternative carbon sources can be reversed, it would immensely bolster the strength of the claims.

   Following Reviewer's suggestion, we generated ST metabolic mutants unable to metabolize glycerol, galactose or fructose. As seen in the Figures below, during infection, the supplementations with glycerol/galactose does not boost Salmonella replication in metabolic mutants as in WT conditions, demonstrating that supplemented carbon sources indeed arrive to bacteria within the SCV and are used by intracellular Salmonella to grow. This Figures will be now Future Figure 4J-N.

   I think it would be useful to include the meaning of this work for other intracellular pathogens in the discussion section: Do the authors believe that this phenotype is Salmonella-specific? If the pathogens are at hand, it might be interesting to infect with other intracellular bacteria, such as Shigella or Francisella to investigate if the boosting of growth by glycerol also holds true for these.

   We have performed experiments with Legionella pneumophila and galactose (see figure below), showing that this carbon source is specific of Salmonella (as shown in Figure 4F in the manuscript). We could perform experiments also with L. pneumophila and glycerol to answer the Reviewers question. However, we think that the results with Legionella might be out of the focus of this article and would constitute themselves a new article, as both pathogens have a very different, non-comparable intracellular metabolism. Thus, the experiment suggested by Reviewer #1 using E. coli K12 (ECK12) while culturing macrophages on different carbon sources (glucose, glycerol, galactose, fructose) is in our opinion a better fit. We will monitor intracellular bacterial growth and, by comparing bacterial growth of the ST-ΔprgH/ΔssaV double mutant with ECK12-WT-GFP under favorable carbon sources such as glycerol, the results will be definitive to answer whether this phenomenon is unique to Salmonella or not.

   Minor comments:

   • Line 41: The authors write "are required for", but given their findings, it might be more accurate to phrase this as "have previously been described to be required for" or "have previously been described essential for".

   We will change it.

   • Line 86: Is the referencing of Figure S1C correct or should it be S1A?

   Yes, thank you, it is S1A, we will change it.

   • Lines 119,120: Related to what is displayed in Figure 2G: Are these differences significant?

   Glucose, galactose and lactate curves are significantly different compared to control (p

   • Lines 126,127: What is the change for glycerol, and is the intracellular growth significantly higher compared to the control?

   6,2 {plus minus} 1.9% in glycerol vs. 2 {plus minus} 1% in control, p

   • Figure 1E&F: Related to one of the major comments: Would it be possible to quantify this at timepoint 0 to ensure that the initial infection rates are the same across conditions?

   As outlined above, we will add the graphs of % of infected cells at t=0 as supplementary Figures S1G-K (Major Comment number 2 from this Reviewer)

   • Figure 3E,F: Why does the sum of the curves not add up to 100% (especially in the beginning)? And related to that, why do both the percentage of cytosolic and vacuolar cells grow over time? Since this infection is performed with gentamycin present, re-infection should not be possible.

   The localization module of the SINA plasmid relies on transcriptional reporters, whose expression requires time for induction and detection. Therefore, at early time points, infected cells are not classified as vacuolar or cytoplasmic because the reporters have not yet been expressed (as described in PLoS Pathog. 2021;17(4):e1009550, PMID: 33930101).

   At later time points, a subset of cells harbors bacteria that do not express any of the reporters. These bacteria are considered dormant, representing about 10% of the population, as detailed in the same article. In addition, a small percentage of infected cells simultaneously contain both STvac and STcyt. Such cells are subclassified as harboring STcyt but also STvac. Consequently, the total proportion of infected cells carrying STvac and STcyt may also exceed 100%.

   • Figure S1A: While significance testing is described in the legend, there are no indications of significance in the figure panels.

   The Reviewer is right, there is no significant changes between conditions, we will change the significance testing to ns=non-significant.

   • Figure S1B: Due to the stark discrepancies between hMDMs and RAW264.7, it might make sense to plot them on two different y-axes. Furthermore, I would clarify the y-axis: In the legend, it seems as CFU counts are shown, while CFU/ml/t2 rather describes a change over time.

   We agree. However, we will maintain the scale of the Y-axis as it was required by Reviewer #1 to be consistent with Y-axis. We will change the legend to indicate that we plot CFU/ml/t2.

   • Figure S1C: The prgH-mutant seems to outperform the wildtype in intracellular proliferation, while the double mutant underperforms compared to the ssaV-mutant. Could you please discuss/explain how the prgH-deletion has seemingly opposite effects on intracellular proliferation, depending on whether it is introduced in a wildtype or ssaV-KO background?

   As T3SS-1 plays a role in inducing macrophage cell death via activation of the NLRC4 inflammasome, macrophages infected with bacteria carrying a functional T3SS-1 (such as WT), are more prone to undergo cell death at late time-points, which disrupts bacterial proliferation and reduces the proportion of infected cells. Thus, these dead cells were not considered in the analysis. Even if cell death of ST-WT-infected RAW macrophages remains below 5%, more ΔprgH-infected cells are considered in the analyses at late time-points, and ST-ΔprgH continue replicating (and growing in ST area).

   • Figure S2A: As for the comments related to Figure 3, I am unsure how the sum of STvac and STcyt can deviate from 100. This is especially puzzling for the red curve (glycerol) at e.g. 3hpi, when the sum of the two clearly seems to be larger than 100.

   At early time points, no infected cells are classified as vacuolar or cytoplasmic because the reporters have not yet been expressed. At later time points, a subset of cells harbor bacteria that do not express any of the reporters, which are considered dormant (10% of the population). Finally, a small percentage of infected cells simultaneously contain both STvac and STcyt, therefore the total proportion of infected cells carrying STvac and STcyt may also exceed 100%.

   **Cross-commenting** I agree in principle with the comments raised by Reviewer #1 - especially when it comes to the enhancement in significance if the authors assess the species specificity. Elucidating whether the growth enhancement is Salmonella-specific, occurs for other intracellular pathogens (e.g. Shigella, Francisella) or also for extracellular bacteria (e.g. E. coli, Yersinia), would definitely strengthen the study.

   As said before, for the revision we are going to perform the experiments suggested by Reviewer #1 of using E. coli K12 (ECK12) while culturing macrophages on different carbon sources (glucose, glycerol, galactose, fructose). And to satisfy this Reviewer's curiosity, we are going to perform experiments also with L. pneumophila and glycerol.

   Reviewer #2 (Significance):

   General assessment:

   As the authors write in their discussion, the strength of this study is also it's limitation: Using single cell tracking in microscopy is a very elegant and powerful approach, yet conversely, it limits the scope of the study to in vitro approaches. While it enables assessment of bacterial pathogenicity and host-dependence on a single-cell level, it remains to be investigated whether the conclusion that the authors draw from their work will hold in more complex or physiologically relevant models.

   During the preparation of this Revision Plan, we discovered the article published in PLoS Pathogens by Andrew Grant and Pietro Mastroni "Attenuated Salmonella Typhimurium Lacking the Pathogenicity Island-2 Type 3 Secretion System Grow to High Bacterial Numbers inside Phagocytes in Mice" (*PLoS Pathog *2012 8(12): e1003070, PMID: 23236281). In this article, authors showed that our main conclusion is also relevant in vivo (Salmonella Typhimurium can replicate within macrophages in the absence of T3SS). This will be addressed in the Discussion of the revised manuscript. Our study provides a metabolic explanation, at the single cell level for those observations.

   A further small shortcoming of the study is the heavy focus on the bacterial aspect in this host-pathogen interaction. While the authors do link the proliferative potential of the intracellular bacteria to the metabolic status of the individual host cell, more could be done with respect to host responses in the varying media compositions, including investigating alterations to the cell cycle, induction of cell death, or the ability to activate inflammatory signaling.

   We agree, and we are actively investigating how restricting macrophages to specific carbon sources impact other host responses, such as cytokine production. For the revised manuscript, we will add the results on the induction of cell death.

   Nonetheless, this study is of large interest to the field and the systematic approach to addressing their hypotheses speaks to the scientific excellence of the investigators.

   Thank you.

   3. Description of the revisions that have already been incorporated in the transferred manuscript

   N/A

   4. Description of analyses that authors prefer not to carry out

   N/A

   Read the original source
2. 

   Review Commons

   Sep 15, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   In their study titled "Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems", Dr. Garcia-Rodriguez et al. describe the influence of the host cell metabolism on the intracellular proliferation potential of Salmonella during infection. The authors investigate whether the supplementation of the media with different carbon sources has an impact on the intracellular lifestyle of Salmonella. By using single cell tracking in live-cell microscopy, including the use of different reporter strains, they describe that glycerol benefits Salmonella's …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   In their study titled "Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems", Dr. Garcia-Rodriguez et al. describe the influence of the host cell metabolism on the intracellular proliferation potential of Salmonella during infection. The authors investigate whether the supplementation of the media with different carbon sources has an impact on the intracellular lifestyle of Salmonella. By using single cell tracking in live-cell microscopy, including the use of different reporter strains, they describe that glycerol benefits Salmonella's ability to grow within its vacuolar niche, in part, interestingly, in a Type-3-Secretion System independent manner.

   They furthermore highlight the dependence on host background for this observation by showing that effects differ between cells of varying metabolic activity. Throughout their study, they use cutting-edge methodologies, as well as Salmonella strains that could be of versatile use in other investigations. This work, while limited to in vitro models for now, has implications for the better understanding of how pathogens and their host are intertwined. This, in turn, has significance for the development of new anti-infective strategies further down the line. I therefore believe that it should be disseminated to the research community. The following comments summarize ideas how the quality of the study could be improved:

   Major comments:

   1. Salmonella, especially when cultured to activate the SPI-1 T3SS, introduce rapid cell death in their host - most commonly through activation of the NLRC4 inflammasome and downstream pyroptotic signaling. The authors don't describe the effect of the infection in differently supplemented media on host cell death, yet it would be important to elucidate whether this cellular response is also altered.
   2. The aspect of partially T3SS-independent growth enhancement by glycerol (and depending on the host background glucose) is most curious. The authors quantify this by determining the percentage of cells containing proliferating Salmonella and by tracking individual cells over the time course of the infection. I am missing a general statement on whether the initial infection rate (i.e. timepoint 0) is comparable across conditions and mutants, and whether possible discrepancies in the infection rate could have downstream effects on the statements and claims made in the manuscript. This is, to my mind, also important for the quantification of cytosolic and vacuolar bacteria. There, the authors always speak in "percent of infected cells", so it is relevant whether the number of infected cells varies among conditions (see e.g. Figure 3).
   3. The authors use a concentration of 10mM for all supplemented alternative carbon sources. It would be useful to discuss the rationale behind this approach, including whether all chemicals have the same ability to be taken up by the cell. A concentration series (at least for some of the tested compounds) may be beneficial to bolster the conclusions that the authors make.
   4. I think it would strengthen the study, if the authors used host cell mutants in certain metabolite transporters, or alternatively Salmonella mutants that are deficient in uptake or metabolism of some of the compounds used in this study. This point is alluded to in the discussion, and I believe if the authors could show that in certain host mutant backgrounds the impact of supplementation with alternative carbon sources can be reversed, it would immensely bolster the strength of the claims.
   5. I think it would be useful to include the meaning of this work for other intracellular pathogens in the discussion section: Do the authors believe that this phenotype is Salmonella-specific? If the pathogens are at hand, it might be interesting to infect with other intracellular bacteria, such as Shigella or Francisella to investigate if the boosting of growth by glycerol also holds true for these.

   Minor comments:

   • Line 41: The authors write „are required for", but given their findings, it might be more accurate to phrase this as „have previously been described to be required for" or „have previously been described essential for".
   • Line 86: Is the referencing of Figure S1C correct or should it be S1A?
   • Lines 119,120: Related to what is displayed in Figure 2G: Are these differences significant?
   • Lines 126,127: What is the change for glycerol, and is the intracellular growth significantly higher compared to the control?
   • Figure 1E&F: Related to one of the major comments: Would it be possible to quantify this at timepoint 0 to ensure that the initial infection rates are the same across conditions?
   • Figure 3E,F: Why does the sum of the curves not add up to 100% (especially in the beginning)? And related to that, why do both the percentage of cytosolic and vacuolar cells grow over time? Since this infection is performed with gentamycin present, re-infection should not be possible.
   • Figure S1A: While significance testing is described in the legend, there are no indications of significance in the figure panels.
   • Figure S1B: Due to the stark discrepancies between hMDMs and RAW264.7, it might make sense to plot them on two different y-axes. Furthermore, I would clarify the y-axis: In the legend, it seems as CFU counts are shown, while CFU/ml/t2 rather describes a change over time.
   • Figure S1C: The prgH-mutant seems to outperform the wildtype in intracellular proliferation, while the double mutant underperforms compared to the ssaV-mutant. Could you please discuss / explain how the prgH-deletion has seemingly opposite effects on intracellular proliferation, depending on whether it is introduced in a wildtype or ssaV-KO background?
   • Figure S2A: As for the comments related to Figure 3, I am unsure how the sum of STvac and STcyt can deviate from 100. This is especially puzzling for the red curve (glycerol) at e.g. 3hpi, when the sum of the two clearly seems to be larger than 100.

   Cross-commenting

   I agree in principle with the comments raised by Reviewer #1 - especially when it comes to the enhancement in significance if the authors assess the species specificity. Elucidating whether the growth enhancement is Salmonella-specific, occurs for other intracellular pathogens (e.g. Shigella, Francisella) or also for extracellular bacteria (e.g. E. coli, Yersinia), would definitely strengthen the study.

   Significance

   General assessment:

   As the authors write in their discussion, the strength of this study is also it's limitation: Using single cell tracking in microscopy is a very elegant and powerful approach, yet conversely, it limits the scope of the study to in vitro approaches. While it enables assessment of bacterial pathogenicity and host-dependence on a single-cell level, it remains to be investigated whether the conclusion that the authors draw from their work will hold in more complex or physiologically relevant models.

   A further small shortcoming of the study is the heavy focus on the bacterial aspect in this host-pathogen interaction. While the authors do link the proliferative potential of the intracellular bacteria to the metabolic status of the individual host cell, more could be done with respect to host responses in the varying media compositions, including investigating alterations to the cell cycle, induction of cell death, or the ability to activate inflammatory signaling.

   Nonetheless, this study is of large interest to the field and the systematic approach to addressing their hypotheses speaks to the scientific excellence of the investigators.

   Advance:

   The advance this study makes is rather on the foundational than the applied side - which does not mean that conclusions drawn in this work are not of interest to a wider field. By investigating the intracellular lifestyle on a single-cell level, the authors were able to observe a striking and curious phenotype: that certain alternative carbon sources can enhance intracellular proliferation in a T3SS-independent manner. By further dissecting the reason for this observation, they create a stronger base for their conclusion in what can be described as an overall comprehensive study.

   Audience:

   As outlined in the description of the main advances, this study will be of largest interest to members of the basic research community in host-pathogen interactions. While the study so far focuses on Salmonella, a well-described and genetically accessible intracellular model pathogen, it could also be of interest to a broader community of researchers investigating bacterial pathogenicity, as well as those that are interested in the host metabolism.

   Describe your expertise:

   I have a background in bacterial pathogenicity in Salmonella infection, and have since expanded to other pathogens, as well as co-infections with viruses. In addition to investigating the pathogens, I have expertise in dissecting the host response, with a focus on innate immunity, inflammasome activation and host cell death. Overall, I am accustomed to unbiased screening approaches, which are followed by the formulation and assessment of hypotheses to unravel the molecular mechanisms underlying the host-pathogen interface.

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   In this manuscript, the authors investigate how host cell metabolic heterogeneity influences the intracellular replication of Salmonella enterica serovar Typhimurium. They use live-cell imaging of infected human primary macrophages to reveal that bacterial replication does not occur uniformly across infected cells. They demonstrate that supplementation with specific carbon sources-used by Salmonella during infection-promotes bacterial replication and increases the proportion of macrophages supporting intracellular growth. These effects are seen even in the absence of functional Type III Secretion Systems (T3SS), using a …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   In this manuscript, the authors investigate how host cell metabolic heterogeneity influences the intracellular replication of Salmonella enterica serovar Typhimurium. They use live-cell imaging of infected human primary macrophages to reveal that bacterial replication does not occur uniformly across infected cells. They demonstrate that supplementation with specific carbon sources-used by Salmonella during infection-promotes bacterial replication and increases the proportion of macrophages supporting intracellular growth. These effects are seen even in the absence of functional Type III Secretion Systems (T3SS), using a ΔprgH/ΔssaV double mutant. The authors further suggest that this replication enhancement is not strictly dependent on host glycolytic activity but rather on the host cell's ability to import nutrients. Their findings imply that intracellular Salmonella can exploit host cell metabolism to grow, even without its canonical virulence secretion systems, under nutrient-favorable conditions.

   Major Concern:

   While the topic is potentially interesting, the novelty is not fully clear. The concept that nutrient availability impacts intracellular Salmonella replication, largely via T3SS2 function, has been addressed previously (e.g., Liss et al., 2017). The finding that added exogenous carbon sources can enhance bacterial growth is thus not unexpected. The key claim-that Salmonella can replicate intracellularly even in the absence of T3SS function-would be significantly strengthened by demonstrating whether this is specific to Salmonella, or whether similar effects are seen with non-intracellular organisms such as E. coli K-12. If the phenomenon is unique to Salmonella, this would suggest a pathogen-specific mechanism beyond general metabolic support.

   Specific Comments:

   1. Figure 1H: The effect shown here is not compelling due to inconsistent y-axis scaling. Panels 1B, 1C, and 1D should use a unified axis range with 1H to allow direct visual comparison of growth dynamics.
   2. Figures 1B, 1C, 1G, 1H: The current presentation of individual growth traces makes it difficult to appreciate the population-level trend. A smoothed average line overlaid on these plots could better represent the average dynamics of replicative vs. non-replicative infections. Or alternatively the total fraction of cells that proliferate summarized as a segmented barplot (possibly binned per time point).
   3. Figure 2G: This panel would benefit from including a comparable condition with the SPI-1/SPI-2 double mutant to aid interpretation. Additionally, the authors should explore whether this nutrient-supported replication is seen in non-phagocytic cells such as HeLa or Caco-2, which would help delineate whether the observed phenomenon is macrophage-specific.
   4. Line 117: The sentence stating that the double mutant can undergo "exponential intracellular growth even in the absence of T3SS-dependent secretion" is an overstatement. The data suggest only a modest improvement in growth, restricted to a minority of infected cells. This claim should be revised accordingly, as should similar overstatements in the discussion (e.g., lines 203-204).
   5. Line 162: The authors should clarify that glycerol had the strongest effect in primary macrophages, while multiple alternative carbon sources had notable effects primarily in RAW cells.
   6. Lines 198-201: This relates to the major concern. The authors should assess whether the observed growth enhancement is unique to Salmonella by testing other bacteria not known for intracellular replication. This would clarify whether the effect is due to general nutrient-driven host cell permissivity or a pathogen-specific adaptation.
   7. RAW 264.7 Observations: The modest intracellular growth of SPI-1/SPI-2 double mutants in RAW cells is consistent with prior observations in the field. The idea that nutrient availability explains this is noteworthy. The authors might consider whether differences in standard culture media (e.g., glucose concentration) influence these outcomes. This could have broader implications for reproducibility in infection models.

   Significance

   This manuscript highlights how host cell metabolism and nutrient availability can influence intracellular Salmonella replication. While the findings are intriguing, the current framing overstates their novelty and impact. Key revisions-such as comparative experiments with non-pathogenic bacteria and non-phagocytic cells, consistent figure scaling, and more measured language-would improve the clarity and significance of the work. If the authors can show Salmonella-specific mechanisms at play, the study could offer important insights into host-pathogen metabolic interactions.

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4. 

   Version published to 10.1101/2025.05.15.653970 on bioRxiv

   May 15, 2025