Salmonella exploits host- and bacterial-derived β-alanine for replication inside host macrophages

Reviewer #1 (Public review):

Summary:

Ma, Yang et al. report a new investigation aimed at elucidating one of the key nutrients S. Typhimurium (STM) utilizes with the nutrient-poor intracellular niche within the macrophage, focusing on the amino acid beta-alanine. From these data, the authors report that beta-alanine plays an important role in mediating STM infection and virulence. The authors employ a multidisciplinary approach that includes some mouse studies and ultimately propose a mechanism by which panD, involved in B-Ala synthesis, mediates the regulation of zinc homeostasis in Salmonella. The impact of this work is questionable. There are already many studies reporting Salmonella-effector interactions, and while this adds to that knowledge it is not a significant advance over previous studies. While the authors are investigating an interesting question, the work has two important weaknesses; if addressed, the conclusions of this work and broader relevance to bacterial pathogenesis would be enhanced.

Strengths:

This reviewer appreciates the multidisciplinary nature of the work. The overall presentation of the figure graphics are clear and organized.

Weaknesses:

First, this study is very light on mechanistic investigations, even though a mechanism is proposed. Zinc homeostasis in cells, and roles in bacteria infections, are complex processes with many players. The authors have not thoroughly investigated the mechanisms underlying the roles of B-Ala and panD in impacting STM infection such that other factors cannot be ruled out. Defining the cellular content of Zn2+ STM in vivo would be one such route. With further mechanistic studies, the possibility cannot be ruled out that the authors have simply deleted two important genes and seen an infection defect - this may not relate directly to Zn2+ acquisition.

Second, the authors hint at their newly described mechanism/pathway being important for disease and possibly a target for therapeutics. This claim is not justified given that they have employed a single STM strain, which was isolated from chickens and is not even a clinical isolate. The authors could enhance the impact of their findings and relevance to human disease by demonstrating it occurs in human clinical isolates and possibly other serovars. Further, the use of mouse macrophage as a model, and mice, have limited translatability to human STM infections.

Reviewer #2 (Public review):

Summary:

Salmonella exploits host- and bacteria-derived β-alanine to efficiently replicate in host macrophages and cause systemic disease. β-alanine executes this by increasing the expression of zinc transporter genes and therefore the uptake of zinc by intracellular Salmonella.

Strengths:

The experiments designed are thorough and the claims made are directly related to the outcome of the experiments. No overreaching claims were made.

Weaknesses:

A little deeper insight was expected, particularly towards the mechanistic aspects. For example, zinc transport was found to be the cause of the b-alanine-mediated effect on Salmonella intracellular replication. It would have been very interesting to see which are the governing factors that may get activated or inhibited due to Zn accumulation that supports such intracellular replication.

Reviewer #3 (Public review):

Summary:

Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focussed on beta-alanine. It is also known from many other studies that Salmonella requires beta-alanine. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which are required for pathogenesis.

Strengths:

This study made a couple of knockouts in Salmonella and did a transcriptomic investigation to understand the global gene expression pattern.

Weaknesses:

The following questions are unanswered:

(1) It is not clear how the exogenous beta-alanine is taken up by macrophages.

(2) It is not clear how the Beta-alanine from the cytosol of the macrophage enters the SCV.

(3) It is not clear how the beta-alanine from SCV enters the bacterial cytosol.

(4) There is no clarity on the utilization of exogenous beta-alanine of the host and the de novo synthesis of beta-alanine by panD of Salmonella.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Ma, Yang et al. report a new investigation aimed at elucidating one of the key nutrients S. Typhimurium (STM) utilizes with the nutrient-poor intracellular niche within the macrophage, focusing on the amino acid beta-alanine. From these data, the authors report that beta-alanine plays an important role in mediating STM infection and virulence. The authors employ a multidisciplinary approach that includes some mouse studies and ultimately propose a mechanism by which panD, involved in B-Ala synthesis, mediates the regulation of zinc homeostasis in Salmonella. The impact of this work is questionable. There are already many studies reporting Salmonella-effector interactions, and while this adds to that knowledge it is not a significant advance over previous studies. While the authors are investigating an interesting question, the work has two important weaknesses; if addressed, the conclusions of this work and broader relevance to bacterial pathogenesis would be enhanced.

Strengths:

This reviewer appreciates the multidisciplinary nature of the work. The overall presentation of the figure graphics are clear and organized.

Weaknesses:

First, this study is very light on mechanistic investigations, even though a mechanism is proposed. Zinc homeostasis in cells, and roles in bacteria infections, are complex processes with many players. The authors have not thoroughly investigated the mechanisms underlying the roles of B-Ala and panD in impacting STM infection such that other factors cannot be ruled out. Defining the cellular content of Zn2+ STM in vivo would be one such route. With further mechanistic studies, the possibility cannot be ruled out that the authors have simply deleted two important genes and seen an infection defect - this may not relate directly to Zn2+ acquisition.

Thank you for your patient and thoughtful reading as well as the constructive comments and advice about our manuscript. We will revise the manuscript based on your comments and suggestions.

You are right that this work have not thoroughly investigated the mechanisms underlying the roles of β-Ala, panD and zinc in impacting Salmonella infection. We will perform additional experiments to detect the content of zinc during Salmonella infection in vivo and in vitro, according to your suggestions.

We agree that other unknown mechanism(s) are also involved in the virulence regulation by β-Ala in Salmonella, as our results showed that the double mutant ΔpanDΔznuA (cannot synthesis of β-Ala and uptake of zinc) is more attenuated than the single mutant ΔznuA (Figure 5D), suggesting that the contribution of β-Ala to the virulence of Salmonella is partially dependent on zinc acquisition_._ We will reword the related description throughout the manuscript for clarity.

Second, the authors hint at their newly described mechanism/pathway being important for disease and possibly a target for therapeutics. This claim is not justified given that they have employed a single STM strain, which was isolated from chickens and is not even a clinical isolate. The authors could enhance the impact of their findings and relevance to human disease by demonstrating it occurs in human clinical isolates and possibly other serovars. Further, the use of mouse macrophage as a model, and mice, have limited translatability to human STM infections.

We thank your comments and advice regarding our manuscript and are delighted to accept them.

You are right that our current findings are relatively limited and not sufficient for disease therapeutics. We will reword the related description throughout the manuscript. Based on this comment, we will also use Salmonella Typhi and human macrophages to perform additional experiments to extend our findings. Salmonella Typhi is a human-limited Salmonella serovar and the cause of typhoid fever, a severe lethal systemic disease. Salmonella Typhimurium (STM) cause systemic disease in mice, which is similar to the symptoms of typhoid fever in human and has been widely used to explore the pathogenesis of Salmonella.

Reviewer #2 (Public review):

Summary:

Salmonella exploits host- and bacteria-derived β-alanine to efficiently replicate in host macrophages and cause systemic disease. β-alanine executes this by increasing the expression of zinc transporter genes and therefore the uptake of zinc by intracellular Salmonella

Strengths:

The experiments designed are thorough and the claims made are directly related to the outcome of the experiments. No overreaching claims were made.

Weaknesses:

A little deeper insight was expected, particularly towards the mechanistic aspects. For example, zinc transport was found to be the cause of the b-alanine-mediated effect on Salmonella intracellular replication. It would have been very interesting to see which are the governing factors that may get activated or inhibited due to Zn accumulation that supports such intracellular replication.

We appreciate your review and advice. We will design and perform additional experiments to further investigate the mechanisms by which β-Ala, panD and zinc influence Salmonella infection, according to your suggestions. For example, we will detect the content of zinc during Salmonella infection in vivo and in vitro.

Reviewer #3 (Public review):

Summary:

Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focussed on beta-alanine. It is also known from many other studies that Salmonella requires beta-alanine. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which are required for pathogenesis.

Strengths:

This study made a couple of knockouts in Salmonella and did a transcriptomic investigation to understand the global gene expression pattern.

Weaknesses:

The following questions are unanswered:

(1) It is not clear how the exogenous beta-alanine is taken up by macrophages.

We thank the reviewer for the question. It is reported that β-alanine is delivered to eukaryotic cells through TauT (SLC6A6) and PAT1 (SLC36A1) transporters (Am J Physiol Cell Physiol. 2020 Apr 1;318(4):C777-C786; Br J Pharmacol 161: 589 –600, 2010; Biochim Biophys Acta 1194: 44 –52, 1994). We will add this information in the revised manuscript.

(2) It is not clear how the Beta-alanine from the cytosol of the macrophage enters the SCV.

Thank you for pointing it out. You are right that the above question is not clear. We will do our best to achieve this issue, via reviewing literature, designing and performing additional experiments.

(3) It is not clear how the beta-alanine from SCV enters the bacterial cytosol.

Thank you for the question. We have attempted to find the transporter of β-alanine in Salmonella, but we found that the CycA transporter transports β-alanine in Escherichia coli but not in Salmonella, despite Salmonella is the closely related species of E. coli.

According to your suggestion, we will perform additional experiments to verify whether BasC is involved in the transport of β-alanine into Salmonella cytosol.

(4) There is no clarity on the utilization of exogenous beta-alanine of the host and the de novo synthesis of beta-alanine by panD of Salmonella.

Thank you for the question. Our results showed that β-alanine concentrations were downregulated in the Salmonella-infected RAW264.7 cells, and the replication of Salmonella in RAW264.7 cells was significantly increased with the addition of β-alanine to the culture medium (RPMI) of RAW264.7 cells, implying that intracellular Salmonella use host-derived β-alanine for growth. Unfortunately, we have not found the transporter of exogenous β-alanine into Salmonella cytosol. We will perform additional experiments to verify whether BasC is involved in the transport of β-alanine into Salmonella cytosol, or search for other transporters that are responsible for the uptake of β-alanine into Salmonella.

Upon confirming the β-alanine transporter in Salmonella, we will compare the intracellular replication and virulence between WT and the transporter mutant strain, via cell and mice infection assays. If the replication ability and virulence of the mutant strain decreases relative to WT, suggesting that Salmonella uptakes the exogenous beta-alanine of the host to enhance intracellular replication and its virulence in mice.

We have found that the replication of Salmonella panD mutant in macrophages and the virulence in mice were significantly decreased relative to WT, suggesting that the de novo synthesis of β-alanine is important for Salmonella intracellular replication and virulence_._ To further confirm that both uptake of host-derived β-alanine and de novo synthesis of β-alanine are critical for the full virulence of Salmonella, we will generate the double mutant of panD and β-alanine transporter gene. If the replication ability and virulence of the double mutant decreases compared with each of the single mutant, suggesting that Salmonella both utilizes the exogenous beta-alanine of the host and de novo synthesis of β-alanine for full virulence.