Loss of Fsr quorum sensing promotes biofilm formation and worsens outcomes in enterococcal infective endocarditis

Domain: biology
Published: 2026-01-14
Editorial outcome: minor_revision

Abstract
Infective endocarditis (IE) is a severe heart infection caused predominantly by Gram-positive bacteria forming biofilm on heart valves. While biofilm formation is central to disease progression, the underlying bacterial mechanisms remain poorly understood. Here, we identify the Fsr quorum sensing (QS) system of Enterococcus faecalis as an unexpected negative regulator of biofilm and pathogenesis in IE. Using microfluidic and in vivo models, we show that blood flow prevents Fsr activation in early IE, with Fsr induction occurring only later, once bacteria form biofilm microcolonies and become shielded from flow. Deletion of Fsr promotes robust biofilm growth, driven partly through the downregulation of GelE and SprE proteases, reprograms metabolism by upregulating lrgAB to enhance pyruvate utilization, and increases gentamicin tolerance in vivo. Furthermore, we show that GelE cleaves the human pro-IL-1β into an active form, suggesting a species-specific mechanism for inflammation modulation by QS. In support of these findings, analysis of IE patient cohorts shows that naturally occurring Fsr-deficient E. faecalis strains are associated with prolonged bacteremia. Overall, our findings provide insights into how host blood flow impacts QS activation, which, in turn, regulates pathogenesis in IE, and highlight the Fsr QS as a potential determinant of clinical disease course.

Introduction
Quorum sensing (QS) is an interbacterial communication system that regulates biofilm formation and other group behaviors through the production, secretion, and sensing of autoinducer signaling molecules1. QS is activated when autoinducers accumulate to a threshold concentration, correlated with increased bacterial population density1, a condition commonly encountered in biofilms within natural habitats. When activated, QS can promote or inhibit gene expression involved in the extracellular matrix polysaccharide and protein synthesis, protease secretion, and eDNA release2,3. This regulation can drive the maturation or dispersal of biofilms2. However, QS is not solely regulated by population density. Mechanical and chemical cues, such as fluid flow, habitat architecture, and nutrient availability, also impact its activation4–10. For instance, fluid flow can disperse autoinducers through advection in vitro, thereby preventing QS activation4,5. Conversely, bacteria growing in confined and structurally complex in vitro habitats can entrap autoinducers, facilitating QS activation4,6–8. Furthermore, despite QS being widespread across the bacterial kingdom, QS-defective mutants in bacterial species such as Enterococcus faecalis, Staphylococcus epidermidis, and Pseudomonas aeruginosa have been isolated from patients, indicating that QS activation may not always confer an advantage11–14. This genomic variation combined with the diverse mechanical and chemical cues present in the host, raises a central question: How does QS regulate biofilm formation across different host microenvironments, and how does this regulation shape infection outcomes?

During infection, bacteria encounter diverse cues from the host microenvironment, such as heterogeneous fluid flow, nutrient availability, and tissue architecture15. One host microenvironment where bacteria encounter all these cues is the heart valves, which direct blood flow through the heart chambers and major arteries16. Despite experiencing one of the highest blood flow rates in the body, heart valves are not exempt from bacterial colonization and infection. Mechanical or inflammatory lesions on the heart valves can render them susceptible to infective endocarditis (IE)17. These lesions trigger the accumulation of platelets and fibrin, leading to thrombus formation at the site of the valvular damage18. Thrombi serve as a substrate for adhesion by Gram-positive bacteria, such as E. faecalis, S. aureus, and Streptococcus spp19–23. Once bacteria attach to the thrombus, they can proliferate into a biofilm, leading to the infiltration of inflammatory cells and eventually to the formation of a septic vegetation24. The inflammatory response is often insufficient to clear the infection25–27. Treatment of IE typically requires a combination of intravenous antibiotic therapy for up to 6 weeks28, but due to the high prevalence of multidrug-resistant bacteria and recalcitrance of biofilms to antibiotics, 50% of patients will also undergo surgery to remove infected tissue and replace the damaged valves29. Despite these treatment approaches, global IE deaths have risen from 28,754 in 1990 to 66,322 in 2019, with age-standardized mortality of IE increasing from 0.73 to 0.87 per 100,000 people over the same period30. This alarming increase highlights the need to better understand how bacteria adapt to the vegetation microenvironment to improve the diagnosis and treatment of IE.

Limited insights into the biofilm-host interplay on cardiac vegetations constrain advances in the diagnosis and treatment of IE. Most studies have focused on initial bacterial adhesion to the thrombus exterior, a microenvironment that differs significantly from the conditions encountered within the vegetation during biofilm maturation. The vegetation exterior is exposed to intense shear stress forces due to blood flow, while the interior is shielded from them. This differential exposure to blood flow may influence QS activity and nutrient availability, depending on bacterial location at the different stages of infection. In this study, we investigated the hypothesis that the heterogeneous microenvironment of IE vegetations critically influences biofilm development and infection outcome in IE caused by E. faecalis, the third most common causative agent of this infection31,32. While the virulence factors that facilitate initial adhesion to thrombus in enterococcal IE have been identified33–40, the regulation of biofilm formation and its impact on disease progression remain largely overlooked. Here, using microfluidic and in vivo models, we show that fluid flow prevents Fsr activation, revealing a spatiotemporal regulation of QS as vegetations grow. We identify Fsr as a negative regulator of biofilm growth in vivo, exerting its control by upregulating gelE and sprE and by limiting lrgAB expression. We also demonstrate that loss of the Fsr QS system, commonly observed in IE clinical isolates12,13, increases tolerance to gentamicin treatment in vivo and correlates with prolonged bacteremia and high disease severity in individuals with enterococcal IE. Collectively, these findings provide new insights into the role of QS in IE biofilm growth, antibiotic tolerance, and infection outcomes.

Results
Fluid flow and microcolony size regulate Fsr QS activation in IE
Vegetations in IE present a heterogeneous microenvironment, where the exterior is exposed to high shear stress and advection from blood flow, while the interior remains shielded. At the onset of IE, bacteria in the bloodstream attach to the vegetation exterior and become directly exposed to blood flow. We therefore hypothesized that blood flow might trigger physiological adaptations in E. faecalis that favor the establishment of IE. To identify these adaptations, we exposed surface-attached E. faecalis OG1RF to a pulsatile flow of media for 30 minutes in a microfluidic system. This flow generated a shear stress of 20 dynes/cm2, simulating the shear stress on aortic valve leaflets41–43. Compared to surface-attached bacteria that were incubated without flow, transcriptomic analysis revealed 59 genes upregulated and 167 genes downregulated under flow (log FC > 1.0, FDR < 0.05) (Fig. 1A, Supplementary Data 1). Gene ontology enrichment analysis revealed an overrepresentation of genes linked to ATPase activity, glycolytic processes, and oxidative stress (Fig. 1B), suggesting that E. faecalis adapts its metabolism to meet energy demands and adapt to different oxygen levels under flow. Notably, transcription of the fsr locus (fsrABDC) and its associated regulon (gelE, sprE, entV, RS04585)38,44,45 were significantly decreased in the presence of fluid flow (Fig. 1A). The fsr locus encodes the Fsr quorum sensing (QS) system, which is activated when the extracellular concentration of the autoinducer peptide (AIP) gelatinase biosynthesis-activating pheromone (GBAP) exceeds a critical concentration and is therefore typically activated in direct proportion with bacterial population density46. Given the role of GBAP concentration in Fsr activation, we investigated whether fsr downregulation under fluid flow was attributed to GBAP advection. To distinguish between advection- and shear stress-mediated transcriptional regulation, we exposed surface-attached bacteria to low (1 dynes/cm2), intermediate (10 dynes/cm2), and high shear stress (20 dynes/cm2). Quantitative PCR showed that transcription of fsr-associated genes was decreased 3- to 4-fold after 30 min, regardless of the shear stress magnitude applied (Fig. 1C), demonstrating that advection rather than mechanical forces regulates their expression. Taken together these data show that fluid flow downregulates quorum sensing gene expression and triggers physiological adaptations in E. faecalis.

To directly demonstrate the effect of flow on QS activation in vivo, we constructed a Dasher GFP reporter plasmid under the control of the gelE promoter (PgelE-GFP) (Figure S1A-H), which is transcriptionally upregulated by FsrA47. Using a rat model of IE, we infected animals with OG1RF WT transformed with PgelE-GFP (WT PgelE-GFP) or with the constitutive promoter control plasmid Pcfb-GFP (WT Pcfb-GFP)48, followed by an IV injection of spectinomycin to select for plasmid maintenance in vivo (Figure S1I, J). Microcolonies in aortic valve vegetations harvested at 24 h post-infection (hpi) were examined by microscopy, and GFP expression, size, and distance from the vegetation surface were quantified. Surface microcolonies exhibited lower gelatinase promoter expression compared to embedded microcolonies (Fig. 1D, E, p=0.0239), and small microcolonies exhibited lower gelatinase promoter expression compared to large microcolonies (Fig. 1F, p=0.001). In WT Pcfb-GFP infection, only small aggregates were detected and CFU counts in vegetations were reduced compared to WT PgelE-GFP infection (Figure S1I, J), suggesting that constitutive GFP overexpression impaired strain fitness in the host. Nevertheless, these small aggregates fluoresced brightly regardless of their location (Figure S1K), confirming the specificity of the PgelE-GFP reporter. Overall, these findings show that exposure to fluid flow and microcolony size regulate QS activation in IE.

Early colonization of vegetation is independent of the Fsr QS system
Fluid flow restricts Fsr activation in superficial microcolonies, suggesting that the QS system may be dispensable for E. faecalis to

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors characterize the role of the fsr quorum sensing in infective endocarditis caused by Enterococcus. Their first major claim is that fluid flow negatively impacts the accumulation of quorum signal, which is established in other systems, and they provide convincing transcriptomic evidence for in vitro. QS is known to impact the formation of biofilms and vegetation; using a rat model, they found QS-mutant bacteria develop greater biofilm by 72 hrs, with no difference at 6 hrs. This is put together into a plausible but undersupported model by which fluid flow prevents the induction of the Fsr QS system in E. faecalis until biofilms become shielded from fluid flow. They go on to show bacteria mutant for gelatinase, one of the major known QS-regulated virulence factors, also develop greater biofilm, a possible advance over prior studies that have examined this in more reductionist systems to different conclusions. Gelatinase cleaves many substrates, and using an in vitro assay the authors newly describe the cleavage of the host cytokine IL-1β. Some cleavage products of this cytokine are proinflammatory, and some lose activity, but the authors ultimately observe no difference that could be attributable to this in the infection model. This is in contrast to the title, that claims it activates inflammation. Finally, the authors perform RNAseq and across additional in vitro and in vivo assays describe fsr effects on metabolism, antibiotic tolerance, disease severity, and other broad effects other than gelatinase expression. Overall, I found the work to describe an advance to the enterococcal infective endocarditis field, with elements that will appeal to those with interests in quorum sensing and host pathogen interactions. However, significant work is needed to develop a “main story”. The effect of flow and biofilm on fsr during actual infection is not fully explored, and the effects of gelatinase and other elements regulated by fsr are ultimately descriptive and do not reach a mechanistic conclusion – see below.\n1) The major question proposed is does flow impacts the fsr regulon in vivo? It is not clear why 30 min is sufficient for major QS regulation changes in vitro (Fig 1), but 6 hr is not in vivo (Fig 2) and requires 72 hr for phenotypic (Fig 3) and expression (Fig 6) differences. Is this phenotypic difference just from a cumulative effect of expression differences? This transition relating the in vitro flow condition to an in vivo phenotype where flow is not under control requires additional measures to make sense. It would make sense to examine expression in bacteria exposed to flow, eg, valve surface, vs deeper tissue or other appropriate control. Altogether, these data support the simple, established fact that QS plays a role during infection. It falls short in showing when QS is not important during infection due to flow, the part of the overall proposed mechanism that carries the most novelty. Reporters, coinfections, and inducible systems (pTCV-Ptet::gelE) may be useful for teasing out the in vivo relevance.\n2) No evidence is provided on a role for IL1b in this model. Since gelatinase is only one part of the fsr response (Fig 1), and IL1b cleavage likely a minor part of the gelatinase response (due to its broad activity), this connection is tenuous. IL-1b cleavage is purported to promote host cell infiltration (lines 250), yet no significant cleavage is observed during infection (Fig 5B), and lack of immune cell difference in the response to vegetations (lines 225) both argue against IL-1b playing an important role. Fig 5B shows the active products observed in fig 5C do not happen during infection and not in an fsr-variable manner. Is this cleavage conserved between species? Additionally, no support given that the IL-1b is extracellular as claimed in line 237. Most of this is shown to be pro-IL-1b, which is expected to be intracellular, and inaccessible to gelatinase. How does gelatinase access the pro-IL1b?\n3) Additionally, this IL1b cleavage event needs additional documentation. Data supporting the F104 cleavage site is absent (Line 246), so its not clear how cleavage site was identified. The gelantinase motif claimed is an identical match to additional residues in this region (FF) – why aren’t they cleaved? Fig 5C observes degradation of IL1b, not a stable mature product. Additionally, can the authors explain the loss of pro-IL1b across this experiment? This is fsr- and gelE- independent, and would appear to be the major driver of IL1b signaling potential in these conditions. The mass spec analysis of Fig 5E is also unconventional, and confusing. The model proposes a cleavage removing the N-terminus, but not clear why these peptides wouldn’t be found anymore. Cleavage is expected to lead to smaller products from larger, not their complete destruction. Possibly something in the preparation is leading to this artifact where the N-terminus can no longer be observed, but at the least, controls should be included for this such as analysis with similar proteins, such as LasB or SpeB. Also, 1-51 should be examined too, to support this mechanism. Alternatively, conventional measure include N-terminal protein sequencing, or various labeling technologies, to positively identify specific sites. Protein staining, rather than immunoblotting, is recommended to detect all cleavage products formed, not just those retaining the antibody epitope.\n4) Fig 7F appears under-powered, and an unexpected distribution. 7E shows ~1/2 are fsrA+/-, but 7F mostly focuses on isolates that are fsr+. Significance is reached by a sole individual with a remarkably long bacterium by an fsr- isolate. With a more representative sampling, this very likely is not accurate. What are the inclusion/exclusion criteria? Where any fsr- by means other than regional deletion? A heterogenous population is possible - how many clones were examined for each patient? Is it fsr+ if even a tiny fraction of the population retains the gene, or fsr- if even a tiny fraction of the population mutates it?\nMinor\nLine 167: References 49 and 50 are redundant, and do not offer sufficient support that fibrin is a major component of vegetation. This offered mechanism that gelatinase activity on fibrin is a contributor is not obviously supported by the experiments of Fig 4, showing differences in CFU but not biomass in a gelatinase-dependent manner. This model should be better explained.\nBiofilm polysaccharides should be imaged between wt, gelE, and fsr conditions since this is a known QS-regulated phenotype and known to be responsible for the purported phenotypes.\nHow are differences in the number of microcolonies per mouse accounted for in Fig 3E? What is the limit of detection? How does this compare to 4H where only one mouse is examined for the gelB mutant? How much variability between animals is expected?\nThe conditions for extended Fig 3C are unclear\nExtended Fig 3D requires a negative control\nNeutrophils are proposed to be a source of pro-IL1b for gelE cleavage – does this occur during enterococcus infection of neutrophils?", "reviewer_2": "This is an interesting manuscript by Antypas et al. that sheds new light on the role of the Fsr quorum sensing system in infective endocarditis caused by Enterococcus faecalis. The paper highlights a lack of knowledge about Fsr activity during fluid flow and how that contributes to vegetation development during endocarditis. The authors make the important point that quorum sensing is not only regulated by cell density, as shear/flow and spatial constraints can affect accumulation of signaling molecules. They also highlight and build upon the prior paradoxical observation from many groups that a large number of Ef clinical isolates have mutations in the Far quorum sensing system, despite the importance of this system (and the Fsr-regulated proteases GelE and SprE) for virulence in multiple animal models.\nThe authors first use RNAseq to profile transcriptional differences between Ef grown statically and with shear stress/flow to model blood flow. They find that Fsr and multiple Fsr-regulated genes are downregulated with fluid flow. From this, they confirm prior results showing that the Fsr system does not contribute to development of early vegetation development in a rat model of endocarditis, and build upon this by examining a later stage of infection. Strikingly, vegetations from animals infected with the Fsr mutant were significantly larger than animals infected with OG1RF. Next, they examine the role of the Fsr-regulated proteases GelE and SprE in modulating the host immune response during endocarditis and show that IL-1B co-localizes with Ef vegetations. Cleavage/generation of the active IL-1B fragment requires functional/active GelE (shown using multiple complementary approaches), providing insight into how Fsr/GelE may modulate the immune response during infection. The authors then investigated the impact of Fsr on Ef gene expression in late (72 hr) vegetations and found that absence of Fsr led to upregulation of lrgAB and differential expression of sugar/carbohydrate metabolic pathways, suggesting that Fsr quorum sensing in Ef is important for metabolism during endocarditis. The authors also demonstrate that the fsr mutant is more tolerant to gentamicin treatment, underscoring how loss of Fsr may provide a protective effect in vivo. Finally, the authors seek to understand why many Ef clinical isolates have a mutation disrupting Fsr, an interesting phenomenon that has been documented previously. Using existing patient records, they show a minor difference between bacteremia duration and disease severity score based on intact fsrA in clinical isolates.\nOverall, this paper will be impactful as it aims to provide context for why a large fraction of Ef isolates have mutations that disrupt Fsr expression and activity when Fsr is important for virulence and disease progression. This has been an outstanding question in the field for some time, and this paper is the first to provide evidence for why these genotypes may be detected. The methods provide an appropriate level of detail. Noteworthy results are that the loss of Fsr quorum sensing prevents control of biofilm/vegetation size and promotes tolerance to antibiotics in vivo. The in vivo RNAseq is an important data set for the field, and the fluid flow RNAseq is relevant not only for the study of endocarditis, but also other infection sites. The approaches for immunofluorescence and in vivo examination of vegetation size/structure will be relevant for other groups interested in host-pathogen dynamics during endocarditis. The authors nicely connect their results to what is known about biofilm formation and endocarditis in other Gram-positive pathogens. However, there are some sections where additional replicates, complementation, statistical analysis, and comparison to existing literature would strengthen the conclusions. My overall comments for the authors are below:\n1) In some places, additional replicates and statistical comparisons would strengthen the conclusions. Statistics are missing from several panels in Extended Data Fig 2 and in Figure 4G. In Figure 4H, the microcolony areas were measured from 1 animal in 1 experiment, and it is unclear whether a direction comparison can be made to the parental strain experiments in Figure 3E. Additional animal work would not be required here if images sets from additional animals already exist.\n2) The lrgAB data presented supports the conclusion that the lrgA mutant has a growth defect in M1+pyruvate, but the direct link between lrgAB and pyruvate transport would be strengthened by inclusion of pyruvate accumulation assays or measure of intracellular pyruvate. Additionally, the lrgAB experiments would be strengthened by complementation of lrgA and lrgB and additional replicates for the experiments shown in Extended Data Fig 4 (as only 1-2 replicates were done).\n3) Intensity of the 35 kDa band corresponding to pro-IL-1B in Figure 5C is reduced in the gelE deletion strain relative to the fsr mutant at 4 and 6 hours. Is this due to non-specific cleavage/degradation?\n4) As the authors note, as vegetations develop and interior cells become protected from/less exposed to fluid flow, this should lead to activation of quorum sensing. Presumably cells that are on the exterior of the vegetation would still experience fluid flow, less Fsr activation, and lower production of GelE. Does GelE that would be produced by these interior cells have access to substrates like IL-1B given the spatial distribution of the vegetation? Can GelE diffuse out of the core of the vegetations to the surface or can IL-1B reach the interior of the vegetation?\n5) Previous microarray work identified numerous genes and metabolic pathways differentially regulated in fsrB and gelE deletion mutants depending on growth phase (PMC1446981). Is there overlap between these genes and the fsr-regulated genes identified in the RNAseq sets in this study? This could be interesting to consider given the shift in metabolism that occurs in different growth phases.\n6) The link between loss of gelE/fsr and accumulation of Ace was previously published. Did the authors see any changes in Ace expression in their RNAseq? Is Ace likely to contribute to overall vegetation structure? It might be beneficial to mention briefly given that Ace contributes to endocarditis pathogenesis (PMC3165527).\n7) Figure 7F is somewhat confusing as it is not clear what each data point represents. Are only 3 patients with fsrA-negative isolates shown? It would be helpful to see bacteremia duration data points for all patients (if available).\n8) Based on Supp Data 3, it looks like the proportion of patients with loss of fsrA is opposite in the Zurich and Pittsburgh cohorts (fsrA not present in a majority (17/24) patients from Zurich cohort, fsrA not present in a smaller fraction (21/57) patients from Pittsburgh cohort). Can the authors speculate on why that might be the case? Are there different predominant circulating strain types in Switzerland vs USA? If so, given that bacteremia duration is similar between fsrA present/absent patients in each independent cohort, does that suggest there are other Ef genomic features driving bacteremia duration?\n9) It looks like the absence of fsrA in isolates from patients (Figure 7G) results in bimodal distribution of disease severity score (1 cluster with scores 2-3, 1 cluster with scores 5-6) while presence of Fsr results in normal distribution of this metric, which is very interesting. Can the authors speculate on what might drive this?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript by Antypas et al. explores how the Fsr (quorum sensing (QS) system influences biofilm formation and inflammation in infective endocarditis (IE) using Enterococcus faecalis as model organism. QS is a bacterial communication mechanism that regulates biofilm development and other collective behaviors through autoinducer signaling molecules. QS activation, driven by increased bacterial density and various environmental factors, can either promote or inhibit gene expression related to biofilm maturation or dispersal. This study highlights that fluid flow can prevent Fsr activation, while confined environments facilitate it. The research demonstrates that the Fsr system restricts biofilm growth and triggers inflammation by upregulating gelatinase, which activates IL-1β. Loss of the Fsr system results in unchecked biofilm growth, reduced inflammation, and increased antibiotic tolerance, correlating with severe disease outcomes.\nThe authors provided solid in vitro and in vivo data to support their major conclusions; the large amount of work involved in the project should also be appreciated. However, the study lacks novelty as it reiterates well-established concepts in QS and biofilm research. The role of QS in regulating biofilm formation and its impact on infection outcomes has been extensively studied in various human pathogens, including E. faecalis. While the specific focus on the Fsr QS system in the context of IE is valuable, the overall findings do not significantly advance our current understanding of QS mechanisms or their implications in biofilm-associated infections. General and more specific comments follow.\n1. A rationale should be provided for using stationary-phase cells when incubating bacteria under fluid flow conditions.\n2. Since the fsrA gene is under the control of a constitutive promoter, how do you explain the results obtained after exposure to fluid flow conditions? Still related to the gene expression data, it is not clear why the authors normalized Ct values obtained to the expression of recA gene (used as reference) instead of normalizing to the number of cells, since we can assume that at 20 dynes/cm2 there are fewer cells attached.\n3. While the authors concluded their first section by stating that fluid flow impacts QS and triggers physiological adaptations in E. faecalis, it is unclear how they formulated the hypothesis presented in the second section based on the results shown in Figure 1. The connection between the observed effects of fluid flow on QS and the proposed limitations on Fsr activation during early colonization required further clarification.\n4. The section describing the impact of the absence of gelE and sprE genes on biofilm growth could be shortened. In fact, the data presented could be merged with the previous section describing the impact of the fsr knockout mutant on biofilm growth in late IE.\n5. IE typically affects injured or abnormal heart valves, which often remain asymptomatic until a blood-borne infection occurs. The most common etiological agents are oral streptococci, S. aureus, and E. faecalis. In the case of E. faecalis, previous studies have found that neither the fsr nor gelatinase production is more common in disease-associated isolates than in isolates colonizing healthy individuals. A surface protein, Esp, has been described as being enriched among endocarditis and bacteremia isolates of E. faecalis but is rare among fecal isolates. Clinical isolates of E. faecalis found in urine often exhibit a chromosomal deletion involving the fsr locus, while some other studies have reported the presence of the fsr locus in endocarditis isolates. Without a deeper genomic analysis of the 81 enterococcal isolates, the results presented in Figure 7 and Extended Figure 5 add little to the manuscript.\n6. Although the authors performed gene ontology enrichment analysis (Fig. 1B), it would be beneficial to provide details regarding the most important factors involved in E. faecalis biofilm patterns, including initial attachment, biofilm maturation, dispersal. Specifically, the prominent adhesive factor Ebp (endocarditis and biofilm-associated pilus) should be highlighted for its similarity to the fibronectin-binding protein found in S. aureus, another agent involved in IE. Other factors of interest include the cell wall anchored collagen adhesin, EbfA, BgsA, and SagA adhesins."}, "author_response": "We would like to thank the reviewers for their thorough evaluation of our study and for providing constructive feedback that has greatly strengthened this manuscript. In the revised manuscript, we introduced several changes, including:\n-Demonstrating how fluid flow and microcolony size regulate QS activation with spatial resolution in vivo\n-Strengthening our conclusion that loss of gelatinase promotes larger microcolonies by including additional biological replicates in our analysis\n-Expanding our investigation on lrgAB by including GC-MS and in vivo data\n-Providing species-specific data on pro-IL-1β processing by gelatinase and clarifying our hypothesis on its impact on inflammation\n-Deepening our genomic analysis on the clinical isolates by performing WGS on the Swiss cohort strains\nThese represent some of the major revisions, alongside additional clarifications and improvements throughout the manuscript, which together make the main story more explicit, add mechanistic detail, strengthen our conclusions, and temper certain claims where warranted.\nBelow, we provide a point-by-point response to all reviewer comments, with changes indicated in the revised manuscript in red.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors characterize the role of the fsr quorum sensing in infective endocarditis caused by Enterococcus. Their first major claim is that fluid flow negatively impacts the accumulation of quorum signal, which is established in other systems, and they provide convincing transcriptomic evidence for in vitro. QS is known to impact the formation of biofilms and vegetation; using a rat model, they found QS-mutant bacteria develop greater biofilm by 72 hrs, with no difference at 6 hrs. This is put together into a plausible but undersupported model by which fluid flow prevents the induction of the Fsr QS system in E. faecalis until biofilms become shielded from fluid flow. They go on to show bacteria mutant for gelatinase, one of the major known QS-regulated virulence factors, also develop greater biofilm, a possible advance over prior studies that have examined this in more reductionist systems to different conclusions. Gelatinase cleaves many substrates, and using an in vitro assay the authors newly describe the cleavage of the host cytokine IL-1β. Some cleavage products of this cytokine are proinflammatory, and some lose activity, but the authors ultimately observe no difference that could be attributable to this in the infection model. This is in contrast to the title, that claims it activates inflammation. Finally, the authors perform RNAseq and across additional in vitro and in vivo assays describe fsr effects on metabolism, antibiotic tolerance, disease severity, and other broad effects other than gelatinase expression. Overall, I found the work to describe an advance to the enterococcal infective endocarditis field, with elements that will appeal to those with interests in quorum sensing and host pathogen interactions. However, significant work is needed to develop a “main story”. The effect of flow and biofilm on fsr during actual infection is not fully explored, and the effects of gelatinase and other elements regulated by fsr are ultimately descriptive and do not reach a mechanistic conclusion – see below.\n1) The major question proposed is does flow impacts the fsr regulon in vivo? It is not clear why 30 min is sufficient for major QS regulation changes in vitro (Fig 1), but 6 hr is not in vivo (Fig 2) and requires 72 hr for phenotypic (Fig 3) and expression (Fig 6) differences. Is this phenotypic difference just from a cumulative effect of expression differences? This transition relating the in vitro flow condition to an in vivo phenotype where flow is not under control requires additional measures to make sense. It would make sense to examine expression in bacteria exposed to flow, eg, valve surface, vs deeper tissue or other appropriate control. Altogether, these data support the simple, established fact that QS plays a role during infection. It falls short in showing when QS is not important during infection due to flow, the part of the overall proposed mechanism that carries the most novelty. Reporters, coinfections, and inducible systems (pTCV-Ptet::gelE) may be useful for teasing out the in vivo relevance.\nWe understand that the reviewer is asking for experimental evidence on how flow conditions regulate quorum sensing in vivo. We sought to address this question by demonstrating Fsr QS activation with spatial resolution in vegetations. We constructed an E. faecalis strain carrying a reporter plasmid expressing GFP under the control of the gelatinase promoter (WT Pgele-GFP), alongside a control strain carrying the reporter plasmid with a constitutive promoter (WT Pcfb-GFP). We added a new supplementary figure (Extended Data Fig. 1A-H in the revised manuscript) demonstrating that this reporter plasmid is specifically activated by Fsr QS in vitro. Importantly, new in vivo data in Fig. 1D-F show that Fsr QS activation depends on the location and size of the microcolony. Vegetation embedded microcolonies exhibit higher QS activation than superficial ones, and large microcolonies display higher activation than small ones. In contrast, WT Pcfb-GFP bacteria remained fluorescent regardless of location (Extended Data Fig. 1K). We further confirmed plasmid stability at 24 hpi in vivo (Extended Data Fig. 1I–J), ruling out plasmid loss as a cause of false-negative gelatinase expression. The first results section has been renamed to “Fluid flow and microcolony size regulate Fsr QS activation in IE” and the Results section text has been revised in lines 135-151, 154-155, 162-164, and 167-172 to reflect the addition of these new results. Relevant Methods sections have been updated to include the methodology for these new experiments (see Bacterial cultures, DNA manipulation and construction of deletion mutants, Infective Endocarditis rat model, Tissue immunofluorescence staining, Microscopy and image analysis). Finally, we have also added qPCR data for fsrC expression at 24 hpi for a more detailed analysis of its expression in vivo (Fig. 3A in the revised manuscript).\n2) No evidence is provided on a role for IL1b in this model. Since gelatinase is only one part of the fsr response (Fig 1), and IL1b cleavage likely a minor part of the gelatinase response (due to its broad activity), this connection is tenuous. IL-1b cleavage is purported to promote host cell infiltration (lines 250), yet no significant cleavage is observed during infection (Fig 5B), and lack of immune cell difference in the response to vegetations (lines 225) both argue against IL-1b playing an important role. Fig 5B shows the active products observed in fig 5C do not happen during infection and not in an fsr-variable manner. Is this cleavage conserved between species? Additionally, no support given that the IL-1b is extracellular as claimed in line 237. Most of this is shown to be pro-IL-1b, which is expected to be intracellular, and inaccessible to gelatinase. How does gelatinase access the pro-IL1b?\nWe appreciate the reviewer’s constructive criticism of this part of the manuscript, as it has prompted us to further investigate the gelatinase effect on human and rat pro-IL-1β. We also acknowledge that our original presentation of the results may have been confusing. Below, we first clarify the rationale in the original manuscript and then explain how we have revised the manuscript.\nIn the original manuscript, our aim was to suggest that gelatinase could contribute to inflammation modulation, based on our in vitro observation that it cleaves human pro-IL-1β and on prior reports of other bacterial proteases (e.g., SpeB, LasB) exerting similar effects. We did not intend to imply that IL-1β plays a central role in our in vivo model. Consistent with the reviewer’s point, there is no statistically significant difference between the number of immune cells in vegetations between WT and ΔgelE infection (Extended Data Fig. 2G-I in the original manuscript, now Extended Data Fig. 4D-F in the revised manuscript). However, the ΔgelE infection leads to an increase in vegetation CFU (Fig. 4D), which made it puzzling to us that this was not accompanied by a corresponding increase in immune cell infiltration. Hence, in the original manuscript we proposed that ΔgelE biofilms might be less immunostimulatory.\nAlthough the reviewer mentions that no support given that the IL-1β is extracellular, we show that IL-1β is localized extracellularly at the interface of biofilm and neutrophils undergoing NETosis (blue insets in Fig. 5A in the original and Fig. 6A revised manuscript). We also provide further evidence that the neutrophils surrounding the biofilm are undergoing NETosis (Extended Data Fig. 3A in the original manuscript, and now Extended Data Fig. 4A in the revised manuscript). We therefore proposed that pro-IL-1β becomes accessible to gelatinase through release of intracellular contents from neutrophils undergoing NETosis.\nAlso consistent with the reviewer’s point, there is no statistically significant difference in the levels of pro-IL1β between WT and Δfsr infection (Fig. 5B in the original manuscript). If cleaving or degradation of extracellular pro-IL-1β occurs in vivo, it is likely restricted to the interface between biofilm and neutrophils undergoing NETosis, which would be difficult to detect by western blot. Nevertheless, this result provides additional evidence that pro-IL-1β is present within vegetations, as shown in Fig. 5A of the original manuscript (Fig. 6A in the revised manuscript). In the revised manuscript, we have replaced the western blot comparing pro-IL-1β levels between WT and ∆fsr with a western blot comparing WT- and ΔgelE-infected vegetations (Fig. 6B in the revised manuscript), to align directly with Fig. 6A, which shows IL-1β staining in WT- and ΔgelE-infected vegetations.\nTo address the reviewer’s question on whether gelatinase activation of IL-1β is conserved across species, in the revised manuscript we assayed the ability of E. faecalis to cleave rat pro-IL-1β. We show that gelatinase degrades & cleaves rat pro-IL-1β into fragments below 15 kDa (Fig. 6C in the revised manuscript). However, these fragments were not bioactive, as they failed to activate the IL1R reporter cell line (Extended Data Fig. 4C in the revised manuscript).\nIn summary, we show that gelatinase activation of pro-IL-1β does not occur in rats, and we have therefore revised this section to remove any claims attributing our in vivo observations to this mechanism. At the same time, our in vitro findings with human pro-IL-1β remain important. The ability of E. faecalis gelatinase to generate bioactive IL-1β fragments in humans suggests a potential biofilm-specific mechanism of inflammation modulation that warrants further investigation. We believe this represents a novel and relevant finding for IE that should be explored in in future studies, particularly in the context of host–biofilm interactions.\nPlease also note that:\n-Results section “E. faecalis gelatinase cleaves and activates human IL-1β” is moved after result section describing lrgAB findings in the revised manuscript to improve the flow of the manuscript\n-The text in Results section “E. faecalis gelatinase cleaves and activates human IL-1β” has been fully revised to align with the new findings on rat pro-IL-1β degradation by gelatinase (lines 295-338).\n-We have also revised Fig. 5F of the original manuscript to include a multiple alignment between the cleavage region of human, rat, and mouse IL-1β (Fig. 6F in the revised manuscript).\n-Discussion (lines 406-408, 459-462) and abstract (lines 32-34) have been updated to reflect that activation of pro-IL-1β is species-specific.\n-Methods sections (IL-1β cleaving assay, IL-1 signaling assay) have been updated to include the new experiments.\n3a) Additionally, this IL1b cleavage event needs additional documentation. Data supporting the F104 cleavage site is absent (Line 246), so its not clear how cleavage site was identified.\nWe appreciate this important comment as it prompted us to clarify how the cleavage site was identified. We subjected single bands of human pro-IL-1β and cleaved fragments after incubation with the E. faecalis WT strain for 0, 6, and 18 h to mass spectrometry analysis (See more details in our answer to your next question). We shortlisted peptides that appeared uniquely at 6 and 18 h compared to 0h and further filtered them for abundance (≥10^6). Among these, we identified a peptide beginning at residue 105, which was one of the most abundant new peptides and consistent with the generation of a ~17 kDa fragment observed by western blotting. We therefore infer that cleavage occurs between residues 104 and 105. The cleavage site at F-F also agrees with what has been previously reported in the literature. We have included Supplementary File 4 in the revised manuscript that contains all peptides identified at the different time points, as well as peptides unique to 6- and 18-h incubation compared to 0h. We have also revised the methods section for “IL-1β cleaving assay” with the relevant information (lines 847-855).\n3b) The gelantinase motif claimed is an identical match to additional residues in this region (FF) – why aren’t they cleaved? Fig 5C observes degradation of IL1b, not a stable mature product. Additionally, can the authors explain the loss of pro-IL1b across this experiment? This is fsr- and gelE- independent, and would appear to be the major driver of IL1b signaling potential in these conditions. The mass spec analysis of Fig 5E is also unconventional, and confusing. The model proposes a cleavage removing the N-terminus, but not clear why these peptides wouldn’t be found anymore. Cleavage is expected to lead to smaller products from larger, not their complete destruction. Possibly something in the preparation is leading to this artifact where the N-terminus can no longer be observed, but at the least, controls should be included for this such as analysis with similar proteins, such as LasB or SpeB. Also, 1-51 should be examined too, to support this mechanism. Alternatively, conventional measure include N-terminal protein sequencing, or various labeling technologies, to positively identify specific sites. Protein staining, rather than immunoblotting, is recommended to detect all cleavage products formed, not just those retaining the antibody epitope.\nWe thank the reviewer for seeking additional clarity on the gelatinase-mediated cleaving of pro-IL-1β. Before we address these comments, we would like to clarify that we subjected specific excised protein bands to mass spectrometry analysis, rather than entire gel lanes. Specifically, we analyzed the band corresponding to human pro-IL-1β from the 0-h incubation, and the ~17 kDa bands from the 6- and 18-h incubations, as this size corresponds to the expected bioactive IL-1β generated by canonical caspase-1 cleavage. We also provide below the Coomassie-stained gel highlighting the bands that were excised and submitted for analysis (this figure is not included in the revised manuscript).\nWe also want to clarify that the other 2 potential F-F cleavage sites are upstream the proposed F104F105 site. Below we provide the annotated protein sequence for human pro-IL-1β (not included in the revised manuscript).\n>sp|P01584|IL1B_HUMAN Interleukin-1 beta OS=Homo sapiens OX=9606 GN=IL1B PE=1 SV=2\nMAEVPELASEMMAYYSGNEDDLF23F24EADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKG\nFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTF93F94PFIFEEEPIF104F105DTWDNEAYVHDAPV\nRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKE\nKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYIST\nSQAENMPVFLGGTKGGQDITDFTMQFVSS\nTo address the reviewer’s comment that “the gelatinase motif claimed is an identical match to additional residues in this region (FF) – why aren’t they cleaved?”, we cannot rule out that they could be cleaved. However, such cleaving would be expected to generate smaller fragments (< ~7 kDa), which are not the bioactive form of IL-1β. Our analysis was focused on the ~17 kDa fragment corresponding to the size of mature, bioactive IL-1β. Accordingly, we specifically excised and submitted the 17 kDa band for MS analysis, and therefore do not have data on smaller degradation products. This clarification also relates to the reviewer’s additional comment regarding the absence of detectable N-terminal peptides. We interpret this not as their “complete destruction,” but rather as the result of two factors: (i) small peptides generated by cleavage and potential subsequent degradation are unlikely to be retained and detected under our workflow, and (ii) our experimental design focused on excision of the ~17 kDa band, which by definition excludes low-molecular-weight products such as peptides spanning residues 1–51. We agree that complementary approaches such as N-terminal sequencing, labeling strategies, or analysis with other proteases (e.g., LasB or SpeB) would provide additional validation of the cleavage site, but these are beyond the scope of the present study. Importantly, the converging evidence from western blotting, peptide mapping, fragment size, and bioactivity retainment consistently supports F104–F105 as the most plausible site of gelatinase-mediated cleavage in human pro-IL-1β.\nThe reviewer also points out that “Fig 5C observes degradation of IL1b, not a stable mature product.” We respectfully disagree with that observation. The ~17 kDa fragment appears by 4 h and remains detectable and stable through 24 h when pro-IL-1β is incubated with the WT strain (Fig. 5C in the original manuscript, Fig. 6E in the revised manuscript). This stability is further supported by the Coomassie-stained gel provided above.\nThe reviewer is also requesting to “explain the loss of pro-IL1b across this experiment” and further proposes that “This is fsr- and gelE- independent, and would appear to be the major driver of IL1b signaling potential in these conditions”. We agree that in the absence of fsr, gelE, and proteolytically inactive gelatinase, there is progressive degradation of pro-IL-1β, which could be mediated by other unidentified factors (Fig. 5C in the original manuscript, Fig. 6E in the revised manuscript). Importantly, degradation is also observed when pro-IL-1β is incubated in plain media, suggesting this may represent protein instability rather than a specific bacterial activity. In contrast, when functional gelatinase is present, we observe the generation of a ~17 kDa fragment that remains stable throughout the incubation (Fig. 5C in original manuscript and Fig. 6E in the revised manuscript) and retains bioactivity (Fig. 5D in the original manuscript and Fig. 6F in the revised manuscript). Moreover, overexpression of gelatinase in the ΔgelE pTCV-gelE strain (Extended Data Fig. 3C in the original manuscript, Extended Data Fig. 4G in the revised manuscript) markedly increased cleavage of human pro-IL-1β into the ~17 kDa fragment compared to WT, indicating that the balance between degradation and cleavage is strongly influenced by gelatinase expression levels.\nWe also thank the reviewer for pointing out that “The mass spec analysis of Fig 5E is also unconventional, and confusing.”. We have removed this figure in the revised manuscript. Instead, we provide the Supplementary File 4 in the revised manuscript that lists all peptides detected in the 3 excised bands during the MS analysis, ranked by abundance. We also revised the Methods section for “IL-1β cleaving assay” and the corresponding Results section (lines 326-338) to explain how the cleaving site was determined.\n4a. Fig 7F appears under-powered, and an unexpected distribution. 7E shows ~1/2 are fsrA+/-, but 7F mostly focuses on isolates that are fsr+. Significance is reached by a sole individual with a remarkably long bacterium by an fsr- isolate. With a more representative sampling, this very likely is not accurate.\nWe would like to clarify that Fig. 7F includes data from all 81 patients. In the original version, we used box plots to display the median, as well as the 10th and 90th percentiles, rather than plotting each individual data point. To address the reviewer’s concern about the distribution and representation, we have revised Fig. 7F to display all data points alongside the median. This update allows for a clearer visualization of the data spread and shows that the observed trend is not driven by a single outlier.\n4b. What are the inclusion/exclusion criteria?\nWe have updated the Methods section “Bacterial isolates and patient cohorts” with the inclusion and exclusion criteria (Lines 527-541).\n4c. Where any fsr- by means other than regional deletion?\nWe would like to mention at this point that have performed WGS on all the clinical isolates from the Swiss cohort for the revised manuscript, which together with the WGS data available from the US cohort enabled us to perform a more detailed genomics analysis. To address the reviewer’s question, with the exception of isolate DVT1034, which harbored a highly truncated fsrA gene, the remaining fsr-negative strains lacked both fsrA and fsrB, and exhibited absence or partial presence of fsrC. This pattern is characteristic of strains carrying the regional deletion that encompasses the fsr locus. We have included Extended Data Fig. 6 in the revised manuscript, to illustrate the presence of the fsr and other virulence factor genes across the clinical isolates used in this study. See also updated text in lines 362-363 in Results and lines 439-453 in Discussion.\n4d. A heterogenous population is possible - how many clones were examined for each patient? Is it fsr+ if even a tiny fraction of the population retains the gene, or fsr- if even a tiny fraction of the population mutates it?\nIn the original manuscript, we analyzed one clone per patient, derived from a single blood sample. For the Swiss cohort, this clone derived from the last blood culture obtained from the patient. To address the possibility of within-patient heterogeneity, we have now screened additional isolates from earlier blood samples collected from the same patients during hospitalization. While this approach does not rule out subpopulations with differing fsr genotypes, all colonies tested from the same patient consistently showed the same fsrA genotype across the sampling period (Table S3 in the revised manuscript). See updated text in lines 381-383. Additionally, we have added a statement to the discussion to reflect the limitation of our approach (lines 447-453).\nRegarding classification, we classified isolates as fsr-positive or fsr-negative based on the genotype of the sequenced isolate from the US cohort or PCR-screened colony from the Swiss cohort in the original manuscript. This classification remained accurate even after we performed WGS on the Swiss cohort isolates in the revised manuscript. Given that the manuscript also investigates the role of gelatinase, we have included information on gelE presence for all isolates (Extended Data Fig. 6), as well as information on gelatinase activity for isolates from the American cohort (Methods, lines 545-548), with all fsrA-positive strains exhibiting gelatinase activity, except for DVT988 and DVT1721 strains.\nMinor\n1. Line 167: References 49 and 50 are redundant, and do not offer sufficient support that fibrin is a major component of vegetation. This offered mechanism that gelatinase activity on fibrin is a contributor is not obviously supported by the experiments of Fig 4, showing differences in CFU but not biomass in a gelatinase-dependent manner. This model should be better explained.\nWe cited two representative studies reporting the presence of fibrin in histological analyses of vegetations in the revised manuscript (line 182 in the revised manuscript). Regarding the role of gelatinase activity on fibrin as a potential contributor to dispersal, we would like to clarify that we do not propose this as the underlying mechanism for the observed phenotype of increased vegetation mass and CFU in our study. In fact, our data argue against such a model: we observed no significant differences in bacterial dissemination to the blood between WT and fsr mutants or between WT and gelatinase mutants (Extended Data Fig. 2B, F in the revised manuscript). Thus, we concluded that the increased bacterial load within vegetations is unlikely to result from altered dispersal dynamics driven by gelatinase-mediated fibrin degradation.\nWe understand that the phrasing in that section of the manuscript may have been confusing and have revised the text. See lines 182-186.\n2. Biofilm polysaccharides should be imaged between wt, gelE, and fsr conditions since this is a known QS-regulated phenotype and known to be responsible for the purported phenotypes.\nWe agree this is an interesting direction, but imaging biofilm polysaccharides in vivo is outside the scope of the current study and would require extensive optimization, as it is unclear which carbohydrates might be produced by E. faecalis in IE vegetations.\n3. How are differences in the number of microcolonies per mouse accounted for in Fig 3E? What is the limit of detection? How does this compare to 4H where only one mouse is examined for the gelB mutant? How much variability between animals is expected?\nWe quantified the total number of microcolonies identified across three cross-sections spaced 80 µm apart per vegetation. Due to the heterogeneous distribution of bacteria within vegetations, the number of microcolonies per sample varied between 20 and 65. We have updated Fig. 4H to include data from 3 WT-infected and 4 gelE-infected vegetations. Additionally, both Fig. 3E and Fig. 4H have been updated to scatterplots to display individual microcolony sizes. To address the reviewer’s question on inter-animal variability, we also include below two graphs showing the distribution of microcolony area per vegetation and the mean. These are provided for reference in this response only and are not part of the revised manuscript.\n4. The conditions for extended Fig 3C are unclear\nWe have revised the figure legend (Extended Data Fig. 4G in the revised manuscript) and methods section (lines 838-841) to clarify the conditions.\n5. Extended Fig 3D requires a negative control\nSupernatants used in Extended Data Fig. 3D in the original manuscript (Extended Data Fig. 4H in the revised manuscript) were taken and diluted from the supernatants assayed in Fig. 6F in the revised manuscript. Except for WT supernatant, all supernatants induced no or negligible activation of the IL-1R reporter cell line. Therefore, inclusion of an additional negative control was not necessary.\n6. Neutrophils are proposed to be a source of pro-IL1b for gelE cleavage – does this occur during enterococcus infection of neutrophils?\nWe propose that neutrophils are a source of pro-IL-1β based on direct and indirect evidence in the manuscript. We show that IL-1β colocalized within neutrophils in vegetations (orange inset in Fig. 6A & Extended Data Fig. 4B in the revised manuscript). We also show that neutrophils comprise ~80% of the immune cells present within vegetations (Extended data Fig. 4F in the revised manuscript), which in combination with the western blot detection of pro-IL-1β (Fig. 6B in the revised manuscript) further suggests that neutrophils are a source of pro-IL-1β.\nReviewer #2 (Remarks to the Author):\nThis is an interesting manuscript by Antypas et al. that sheds new light on the role of the Fsr quorum sensing system in infective endocarditis caused by Enterococcus faecalis. The paper highlights a lack of knowledge about Fsr activity during fluid flow and how that contributes to vegetation development during endocarditis. The authors make the important point that quorum sensing is not only regulated by cell density, as shear/flow and spatial constraints can affect accumulation of signaling molecules. They also highlight and build upon the prior paradoxical observation from many groups that a large number of Ef clinical isolates have mutations in the Far quorum sensing system, despite the importance of this system (and the Fsr-regulated proteases GelE and SprE) for virulence in multiple animal models.\nThe authors first use RNAseq to profile transcriptional differences between Ef grown statically and with shear stress/flow to model blood flow. They find that Fsr and multiple Fsr-regulated genes are downregulated with fluid flow. From this, they confirm prior results showing that the Fsr system does not contribute to development of early vegetation development in a rat model of endocarditis, and build upon this by examining a later stage of infection. Strikingly, vegetations from animals infected with the Fsr mutant were significantly larger than animals infected with OG1RF. Next, they examine the role of the Fsr-regulated proteases GelE and SprE in modulating the host immune response during endocarditis and show that IL-1B co-localizes with Ef vegetations. Cleavage/generation of the active IL-1B fragment requires functional/active GelE (shown using multiple complementary approaches), providing insight into how Fsr/GelE may modulate the immune response during infection. The authors then investigated the impact of Fsr on Ef gene expression in late (72 hr) vegetations and found that absence of Fsr led to upregulation of lrgAB and differential expression of sugar/carbohydrate metabolic pathways, suggesting that Fsr quorum sensing in Ef is important for metabolism during endocarditis. The authors also demonstrate that the fsr mutant is more tolerant to gentamicin treatment, underscoring how loss of Fsr may provide a protective effect in vivo. Finally, the authors seek to understand why many Ef clinical isolates have a mutation disrupting Fsr, an interesting phenomenon that has been documented previously. Using existing patient records, they show a minor difference between bacteremia duration and disease severity score based on intact fsrA in clinical isolates.\nOverall, this paper will be impactful as it aims to provide context for why a large fraction of Ef isolates have mutations that disrupt Fsr expression and activity when Fsr is important for virulence and disease progression. This has been an outstanding question in the field for some time, and this paper is the first to provide evidence for why these genotypes may be detected. The methods provide an appropriate level of detail. Noteworthy results are that the loss of Fsr quorum sensing prevents control of biofilm/vegetation size and promotes tolerance to antibiotics in vivo. The in vivo RNAseq is an important data set for the field, and the fluid flow RNAseq is relevant not only for the study of endocarditis, but also other infection sites. The approaches for immunofluorescence and in vivo examination of vegetation size/structure will be relevant for other groups interested in host-pathogen dynamics during endocarditis. The authors nicely connect their results to what is known about biofilm formation and endocarditis in other Gram-positive pathogens. However, there are some sections where additional replicates, complementation, statistical analysis, and comparison to existing literature would strengthen the conclusions. My overall comments for the authors are below:\n1) In some places, additional replicates and statistical comparisons would strengthen the conclusions. Statistics are missing from several panels in Extended Data Fig 2 and in Figure 4G. In Figure 4H, the microcolony areas were measured from 1 animal in 1 experiment, and it is unclear whether a direction comparison can be made to the parental strain experiments in Figure 3E. Additional animal work would not be required here if images sets from additional animals already exist.\nWe thank the reviewers for this comment. In the revised manuscript Fig. 4H now includes data from 3 WT-infected and 4 gelE-infected vegetations, with statistical analysis provided. We have also reformatted Fig. 4H as scatterplots to display individual microcolony sizes and statistical analysis has been applied to Fig. 4G. Regarding Extended Data Fig. 2A–F in the original manuscript, during the revision of the section on the gelatinase effect on pro-IL-1β and the integration of new results showing that rat pro-IL-1β is degraded rather than cleaved by gelatinase, we carefully re-evaluated these figures. In particular, we recognized that the supplementary Western blot panel was a potentially confusing and inaccurate way to estimate the ratio of immune cells to bacteria. As increased bacterial presence is already more appropriately and quantitatively addressed in Fig. 3C and Fig. 4D by CFU quantification, we omitted the panel in the revised version.\n2a) The lrgAB data presented supports the conclusion that the lrgA mutant has a growth defect in M1+pyruvate, but the direct link between lrgAB and pyruvate transport would be strengthened by inclusion of pyruvate accumulation assays or measure of intracellular pyruvate.\nWe thank the reviewer for this suggestion. In the revised manuscript, we have added new GC-MS analyses of extracellular and intracellular pyruvate in bacteria grown in M1 supplemented with pyruvate. Under aerobic conditions, both extracellular and intracellular pyruvate were significantly higher in the lrgA::Tn strain compared to WT and lrgB::Tn (Fig. 5E in the revised manuscript), indicating that lrgA plays a role in pyruvate import and downstream catabolism. We further quantified extracellular and intracellular lactate and alanine and observed no significant differences under either aerobic or anaerobic conditions (Extended Data Fig. 3A, B in the revised manuscript), showing that pyruvate conversion into these metabolites remained intact. Together, these new data strengthen the conclusion that lrgAB functions in pyruvate catabolism rather than transport.\nTo further explore the role of lrgAB in IE pathogenesis, we infected rats with a Δfsr ∆lrgAB double mutant. Vegetation weight and CFU were comparable to WT (Fig. 5G, H in the revised manuscript), indicating that lrgAB contributes to the increased vegetation mass and bacterial burden seen in the absence of QS. No differences were observed in spleen, liver, or blood CFU (Extended Data Fig. 3F–H in the revised manuscript).\nThe Results section presenting the lrgAB findings has been renamed to “fsr locus absence enhances metabolic adaptation via lrgAB and pyruvate catabolism” and the Results text has been revised to reflect the addition of new results (lines 260-277, 282-283, 285-293). Discussion has been revised (lines 473-481) and a new Methods section “Relative metabolite quantification by gas chromatography-mass spectrometry (GC-MS)” has been added.\n2b) Additionally, the lrgAB experiments would be strengthened by complementation of lrgA and lrgB and additional replicates for the experiments shown in Extended Data Fig 4 (as only 1-2 replicates were done).\nWe attempted to complement the lrgA::Tn and lrgB::Tn mutants, but overexpression of lrgAB on a plasmid caused a pronounced growth defect, particularly in lrgB::Tn, even in rich media. This effect may reflect the combined impact of kanamycin selection and lrgAB overexpression. Although this is an interesting finding that could provide further insight on lrgAB function, it requires additional optimization beyond the scope and timeline of the current revision.\nRegarding the Triton X-100 sensitivity assay (Extended Data Fig. 4A in the original manuscript, Extended Data Fig. 3C in the revised manuscript), we have now performed 2 additional replicates, which we include below for the reviewer’s reference. The results are in line with those presented in the manuscript, showing no lysis across strains with or without Triton X-100. We have revised the figure legend to clarify that a representative result from N = 3 is shown.\n3) Intensity of the 35 kDa band corresponding to pro-IL-1B in Figure 5C is reduced in the gelE deletion strain relative to the fsr mutant at 4 and 6 hours. Is this due to non-specific cleavage/degradation?\nWe thank the reviewer for this observation. Our explanation to reviewer #1, question 3b, is highly relevant here:\n“We agree that in the absence of fsr, gelE, and proteolytically inactive gelatinase, there is progressive degradation of pro-IL-1β, which could be mediated by other unidentified factors (Fig. 5C in the original manuscript, Fig. 6E in the revised manuscript). Importantly, degradation is also observed when pro-IL-1β is incubated in plain media, suggesting this may represent protein instability rather than a specific bacterial activity. In contrast, when functional gelatinase is present, we observe the generation of a ~17 kDa fragment that remains stable throughout the incubation (Fig. 5C in original manuscript and Fig. 6E in the revised manuscript) and retains bioactivity (Fig. 5D in the original manuscript and Fig. 6F in the revised manuscript). Moreover, overexpression of gelatinase in the ΔgelE pTCV-gelE strain (Extended Data Fig. 3C in the original manuscript, Extended Data Fig. 4G in the revised manuscript) markedly increased cleavage of human pro-IL-1β into the ~17 kDa fragment compared to WT, indicating that the balance between degradation and cleavage is strongly influenced by gelatinase expression levels.”\nIn addition, we cannot exclude the possibility that loss of gelatinase induces compensatory expression of another protease.\n4) As the authors note, as vegetations develop and interior cells become protected from/less exposed to fluid flow, this should lead to activation of quorum sensing. Presumably cells that are on the exterior of the vegetation would still experience fluid flow, less Fsr activation, and lower production of GelE. Does GelE that would be produced by these interior cells have access to substrates like IL-1B given the spatial distribution of the vegetation? Can GelE diffuse out of the core of the vegetations to the surface or can IL-1B reach the interior of the vegetation?\nThank you for this comment. Before we address it, we would like to add that our hypothesis that microcolony localization in vegetation and exposure to flow will affect QS activity is now supported experimentally in Fig. 1D-F in the revised manuscript. Specifically, we demonstrate that QS expression is higher in microcolonies embedded within the vegetation compared to superficial ones. Moreover, QS activity is also dependent on microcolony size, with larger colonies showing high levels of QS expression regardless of their location.\nTo address the reviewer’s point, Fig. 6A shows that neutrophils infiltrate vegetations and surround biofilms embedded within them. We further show that IL-1β is present extracellularly at the interface between biofilms and neutrophil-derived NETs (Fig. 6A). Thus, while diffusion of gelatinase outward or IL-1β inward may occur, our data support a model in which pro-IL-1β activation is largely localized to sites of direct interaction between host immune cells and biofilm communities. Although in the revised manuscript we show that in the rat model gelatinase degrades pro-IL-1β without generating a bioactive fragment (Fig. 6C and Extended Data Fig. 4C in the revised manuscript), we expect that the comparable distribution of neutrophils around embedded biofilms in human vegetations would provide the spatial conditions for GelE to encounter and cleave human pro-IL-1β.\n5) Previous microarray work identified numerous genes and metabolic pathways differentially regulated in fsrB and gelE deletion mutants depending on growth phase (PMC1446981). Is there overlap between these genes and the fsr-regulated genes identified in the RNAseq sets in this study? This could be interesting to consider given the shift in metabolism that occurs in different growth phases.\nThank you for this comment. We focused our comparison on the set of genes previously reported as negatively regulated by fsr (Bourgogne et al, 2008, PMC1446981), since our study focuses on the impact of the absence of the fsr locus. Out of the 97 negatively regulated genes identified in Bourgogne et al, 2008, 49 overlapped with our RNAseq dataset comparing the transcriptomes of vegetations infected with WT or Δfsr strains at 72 hpi. These overlapping genes did not exhibit strong growth phase–dependent differences in Bourgogne et al, 2008. They were consistently negatively regulated regardless of the growth phase.\nAmong these, we highlight below the genes from our dataset showing >2-fold change in the absence of fsr in vegetations and are negatively regulated by fsr in Bourgogne et al., 2008:\nBourgogne et al., 2008 Antypas et al., 2025\nID old_locus_tag gene_name Description Fold Fold Fold logFC Pvalue FDR\n(Late (Entry (Early\nlog) Stat) Stat)\nPTS system\nEF0292 OG1RF_10235 component -3 -4 4.55023 0.00029 0.007\nornithine\ncarbamoyltransferase,\nEF0105 OG1RF_10100 argF catabolic -6 -2 2.96161 0.00088 0.01\nEF3327 OG1RF_12572 citrate transporter -4 2.784 0.00092 0.01\nEF1662 OG1RF_11374 buk butyrate kinase -11 2.69472 0.00792 0.031\nEF0104 OG1RF_10099 arcA arginine deiminase -5 -3 2.5987 0.00439 0.022\nEF0106 OG1RF_10101 arcC carbamate kinase -3 -3 -4 2.40184 0.00094 0.01\nconserved\nEF0108 OG1RF_10103 hypothetical protein -3 -3 -3 2.15922 9E-05 0.004\nPTS system\nEF1803 OG1RF_11512 component -2 2.07382 0.0122 0.039\nmalate\nEF1206 OG1RF_10978 oxidoreductase -2 -5 2.00827 0.00178 0.015\narcA, arcC, argF genes belong to the arginine deiminase (ADI) pathway which allows bacteria to degrade L-arginine under anaerobic conditions to generate ATP, while others encode sugar transporters (PTS components). Similarly, butyrate kinase is involved in ATP generation under anaerobic conditions. These findings suggest that fsr loss derepresses multiple metabolic functions that could enhance adaptation to nutrient and oxygen availability during infection. We also note that lrgAB was not identified as differentially expressed in the Bourgogne et al. study, despite being upregulated in the absence of fsr in our in vivo dataset. This suggests that some aspects of fsr-dependent regulation may be specific to the host infection environment and not apparent under in vitro growth conditions.\nWe have revised our Discussion to include the comparison with the findings from Bourgogne et al., 2008. See lines 486-493.\n6) The link between loss of gelE/fsr and accumulation of Ace was previously published. Did the authors see any changes in Ace expression in their RNAseq? Is Ace likely to contribute to overall vegetation structure? It might be beneficial to mention briefly given that Ace contributes to endocarditis pathogenesis (PMC3165527).\nWe thank the reviewer for raising this interesting point and for referring us to the relevant publication. Pinkston et al. (PMC3165527) demonstrated that gelatinase cleaves Ace from the cell surface. Regarding transcriptional regulation, they reported that “preliminary analysis of ace transcript abundance at different growth phases comparing the ΔfsrB strain to the wild-type strain showed no obvious differences” and that “a post-transcriptional mechanism might be responsible for the Ace surface display phenotype seen in the fsr mutants.” This is consistent with prior microarray analyses that did not identify ace as transcriptionally regulated by Fsr in vitro (Bourgogne et al., 2008).\nIn our study, we observed changes in ace transcript levels under different conditions. Specifically, RNAseq data revealed that ace expression was downregulated in the Δfsr strain compared to WT in vegetations at 72 hpi (LogFC = –1.8, Supplementary File 2). Taken together with the findings of Pinkston et al. and Bourgogne et al., our results suggest that the impact of fsr on ace expression differs between in vitro and in vivo contexts, potentially reflecting environment-specific regulatory mechanisms.\nWe also found that ace presence was significantly correlated with fsr presence (p = 2e-04) across the clinical isolates in our study, with only 2 fsr-negative strains harbouring ace (Extended Data Fig. 6). Thus, although ace in an important virulence factor in IE, it is unlikely to account for the increased biofilm formation, prolonged bacteremia, and higher disease severity observed in our study, as it was virtually absent in strains lacking fsrA.\n7) Figure 7F is somewhat confusing as it is not clear what each data point represents. Are only 3 patients with fsrA-negative isolates shown? It would be helpful to see bacteremia duration data points for all patients (if available).\nWe have revised Fig. 7F to display all data points alongside the median. Kindly see response to question 4a from reviewer #1.\n8a. Based on Supp Data 3, it looks like the proportion of patients with loss of fsrA is opposite in the Zurich and Pittsburgh cohorts (fsrA not present in a majority (17/24) patients from Zurich cohort, fsrA not present in a smaller fraction (21/57) patients from Pittsburgh cohort). Can the authors speculate on why that might be the case? Are there different predominant circulating strain types in Switzerland vs USA?\nWe appreciate the reviewer’s observation. In the Pittsburgh cohort, the predominant sequence types were ST6 (fsr ), ST179 (fsr ), and ST40 (fsr ), which helps explain the overall lower frequency of fsrA loss. The Zurich cohort is smaller and thus limits our ability to confidently determine dominant strain types. Nevertheless, we note that ST6 (fsr ), which was predominant in Pittsburgh, is absent from the Zurich isolates. This absence may contribute to the higher proportion of fsrA-negative strains observed in the Zurich cohort. We have updated the Results sections (lines 362-363) and added new Extended Data Fig. 5 to illustrate the different sequence types.\n8b. If so, given that bacteremia duration is similar between fsrA present/absent patients in each independent cohort, does that suggest there are other Ef genomic features driving bacteremia duration?\nWe thank the reviewer for this comment. The main reason that the bacteremia duration is similar between fsrA-positive and fsrA-negative infections within each cohort is is likely due to limited sample sizes, particularly in the Zurich cohort. To further address this question, we experimented expanding the Pittsburgh cohort from 57 to 119 isolates by incorporating enterococcal IE genome sequences recently published by two of our co-authors (https://doi.org/10.1093/infdis/jiaf272), while our current manuscript was under review. Within the expanded Pittsburgh cohort, bacteraemia duration was significantly longer in patients infected with fsrA-negative strains compared to fsrA-positive strains (p = 0.02). However, combining this expanded dataset with the Zurich cohort introduced cohort bias, and we therefore chose not to include this analysis in the revised manuscript. However, we include the plot comparing bacteremia duration within the Pittsburgh cohort below for the reviewer’s reference.\nMoreover, to determine whether other E. faecalis genomic features might underlie the observed differences in clinical outcomes, we examined virulence factors that anticorrelate with fsrA presence. We found significant anticorrelations between fsrA and esp, EF_0149, and some cytolysin operon genes (Extended Data Fig. 8 in the revised manuscript). However, none of these genes were significantly associated with prolonged bacteraemia or increased disease severity in our cohort (Extended Data Fig. 9). These findings support the conclusion that the absence of the fsr locus itself, rather than co-occurring virulence factors, is the most consistent genomic feature associated with more severe clinical outcomes and prolonged bacteremia. See also updated results text (lines 381-390).\n9) It looks like the absence of fsrA in isolates from patients (Figure 7G) results in bimodal distribution of disease severity score (1 cluster with scores 2-3, 1 cluster with scores 5-6) while presence of Fsr results in normal distribution of this metric, which is very interesting. Can the authors speculate on what might drive this?\nWe thank the reviewer for this observation. While there is a bimodal distribution of disease severity among patients infected with fsrA-negative isolates, we currently cannot determine the underlying cause. When we examined only the fsrA-negative isolates ordered by disease severity, we found no consistent patterns in the presence or absence of known virulence genes or sequence type. This suggests that the observed heterogeneity in clinical outcomes among fsrA-negative infections may be driven by factors beyond the set of genes we analyzed or patient variables that remain to be elucidated.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Antypas et al. explores how the Fsr (quorum sensing (QS) system influences biofilm formation and inflammation in infective endocarditis (IE) using Enterococcus faecalis as model organism. QS is a bacterial communication mechanism that regulates biofilm development and other collective behaviors through autoinducer signaling molecules. QS activation, driven by increased bacterial density and various environmental factors, can either promote or inhibit gene expression related to biofilm maturation or dispersal. This study highlights that fluid flow can prevent Fsr activation, while confined environments facilitate it. The research demonstrates that the Fsr system restricts biofilm growth and triggers inflammation by upregulating gelatinase, which activates IL-1β. Loss of the Fsr system results in unchecked biofilm growth, reduced inflammation, and increased antibiotic tolerance, correlating with severe disease outcomes.\nThe authors provided solid in vitro and in vivo data to support their major conclusions; the large amount of work involved in the project should also be appreciated. However, the study lacks novelty as it reiterates well-established concepts in QS and biofilm research. The role of QS in regulating biofilm formation and its impact on infection outcomes has been extensively studied in various human pathogens, including E. faecalis. While the specific focus on the Fsr QS system in the context of IE is valuable, the overall findings do not significantly advance our current understanding of QS mechanisms or their implications in biofilm-associated infections. General and more specific comments follow.\nWe thank reviewer #4 for reviewing our manuscript but we respectfully disagree with the overall assessment that “the study lacks novelty as it reiterates well-established concepts in QS and biofilm research” and that “while the specific focus on the Fsr QS system in the context of IE is valuable, the overall findings do not significantly advance our current understanding of QS mechanisms or their implications in biofilm-associated infections”.\nAs we highlighted in our introduction, while the virulence factors that facilitate initial adhesion to thrombus during early enterococcal IE have been identified, the regulation of biofilm formation and its impact on disease progression remain largely overlooked. Moreover, previous in vitro studies established the Fsr QS system as a positive regulator of biofilm formation. Our work directly contradicts this long-standing concept by demonstrating that loss of Fsr promotes biofilm growth and worsens infection outcome. Our study highlights that the role of QS in E. faecalis is context-dependent, and to our knowledge, this is the first study to define the impact of fsr absence in enterococcal IE. Given the homology between Fsr in E. faecalis and the Agr system in S. aureus, our findings also have broader implications for understanding the role of QS in staphylococcal IE, where similar context-dependent regulatory effects may be at play.\nFinally, despite extensive literature proposing anti-QS strategies as potential therapies against biofilm-associated infections, our results demonstrate that QS does not universally promote biofilm formation. In the case of enterococcal IE, Fsr activity actually restricts biofilm growth. This underscores the importance of context when considering QS as a therapeutic target and cautions against assuming that QS inhibition will always be beneficial in treating biofilm-related infections.\nPlease see our answers to the specific comments below.\n1. A rationale should be provided for using stationary-phase cells when incubating bacteria under fluid flow conditions.\nWe thank the reviewer for this comment. We chose to use stationary-phase E. faecalis cells for the flow chamber experiments because vegetations represent a nutrient-limited environment that more closely resembles stationary-phase physiology rather than exponential growth, which primarily reflects an in vitro state in nutrient-rich media.\n2. Since the fsrA gene is under the control of a constitutive promoter, how do you explain the results obtained after exposure to fluid flow conditions? Still related to the gene expression data, it is not clear why the authors normalized Ct values obtained to the expression of recA gene (used as reference) instead of normalizing to the number of cells, since we can assume that at 20 dynes/cm2 there are fewer cells attached.\nfsrA is under a constitutive promoter that sustains basal expression, but further upregulation of the Fsr QS system requires accumulation and sensing of the GBAP autoinducer. Fluid flow reduces GBAP accumulation through advection, thereby preventing upregulation of the fsr regulon, explaining our results in Fig. 1.\nRegarding the question about qPCR, we understand that the reviewer refers to Fig. 1C. To clarify, we used the geometric mean of recA and dnaB, based on their stable expression under both static and fluid flow conditions as indicated by transcriptomic data and validation using the Bestkeeper v1 analysis tool. This information is present in the original and revised manuscript in Methods section “Quantitative PCR”.\nSecond, we used equal amounts of total RNA from static and shear-stress samples for cDNA synthesis (we have emphasized that in the qPCR result section in the revised manuscript). Moreover, normalization to stably expressed reference genes such as recA and dnaB is required to compare gene expression across different conditions, because it corrects for differences in RNA yield, cDNA synthesis efficiency, and template input. In contrast, normalizing to cell numbers would not account for these variables and would therefore not provide valid expression comparisons.\n3. While the authors concluded their first section by stating that fluid flow impacts QS and triggers physiological adaptations in E. faecalis, it is unclear how they formulated the hypothesis presented in the second section based on the results shown in Figure 1. The connection between the observed effects of fluid flow on QS and the proposed limitations on Fsr activation during early colonization required further clarification.\nWe thank the reviewer for this comment. In the revised manuscript, we have added experimental evidence on how the fluid flow affects QS expression in vivo to further support the hypothesis formulated in the second results section.\nAs we also replied to question #1 from reviewer #1, “We constructed an E. faecalis strain carrying a reporter plasmid expressing GFP under the control of the gelatinase promoter (WT Pgele-GFP), alongside a control strain carrying the reporter plasmid with a constitutive promoter (WT Pcfb-GFP). We added a new supplementary figure (Fig. S1A-H) demonstrating that this reporter plasmid is specifically activated by Fsr QS in vitro. Importantly, new in vivo data in Fig. 1D-F show that Fsr QS activation depends on the location and size of the microcolony. Vegetation embedded microcolonies exhibit higher QS activation than superficial ones, and large microcolonies display higher activation than small ones. In contrast, WT Pcfb-GFP bacteria remained fluorescent regardless of location (Fig. S1K). We further confirmed plasmid stability at 24 hpi in vivo (Fig. S1I–J), ruling out plasmid loss as a cause of false-negative gelatinase expression.”\nWe have also revised the text in the Results sections (lines 135-151, 154-155, 162-164, 167-171) to provide a clearer transition from the first to the second and third section. Overall, these new data provide support for our hypothesis that the combination of fluid flow and low population density during early colonization restricts GBAP accumulation superficial microcolonies, thereby limiting Fsr activation in superficially adhered bacteria.\n4. The section describing the impact of the absence of gelE and sprE genes on biofilm growth could be shortened. In fact, the data presented could be merged with the previous section describing the impact of the fsr knockout mutant on biofilm growth in late IE.\nWe thank the reviewer for the suggestion but we prefer to keep the sections on downstream genes modulated by the fsr locus (e.g., gelE/sprE, lrgAB) separate, in order to emphasize their individual contributions to IE in the presence or absence of fsr.\n5a. IE typically affects injured or abnormal heart valves, which often remain asymptomatic until a blood-borne infection occurs. The most common etiological agents are oral streptococci, S. aureus, and E. faecalis. In the case of E. faecalis, previous studies have found that neither the fsr nor gelatinase production is more common in disease-associated isolates than in isolates colonizing healthy individuals. A surface protein, Esp, has been described as being enriched among endocarditis and bacteremia isolates of E. faecalis but is rare among fecal isolates.\nAs noted in the Discussion section of the manuscript, we do state that the fsr locus is variably present among E. faecalis strains isolated from infective endocarditis, blood, urine, and feces, and we cite the study the reviewer refers to (lines 433-435). However, we also emphasize that the role of fsr in influencing disease severity has not been previously explored (lines 433-435). Our study explicitly addresses this gap by demonstrating that the absence of the fsr locus correlates with larger biofilms and greater antibiotic tolerance in vivo and is also associated with prolonged bacteraemia and higher disease severity scores in patients with infective endocarditis.\nRegarding the reviewer’s comment about esp, we did not observe enrichment of this gene in our IE cohorts, as its frequency was approximately 50% (Extended data Fig. 6 in the revised manuscript). In our analysis, esp was significantly anticorrelated with fsr (p = 4.6 × 10 ⁸, Extended Data Fig. 8 in the revised manuscript), but it was not associated with prolonged bacteremia or high disease severity scores (Extended Data Fig. 8 in the revised manuscript). Moreover, the OG1RF strain used in our in vitro and in vivo experiments does not harbor esp, further supporting that esp is not implicated in the phenotypes we observed.\n5b. Clinical isolates of E. faecalis found in urine often exhibit a chromosomal deletion involving the fsr locus, while some other studies have reported the presence of the fsr locus in endocarditis isolates. Without a deeper genomic analysis of the 81 enterococcal isolates, the results presented in Figure 7 and Extended Figure 5 add little to the manuscript.\nAs part of the revision, we have sequenced all isolates from the Zurich cohort. Combined with the previously sequenced isolates from the Pittsburgh cohort, we now provide a comprehensive genomic analysis of all 81 enterococcal strains. Specifically, we report the presence of additional virulence factors (Extended Data Fig. 6 in the revised manuscript), show that esp and EF0149 are significantly anticorrelated with fsr but do not account for the observed phenotypes (Extended Data Fig. 9 in the revised manuscript), and present the sequence types for all isolates (Extended Data Fig. 5 in the revised manuscript). Collectively, these data strengthen our conclusion that the absence of fsr is associated with prolonged bacteremia and higher disease severity scores.\n6. Although the authors performed gene ontology enrichment analysis (Fig. 1B), it would be beneficial to provide details regarding the most important factors involved in E. faecalis biofilm patterns, including initial attachment, biofilm maturation, dispersal. Specifically, the prominent adhesive factor Ebp (endocarditis and biofilm-associated pilus) should be highlighted for its similarity to the fibronectin-binding protein found in S. aureus, another agent involved in IE. Other factors of interest include the cell wall anchored collagen adhesin, EbfA, BgsA, and SagA adhesins.\nWe thank the reviewer for this comment. In our dataset comparing gene expression between static and fluid flow conditions, these adhesion factors did not show striking changes: ace was modestly upregulated (1.73-fold), while the ebp pilus operon was slightly downregulated (ebpA –0.54, ebpB –0.57, ebpC –0.72). ebfA (OG1RF_11019), bgsA (OG1RF_12193), and sagA-like genes (salA, OG1RF_12331; salB, OG1RF_10281) were not differentially expressed. This data is already provided in Supplementary File 1 in the original manuscript for readers to explore in detail.\nGiven that no major differential expression was observed for these adhesins and that the primary focus of this work is quorum sensing–mediated regulation, we have not expanded this section further. We agree that adhesion under flow is an important aspect of E. faecalis biology and will address it in detail in a future manuscript currently in preparation."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My comments have been addressed", "reviewer_2": "This is a revised manuscript by Antypas et al. investigating the role of the Fsr quorum sensing system in enterococcal infective endocarditis. The authors have thoroughly revised the manuscript and addressed major critiques from the first round of review, resulting in a compelling story. My only comment for the submitted revision is that, from my reading, whole-genome sequencing was not done on the lrgA/B mutants. The authors comment on problems with complementation via overexpression. However, the conclusions from this portion of the paper would be strengthened if whole-genome sequencing were done on all lrgA/B mutants (including the fsr lrgAB deletion strain) to ensure that no additional mutations are present in these strains that may confound the results. Otherwise, I think the authors have addressed all major points from the first round of reviews.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter