Navigating contradictions: Salmonella Typhimurium chemotactic responses to conflicting effector stimuli
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eLife Assessment
In this manuscript, Franco and colleagues describe valuable findings about the chemotactic response of Salmonella to serine and indole, conflicting chemotactic signals. Although the evidence presented is solid, concerns were raised about the novelty of the chemotactic phenomena observed with these two compounds. Also, although the induction of invasion by feces is a novel and interesting finding, the lack of follow-up to this observation was also noted.
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Abstract
Chemotaxis controls motility and colonization in many enteric pathogens, yet most studies have examined bacterial responses to single effectors in isolation. Previously, we reported that Salmonella Typhimurium uses the chemoreceptor Tsr to detect L-serine (L-Ser) in human blood serum, promoting invasion of damaged vasculature ( Glenn et al., eLife 2024 1 ). Tsr also mediates sensing of indole, a microbiota-derived chemorepellent and bactericide proposed to protect against enteric infection by deterring pathogen colonization. The major biological reservoir of indole in the gut is feces, where it accumulates to millimolar levels. Here, we tested whether indole-rich human fecal material is protective against infection and found that exposure to feces instead enhances intestinal invasion in an explant model. Surprisingly, diverse non-typhoidal Salmonella serovars were strongly attracted to feces despite its high indole content. We found that while pure indole is a strong repellent sensed through Tsr, its effects are overridden in the presence of nutrient attractants, including L-Ser. Moreover, indole only minimally impairs growth in the presence of sufficient nutrients. Using video microscopy, we observed that Tsr integrates L-Ser and indole signals in real time, biasing bacterial movement based on the relative concentrations of attractant and repellent. We propose that this chemotactic compromise optimizes pathogen fitness by guiding bacteria to niches with a favorable L-Ser-to-indole ratio, balancing nutrient acquisition and avoidance of high microbial competitor density. These findings highlight the limitations of single-effector studies in predicting bacterial navigation in complex environments, where chemotaxis is shaped by the integration of multiple, often opposing, chemical cues.
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eLife Assessment
In this manuscript, Franco and colleagues describe valuable findings about the chemotactic response of Salmonella to serine and indole, conflicting chemotactic signals. Although the evidence presented is solid, concerns were raised about the novelty of the chemotactic phenomena observed with these two compounds. Also, although the induction of invasion by feces is a novel and interesting finding, the lack of follow-up to this observation was also noted.
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Reviewer #1 (Public review):
Summary:
The study shows, perhaps surprisingly, that human fecal homogenates enhance the invasiveness of Salmonella typhimurium into cells of a swine colonic explant. This effect is only seen with chemotactic cells that express the chemoreceptor Tsr. However, two molecules sensed by Tsr that are present at significant concentrations in the fecal homogenates, the repellent indole and the attractant serine, do not, either by themselves or together at the concentrations in which they are present in the fecal homogenates, show this same effect. The authors then go on to study the conflicting repellent response to indole and attractant response to serine in a number of different in vitro assays.
Strengths:
The demonstration that homogenates of human feces enhance the invasiveness of chemotactic Salmonella …
Reviewer #1 (Public review):
Summary:
The study shows, perhaps surprisingly, that human fecal homogenates enhance the invasiveness of Salmonella typhimurium into cells of a swine colonic explant. This effect is only seen with chemotactic cells that express the chemoreceptor Tsr. However, two molecules sensed by Tsr that are present at significant concentrations in the fecal homogenates, the repellent indole and the attractant serine, do not, either by themselves or together at the concentrations in which they are present in the fecal homogenates, show this same effect. The authors then go on to study the conflicting repellent response to indole and attractant response to serine in a number of different in vitro assays.
Strengths:
The demonstration that homogenates of human feces enhance the invasiveness of chemotactic Salmonella Typhimurium in a colonic explant is unexpected and interesting. The authors then go on to document the conflicting responses to the repellent indole and the attractant serine, both sensed by the Tsr chemoreceptor, as a function of their relative concentration and the spatial distribution of gradients.
Weaknesses:
The authors do not identify what is the critical compound or combination of compounds in the fecal homogenate that gives the reported response of increased invasiveness. They show it is not indole alone, serine alone, or both in combination that have this effect, although both are sensed by Tsr and both are present in the fecal homogenates. Some of the responses to conflicting stimuli by indole and serine in the in vitro experiments yield interesting results, but they do little to explain the initial interesting observation that fecal homogenates enhance invasiveness.
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Reviewer #2 (Public review):
Summary:
The manuscript presents experiments using an ex vivo colonic tissue assay, clearly showing that fecal material promotes Salmonella cell invasion into the tissue. It also shows that serine and indole can modulate the invasion, although their effects are much smaller. In addition, the authors characterized the direct chemotactic responses of these cells to serine and indole using a capillary assay, demonstrating repellent and attractant responses elicited by indole and serine, respectively, and that serine can dominate when both are present. These behaviors are generally consistent with those observed in E. coli, as well as with the observed effects on cell invasion.
Strengths:
The most compelling finding reported here is the strong influence of fecal material on cell invasion. Also, the local and …
Reviewer #2 (Public review):
Summary:
The manuscript presents experiments using an ex vivo colonic tissue assay, clearly showing that fecal material promotes Salmonella cell invasion into the tissue. It also shows that serine and indole can modulate the invasion, although their effects are much smaller. In addition, the authors characterized the direct chemotactic responses of these cells to serine and indole using a capillary assay, demonstrating repellent and attractant responses elicited by indole and serine, respectively, and that serine can dominate when both are present. These behaviors are generally consistent with those observed in E. coli, as well as with the observed effects on cell invasion.
Strengths:
The most compelling finding reported here is the strong influence of fecal material on cell invasion. Also, the local and time-resolved capillary assay provides a new perspective on the cell's responses.
Weaknesses:
The weakness is that indole and serine chemotaxis does not seem to control the fecal-mediated cell invasion and thus the underlying cause of this effect remains unclear.
In addition, the fact that serine alone, which clearly acts as a strong attractant, did not affect cell invasion (compared to buffer) is somewhat puzzling. Additionally, wild-type cells showed nearly a tenfold advantage even without any ligand (in buffer), suggesting that factors other than chemotaxis might control cell invasion in this assay, particularly in the serine and indole conditions. These observations should probably be discussed.
Final comment. As shown in reference 12, Tar mediates attractant responses to indole, which appear to be absent here (Figure 3J). Is it clear why? Could it be related to receptor expression?
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Reviewer #3 (Public review):
Summary:
In this manuscript, Franco and colleagues describe careful analyses of Salmonella chemotactic behavior in the presence of conflicting environmental stimuli. By doing so, the authors describe that this human pathogen integrates signals from a chemoattractant and a chemorepellent into an intermediate "chemohalation" phenotype.
Strengths:
The study was clearly well-designed and well-executed. The methods used are appropriate and powerful. The manuscript is very well written and the analyses are sound. This is an interesting area of research and this work is a positive contribution to the field.
Weaknesses:
Although the authors do a great job in discussing their data and the observed bacterial behavior through the lens of chemoattraction and chemorepulsion to serine and indole specifically, the …
Reviewer #3 (Public review):
Summary:
In this manuscript, Franco and colleagues describe careful analyses of Salmonella chemotactic behavior in the presence of conflicting environmental stimuli. By doing so, the authors describe that this human pathogen integrates signals from a chemoattractant and a chemorepellent into an intermediate "chemohalation" phenotype.
Strengths:
The study was clearly well-designed and well-executed. The methods used are appropriate and powerful. The manuscript is very well written and the analyses are sound. This is an interesting area of research and this work is a positive contribution to the field.
Weaknesses:
Although the authors do a great job in discussing their data and the observed bacterial behavior through the lens of chemoattraction and chemorepulsion to serine and indole specifically, the manuscript lacks, to some extent, a deeper discussion on how other effectors may play a role in this phenomenon. Specifically, many other compounds in the mammalian gut are known to exhibit bioactivity against Salmonella. This includes compounds with antibacterial activity, chemoattractants, chemorepellers, and chemical cues that control the expression of invasion genes. Therefore, authors should be careful when making conclusions regarding the effect of these 2 compounds on invasive behavior. It is important that the word invasion is used in the manuscript only in its strictest sense, the ability displayed by Salmonella to enter non-phagocytic host cells. With that in mind, authors should discuss how other signals that feed into the control of Salmonella invasion can be at play here.
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Author response:
We thank the reviewers of this manuscript for their thoughtful and detailed feedback, and agree that they bring up valid points. We also thank them for their suggestions on how to improve this study. We intend to revise this manuscript to help address these concerns and in the future will submit a revised version that will hopefully be improved in terms of the clarity of the text and rigor of the experimental findings.
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