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Open Access

Peer-reviewed

Research Article

Exploiting peptide chirality and transport to dissect the complex mechanism of action of host peptides on bacteria

Abstract

Elucidation of the complex mechanisms of action of antimicrobial peptides (AMPs) is critical for improving their efficacy. A major challenge in AMP research is distinguishing AMP effects resulting from various protein interactions from those caused by membrane disruption. Moreover, since AMPs often act in multiple cellular compartments, it is challenging to pinpoint where their distinct activities occur. Nodule-specific cysteine-rich (NCR) peptides secreted by some legumes, including NCR247, have evolved from AMPs to regulate differentiation of their nitrogen-fixing bacterial partner during symbiosis as well as to exert antimicrobial actions. At sub-lethal concentrations, NCR247 exhibits strikingly pleiotropic effects on Sinorhizobium meliloti. We used the L- and D-enantiomeric forms of NCR247 to distinguish between phenotypes resulting from stereospecific, protein-targeted interactions and those caused by non-specific interactions such as membrane disruption. In addition, we utilized an S. meliloti strain lacking BacA, a transporter that imports NCR peptides into the cytoplasm. The bacterial protein BacA, plays critical symbiotic roles by possibly reducing periplasmic peptide accumulation and fine-tuning symbiotic signaling. Use of the BacA-deficient strain made it possible to distinguish between phenotypes resulting from peptide interactions in the periplasm and those occurring in the cytoplasm. At high concentrations, both L- and D-NCR247 permeabilize bacterial membranes, consistent with nonspecific cationic AMP activity. In the cytoplasm, both NCR247 enantiomers sequester heme and trigger iron starvation in a chirality-independent but BacA-dependent manner. However, only L-NCR247 activates bacterial two-component systems via stereospecific periplasmic interactions. By combining stereochemistry and genetics, this work disentangles the spatial and molecular complexity of NCR247 action. This approach provides critical mechanistic insights into how host peptides with pleiotropic functions modulate bacterial physiology.

Author summary

Many organisms produce antimicrobial peptides (AMPs) to fight infections, but some legumes have uniquely co-opted these molecules to control their symbiotic partners. During symbiosis between Medicago truncatula and Sinorhizobium meliloti, the plant secreted Nodule-specific Cysteine-Rich (NCR) peptides, transforms free-living bacteria into differentiated bacteroids that fix nitrogen but cannot reproduce outside the host. One such peptide, NCR247, exerts pleiotropic effects on the bacteria, acting on different subcellular locations, including membrane, heme, and proteins. Using a mirror-image (D-form) peptide, we disentangled peptide effects arising from generic physiochemical interactions versus stereospecific binding. The bacterial inner membrane protein BacA is known to play a protective role by importing NCR peptides into the cytoplasm. Using a bacterium lacking BacA, we were able to distinguish the effects of the peptide within and outside the cytoplasm. It was thought that BacA safeguards symbiotic bacteria by internalizing NCR peptides, thereby limiting their toxic membrane-lytic effects; however, this has not been demonstrated. Our results allow us to infer that BacA prevents lethal overstimulation of signaling pathways in the periplasm by internalizing the peptides. Our methods provide a framework for testing the mechanism of action of new peptide-based antibiotics to combat multidrug-resistant bacteria.

Citation: Sankari S, Arnold MFF, Babu VMP, Deutsch M, Walker GC (2025) Exploiting peptide chirality and transport to dissect the complex mechanism of action of host peptides on bacteria. PLoS Genet 21(12): e1011892. https://doi.org/10.1371/journal.pgen.1011892

Editor: Aimee Shen, Tufts University School of Medicine, UNITED STATES OF AMERICA

Received: September 23, 2025; Accepted: December 3, 2025; Published: December 11, 2025

Copyright: © 2025 Sankari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The raw data behind the graphs has been made available as supporting material.

Funding: This work was supported by the National Institutes of Health (NIH) Grant RO1 GM031010 (to G.C.W) and funds from the Stowers Institute for Medical Research https://www.stowers.org/ (to S.S). GCW is an American Cancer Society Professor. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Host secreted antimicrobial peptides (AMPs) constitute a crucial component of the mammalian innate immune system, characterized by their killing activity against pathogenic bacteria, fungi, viruses, and parasites. Beyond pathogen defense, host-secreted peptides also modulate symbiotic relationships [1–3]. One particularly interesting class of defensin-like peptides is secreted by legumes of the Inverted Repeat Lacking Clade and some belonging to the Dalbergoid clade [4]. They are called Nodule-specific Cysteine-Rich peptides (NCRs) and play an important role in modifying the life cycle of endosymbiotic bacteria [5]. The free-living, soil-dwelling bacteria enter developing nodules, chronically infect the plant cell, and endosymbiotically reside inside plant-derived symbiosomes. This symbiotic relationship is agriculturally, ecologically, and economically important. In the partnership between the Inverted Repeat Lacking Clade (IRLC) legume Medicago truncatula and Sinorhizobium meliloti, the free-living monoploid bacteria infect the plant, but the plant takes control over some of the bacteria, turning them into polyploid, terminally differentiated endosymbionts, called bacteroids [5]. During this process, terminally differentiated bacteroids lose their ability to survive outside the host. The plant achieves this extreme control over the life cycle of bacteria by targeting bacteria with an arsenal of ~700 small peptides known as NCR peptides, which contain conserved cysteine motifs. They drive profound physiological, transcriptional, and morphological changes underlying bacteroid differentiation, which include endoreduplication, altered membrane permeability, and cell elongation or branching. NCR production is tightly developmentally coordinated, with successive waves secreted into symbiosomes via an endoplasmic reticulum–dependent pathway requiring the DNF1 nodule-specific signal peptidase [6].

Approximately one-third of NCR peptides in Medicago truncatula are cationic [7] and, at high concentrations, exhibit potent antimicrobial activity against a range of bacteria and fungi [8,9]. In the nodule micro-environment, however, these peptides are non-lethal, instead modulating bacterial metabolism and altering their life cycle [10]. NCR247, a well-studied NCR peptide, exerts complex effects on bacterial physiology through a combination of direct physicochemical interactions with bacterial cell envelopes and specific molecular engagements with intracellular targets [11]. These effects are often pleiotropic, simultaneously involving membrane disruption, modulation of signaling pathways [12], interference with translational machinery [13], and alteration of metal homeostasis [14]. In Sinorhizobium meliloti, BacA, an inner membrane protein critical for bacterial survival within host environments [15], facilitates the uptake of host-derived NCR peptides into the cytoplasm [16,17]. This is presumed to protect the bacterium from the peptides’ deleterious effects, thereby enabling intracellular persistence and symbiotic nitrogen fixation.

Many AMPs combat multidrug-resistant bacteria, making them promising candidates for therapeutic development [18]. While their classical activity involves pore formation or membrane permeabilization [19,20], increasing evidence shows that AMPs also translocate into cells and act on intracellular targets [21,22]. Intracellularly, they are shown to target vital biochemical processes, including DNA [23,24], RNA [25], and protein synthesis [26,27], enzymatic functions [28], and cell wall biosynthesis [29–33], highlighting their multifaceted mechanisms and potential to limit the development of resistance.

A significant obstacle to elucidating the precise mechanisms of NCR peptides and other antimicrobial peptides lies in resolving the multiplicity of subcellular compartments of the Gram-negative symbiotic bacteria—outer membrane, periplasm, cytoplasmic membrane, and cytoplasm—in which they exert their effects. Another difficulty lies in distinguishing whether their biological activities arise from non-specific interactions with membranes and small molecules or from specific protein–peptide engagements. This complexity is compounded by the capacity of these peptides to permeate bacterial barriers and modulate distinct physiological processes in various cellular locales, making it challenging to determine the contribution of each compartment and interaction type to the overall antimicrobial or symbiotic outcome. Disentangling chirality-dependent, target-specific interactions from generic chirality-independent, charge and hydrophobicity-driven effects is crucial for the rational development of these peptides as antimicrobial agents.

Here, we address the complex mode of action of the nodule-specific cysteine-rich peptide NCR247 on the Gram-negative bacterium S. meliloti using two complementary experimental approaches. First, we employed stereospecificity analysis using D-NCR247, the D-enantiomeric form of NCR247, in which all amino acid residues are in the D-configuration, to distinguish phenotypes that arise from stereospecific protein–peptide interactions from those mediated by non-stereospecific membrane or small molecule binding effects. D-enantiomeric peptides are commonly used in studies because their unnatural stereochemistry confers exceptional resistance to proteolytic degradation, allowing them to maintain stability and bioactivity longer in biological environments [34,35]. Here, we took advantage of their mirror-image stereochemistry that renders their three-dimensional structures incompatible with the binding sites of proteins evolved to recognize L-amino acid configurations.

Second, we used a a bacA deletion mutant to differentiate between phenotypes resulting from NCR247 activity within the cytoplasm versus those occurring outside the cytoplasm, given the established role of BacA in peptide uptake and intracellular delivery [16]. Through this dual approach, we aimed to parse chirality-dependent effects from spatial determinants of peptide action, thereby refining our understanding of NCR247’s mechanisms under symbiotically relevant sublethal concentrations. While BacA is postulated to protect bacteria by sequestering peptides away from membrane targets through cytoplasmic internalization, the mechanistic basis for this protective role remains speculative, especially given that NCR peptides are at sublethal concentrations within nodules. Our work here supports a specific role for BacA in shielding bacteroids under sublethal NCR247 exposure, a condition highly pertinent to the symbiotic state.

Results

Achiral membrane disruption by NCR247 at high concentrations

Cationic antimicrobial peptides are known to induce visible alterations such as bleb and blister formation on the cell surface and, eventually, lysis [36]. L- and D-NCR247 are highly cationic peptides with a pI of 10.5. They also have a hydrophobic residue content of approximately 29%, which facilitates their insertion into microbial membranes. Their exceptionally high Boman index of 4.63 kcal/mol, which is the highest among NCR peptides, indicates a strong potential for binding to other proteins and membrane components due to hydrophobic interactions. This balance between hydrophobic and cationic residues is considered to underpin its membrane-disruptive function [37]. L-NCR247, when used at higher concentrations, has been shown to induce membrane blebbing and compromise membrane integrity in bacteria like S. meliloti, Escherichia coli, Bacillus subtilis, and Salmonella enterica, presumably due to non-chiral, direct interaction with the membrane [37,38]. To test if D-NCR247 exhibits the same phenotype on S. meliloti, we treated S. meliloti with 20 μM of L and D-NCR247 and performed a cell viability assay to determine toxicity. As expected, both L and D-NCR247 were equally bactericidal to the cells at higher concentrations (Fig 1A). Scanning electron microscopy (SEM) revealed comparable morphological features such as membrane blebbing, distortion, and cellular shrinkage in 15 μM peptide-treated samples (Fig 1B). This shows that at higher concentrations of the peptide, chirality-independent interactions cause membrane destabilization, resulting in cell death. This aligns with the properties of typical antimicrobial peptides that rely on charge and amphipathy-based interactions with the bacterial membranes rather than specific recognition [39].

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Fig 1. Chirality-independent cell killing and membrane disruption effects.

(A) A 100-fold reduction in colony-forming units when treated with 20 μM of L (blue) or D-NCR247 (green) when compared to untreated cells (black). (B) Scanning electron micrographs showing membrane blebs and blisters in 15 μM L- or D-NCR247 treated cells when compared to untreated cells. Data in A is presented as the mean of three biological replicates ± s.d. ****P < 0.0001 versus untreated samples; two-way analysis of variance (ANOVA) with multiple comparisons. ns-no significant changes noted.

https://doi.org/10.1371/journal.pgen.1011892.g001

L- and D- NCR247 binding to BacA

BacA has previously been shown to transport a wide range of peptides, including bleomycin [16]. The recent structure of SbmA, an E. coli homolog of BacA, reveals how the peptide binding cavity facing the periplasm in an open-outward conformation could allow the protein to promiscuously transport anti-microbial peptides [40]. Due to this promiscuity, it is reasonable to assume D-NCR247 will be transported through BacA, similar to L-NCR247. We tested whether D-NCR247 can bind purified SbmA with a similar binding curve to L-NCR247 in a Biolayer Interferometry experiment. Here, biotinylated L- or D-NCR247 was immobilized on a streptavidin sensor, and purified SbmA was tested for binding to the peptides. Both L and D-NCR247 bound to SbmA to the same extent (Fig 2A and 2B), further bolstering the idea that D-NCR247 could be transported by the same mechanism as L-NCR247.

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Fig 2. Binding of L and D-NCR247 to SbmA (BacA homolog) Similar level of binding of L-NCR247 (A) and D-NCR247 (B) to SbmA in a biolayer interferometry experiment.

Biotin-tagged L- or D-NCR247 was immobilized to the streptavidin sensor, and purified SbmA was loaded. Binding curve of SbmA interacting with the peptides is shown. L-, D- NCR247, and SbmA were loaded separately onto the sensors directly as controls to rule out non-specific binding to the sensor.

https://doi.org/10.1371/journal.pgen.1011892.g002

Testing of symbiotically relevant phenotypes at sub-lethal levels of L- and D-NCR247

Next, we investigated the effects of sublethal concentrations of NCR247, which have previously been shown to modulate several physiologically pertinent processes to symbiosis [12]. To help us infer the underlying mechanisms, we employed both the L- and D-enantiomers of NCR247, and compared their effects on WT vs ΔbacA mutant cells. This approach allowed us to systematically re-examine a variety of phenotypes previously attributed to NCR247, and to identify the contributions of protein specificity and BacA-dependent uptake to these symbiotically relevant responses.

  1. a) Chirality dependent activation of FeuP-FeuQ and ExoS-ChvI Two-Component Systems

Our previous microarray analyses examining the effects of sublethal concentrations of L-NCR247 on synchronized S. meliloti cultures revealed an extensive transcriptional response that included the upregulation of two regulons critical for symbiosis [12]. The FeuQ/FeuP regulon is controlled by a two-component response signaling system in which FeuP is the response regulator and FeuQ is an inner membrane protein with a short N-terminal cytoplasmic domain, a periplasmic sensor domain and C-terminal cytoplasmic histidine kinase response regulator domain [41]. Together they regulate genes, including ndvA, a cyclic beta-glucan exporter [41]. This regulon is usually activated under osmotic stress. The ExoS/ChvI regulon is involved in regulating multiple genes involved in exopolysaccharide production, motility, flagellar biosynthesis, and cell envelope integrity. ExoS has an overall structural organization similar to FeuQ with a periplasmic sensor and a cytoplasmic histidine kinase, while ChvI is the regulator [42–44]. smc01581 is a known direct target of ChvI [45]. To determine whether the activation of these regulons results from chirality-dependent interactions or from a general membrane stress response, we performed RT-qPCR analysis of representative genes from these regulons following treatment with 4 µM of either the L- or D-isoform of NCR247. Treatments were performed as described previously [12]. As expected, genes from both regulons were increased in expression level upon L-NCR247 treatment. Transcript levels for multiple ExoS/ChvI-regulated genes, such as smb21440 and chvI were significantly upregulated upon L-NCR247 treatment. In contrast, there was very little to no increase in gene expression in these regulons when the cells were treated with D-NCR247, showing that these signaling responses are due to stereospecific interactions of the peptide with proteins, rather than general membrane stress due to the cationic nature of the peptide (Figs 3A, 3B, and S1A and S1B). To determine whether signaling due to chirality-dependent interaction occurs in the periplasm or inside the cytoplasm, we tested the same peptide treatments in a ΔbacA mutant. In the ΔbacA mutant, the increase in gene expression of these regulons was significantly higher than that of WT when treated with L-NCR247, suggesting that there may be increased accumulation of peptide in the periplasm, which may lead to the observed overstimulation of these signaling pathways. In contrast, the D-NCR247 elicited minimal changes in gene expression and remained similar to untreated, confirming that the chirality-dependent interactions within the periplasmic space are responsible for the upregulation of FeuQ/FeuP and ExoS/ChvI two-component signaling by NCR247 (Figs 3A, 3B, S1 A and S1B).

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Fig 3. Chirality-dependent interactions in periplasm stimulate two-component signaling.

Increase in expression of a gene target of FeuP/FeuQ signaling (ndvA) (A) and gene target of ExoS/ChvI signaling (smc01581) (B) upon treatment with 4 µM L-NCR247(blue) when compared to untreated cells (black) as quantitated by RT–qPCR analysis. Treatment with 4 µM D-NCR247 shows a significant reduction in expression in both cases (Green). In ∆bacA mutant there is significant increase in expression when treated with L-NCR247 when compared to wildtype treated at the same condition. The data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene smc00128 and are presented as an average of three technical replicates ± s.d, Two-way analysis of variance (ANOVA) with multiple comparisons was used to calculate p values. ****, p ≤ 0.0001;*, p ≤ 0.05.

https://doi.org/10.1371/journal.pgen.1011892.g003

  1. b) Cell cycle regulation is only partially modified by chirality-dependent interactions

We then focused on the CtrA regulon, given its established role in controlling cell division and polarity [12,46]. Treatment with a sublethal dose of NCR247 leads to expression changes in several genes of the CtrA regulon [12]. However, unlike the FeuP and ExoS regulons, the molecular components involved in CtrA signaling are not well understood. Gene expression analysis demonstrated that treatment with L-NCR247 led to a significant reduction in the expression of ctrA and gcrA, genes involved in cell cycle regulation (Figs 4A and S2A). Upon D-NCR247 treatment, a more modest reduction in expression was observed, which was nevertheless significantly different from the untreated samples. Thus, the responses associated with cell division inhibition are only partially reliant on the chirality-dependent interaction of the peptide. Since it has been previously shown that LL37 and Polymyxin B, despite being cationic, cannot elicit cell cycle changes in S. meliloti [12], the partial chirality-independent response may not be due to cationicity but rather another property of NCR247.

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Fig 4. Phenotypes associated with peptide action in cytoplasm.

(A) Decrease in ctrA expression occurs when cells are treated with 4 µM L-NCR247. Comparable treatment with D-NCR247 causes less of a decrease as quantified by RT-qPCR analysis. Same treatments on ∆bacA mutant show a similar but modest response. (B) Increase in expression of heme import gene hmup, that is part of RirA regulon upon L-NCR247 treatment as analyzed by RT-qPCR analysis (blue) as compared to untreated samples(black). Larger increase upon D-NCR247 treatment is noticed (green). Significant reduction of hmuP expression upon both L-NCR247 and D-NCR247 treatment in ∆bacA mutant when compared to treatments on wildtype. For A and B, the data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene smc00128 and are presented as an average of three technical replicates ± s.d. (C) Increase in cellular iron content upon treatment with 4 µM of L-NCR247 (blue) and a larger increase occurs upon treatment with 4 µM of D-NCR247 (green) in the wildtype, measured by ICPMS analysis. No significant change was observed in a ∆bacA mutant upon treatment when compared to the untreated. Data are presented as the mean of three biological replicates ± s.d. (D) Fluorescence measurement (λex = 485 nm, λem = 530 nm) after treatment with lowering concentrations of L (blue) or D (green)-NCR247 in an in vitro translation assay using GFP vector. Data is presented as the mean of five independent reactions ± s.d. In A, B, and C, two-way analysis of variance (ANOVA) with multiple comparisons was used to calculate p values. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

https://doi.org/10.1371/journal.pgen.1011892.g004

To test whether this effect depended on cytoplasmic entry of the peptide, we repeated these experiments using a ΔbacA mutant strain. We observed a modest decrease in ctrA and gcrA gene expression following treatment with both L- and D-NCR247 compared to wild type, indicating that gene regulation by the peptides is partially independent of BacA-mediated import. Additionally, the difference in response between L- and D-NCR247 treatments was also modestly reduced relative to wild type, suggesting that stereospecific effects distinguishing L- and D-forms are at least partially dependent on cytoplasmic localization, with their distinction becoming less pronounced in the absence of BacA.(Figs 4A and S2A). Since the responses are not apparent as in FeuP and ExoS signaling, there are likely some cytoplasmic mechanisms involved directly or indirectly in the control of the CtrA regulon.

  1. c) Cytoplasmic Sequestration of Heme by NCR247 and induction of Iron Starvation Response is not chirality-dependent.

Next, we investigated changes in iron-related gene expression regulated by the iron-responsive transcriptional regulator RirA. Reduced NCR247 binds heme with high affinity [14]. This sequestration of cytoplasmic heme deprives the cell of a critical iron source, thereby inducing genes involved in the iron starvation response. We previously showed that the ∆bacA mutant is defective in inducing iron import genes, indicating that heme sequestration occurs in the cytoplasm [14]. We have biochemically shown that the D-NCR247 is capable of binding heme to the same extent as L-NCR247 [14]. Here, we tested whether D-NCR247 elicits the induction of genes involved in iron import similar to L-NCR247. In contrast to the two-component systems discussed above, when S. meliloti was treated with either L-NCR247 or D-NCR247, we observed upregulation of multiple iron acquisition and transport genes, including hmuP, entC, and fhuP, which was observed with D-NCR247 as well. As expected, this response was abolished in a ∆bacA mutant, showing that indeed iron-related responses are due to the chirality-independent interaction of NCR247 with heme in the cytoplasm (Figs 4B, S2B and S2C). To confirm that the iron content increases upon an increase in expression of iron import genes, we measured cellular iron content using ICP-MS following treatment with L- or D-NCR247. Similar to its effect on gene expression, D-NCR247 induces an increase in iron content of the cells when compared to untreated cells, and this response is greatly diminished in a ∆bacA mutant (Fig 4C). Interestingly, we noticed that there was a significant increase in the expression of iron uptake genes and iron content upon treatment with D-NCR247 compared to the increase observed with L-NCR247. This suggests that L-NCR247 may undergo either degradation or modification after entering the cytoplasm. To test this, we performed a pull-down assay with biotinylated L- and D-NCR247-treated cell cytoplasmic extracts. After incubation for 3 hours at room temperature, a silver stain of the pull-down clearly showed a band of the expected size (~3 kDa) of NCR247 when D-NCR247 was used. Yet this band was not seen in extracts after the L-NCR247 pull-down. This suggests that there may be cellular factors, such as proteases, that act on L-NCR247 to modify or degrade the same (S2D Fig). This is important in the context of anti-microbial peptides, where BacA is suggested to play a role in protection against host peptides.

BacA is not only essential for peptide transport but also influences the modification of very long chain fatty acids (VLCFAs) in the outer membrane, affecting membrane stability [47,48]. To distinguish between these roles, a ΔbacA mutant complemented with a construct encoding a R389G BacA variant—defective in VLCFA modification but proficient in peptide uptake [16]—showed restoration of iron-related phenotypes similar to wild-type bacA complementation. Structural studies of the homologous transporter SbmA by cryo-EM revealed an outward-open conformation with a conserved glutamate (E203 in SbmA, E207 in S. meliloti BacA) essential for proton binding and transport activity (36). Mutation of this residue (E207A) resulted in loss of BacA function and recapitulated the ΔbacA phenotype, underscoring the necessity of BacA’s peptide transport activity, rather than VLCFA modification, for mediating NCR247’s iron-related phenotypes (S2E Fig).

  1. d) Inhibition of Translation by NCR247 Is Partially Dependent on Peptide Chirality

Components of the protein translation machinery are the targets of some anti-microbial peptides [26,49]. Pull-down experiments with NCR247 have shown that many ribosomal proteins are capable of interacting with the peptide [11]. Additionally, previous in vitro translational assays have shown that L-NCR247 is capable of inhibiting translation [13]. To determine whether this inhibition is chirality-dependent, we performed in vitro coupled transcription-translation assays using GFP as a reporter. Reactions with L-NCR247 showed a dose-dependent reduction in GFP fluorescence intensity, indicating a reduction in protein synthesis. D-NCR247 treatment resulted in a reduction in translation efficiency, but the effect was smaller when compared to L-NCR247, suggesting that both chiral and achiral mechanisms may contribute to translational inhibition by NCR247(Fig 4D). We also confirmed these results by performing a western blot analysis against GFP which showed that changes in protein levels are comparable to the measurements using fluorescence (S3A and S3B Fig). These results support the model that, after NCR247 is internalized by BacA-mediated import, NCR247 may interact with cytoplasmic targets beyond heme, such as ribosomal components and RNA-binding proteins, in a manner that is partially chirality dependent. We had previously reported that one of the three regioisomers of disulfide cross-linked NCR247 was especially potent at inhibiting translation in vitro, and the cysteine residues are important for this activity [13]. To test if the partial chirality independent phenotype is caused by cationic nature of the peptide, we tested the ability of LL37 to inhibit in vitro translation at same molarity. LL37 showed a very diminished phenotype as compared to the L-NCR247 and was lesser than the inhibition by D-NCR247 (S3C Fig). This shows that cationic nature of the peptide may not be fully responsible for the phenotypes observed.

Mechanistic insights into protection of bacteroids by BacA using chiral peptides

BacA carries out two conceptually distinct critical roles in symbiosis. One role is to deliver certain NCR peptides into the cytoplasm where they can exert their critical intracellular symbiotic roles, for example, the induction of iron import via heme-sequestration in the case of NCR247 [14]. The other is to protect the endocytosed bacteria within the nodule cells from the potentially lethal anti-microbial peptide like effects of certain NCR peptides, including NCR247, by importing them into the cytoplasm, thereby reducing their concentration in the periplasm and the cytoplasmic membrane [50]. A BacA-deficient mutant must experience higher levels of NCR peptides in its periplasm than a wildtype during symbiosis, which can account for the increased cell death in ∆bacA mutant during symbiosis. This increased cell death of ∆bacA mutant cells has generally been attributed to increased membrane damage caused by the higher levels of NCR peptides in the periplasm [50,51], but this has not been tested to date.

A second possible death mechanism is suggested by: i) our observation of chirality-dependent periplasmic overstimulation of FeuP and ExoS signaling, and ii) a previous report that the absence of negative regulation of FeuP, which is mediated by FeuN, results in hyperactivation of FeuP/FeuQ signaling that leads to loss of S. meliloti viability [52]. Connecting these two observations, we hypothesized that the hyperactivation of the two-component signaling caused by possible increased levels of NCR247 in the periplasm of the ΔbacA mutant may lead to detrimental effects on cell growth. To test this hypothesis, we compared the survival of wild-type and ΔbacA strains treated with sub-lethal doses of L- and D-NCR247 and observed growth over a period of 24 hrs. ΔbacA mutant exhibited inhibition of cell growth to L-NCR247 when compared to the wild type, even at a sublethal concentration of 4 μM. This concentration was used because at concentrations above 6 µM we start to see bacteriostatic effects when using L-NCR247 in wildtype (S4 Fig). However, ΔbacA was less sensitive to treatment with the same concentrations of D-NCR247 when compared to the wildtype (Fig 5A and 5B). This suggests that in ΔbacA, when NCR247 cannot enter the cytoplasm, the peptide possibly accumulating in the periplasm is detrimental to the cell. These findings may explain the activity of NCR247 even at very low, sublethal concentrations. Additionally, this inhibition of cell growth is due to the stereospecific interaction of the peptide with a periplasmic or outer membrane protein, since this inhibition was absent during D-NCR247 treatment. We then tested the cell viability/ bactericidal effects at a concentration of 8 μM. While the cell viability of wildtype was reduced to the same extent with treatment of L- and D- NCR247, in a ∆bacA mutant, treatment with L-NCR247 is significantly more bactericidal than treatment with D-NCR247 (Fig 5C). This suggests that possible overaccumulation of L-NCR247 outside the cytoplasm imposes a severe bactericidal activity in a ∆bacA mutant in addition to the chirality-independent toxicity due to membrane alterations. In contrast, the cell growth inhibition caused by D-NCR247 on wild-type cells can be recovered by the addition of increasing concentrations of iron (Fig 5D,5E and 5F). This suggests that both L- and D-NCR247 bind to heme in the cytoplasm and cause a bacteriostatic effect that can be recovered at high concentrations of iron.

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Fig 5. Possible accumulation of peptide in periplasm leads to reduction in cell growth of ∆bacA mutant.

(A) Growth curve analysis over a period of 24 hours on wildtype cells treated with 4 µM L- or D- NCR247showing severe growth inhibition upon treatment with D-NCR247 when compared to treatment with L-NCR247. (B) Growth curve analysis over a period of 24 hours on ∆bacA mutant cells treated with 4 µM L- or D- NCR247 showing severe growth inhibition upon treatment with L-NCR247 when compared to D-NCR247. A and B, Data is presented as the mean of three biological replicates ± s.d. (C) Cell viability assay of 8 µM L- and D-NCR247 treated wildtype and ∆bacA mutants. After treatment, cells were serial diluted, spotted and colonies were counted. % survival cells when compared to wildtype untreated cells are plotted. Data is presented as the mean of three biological replicates ± s.d. Two-way analysis of variance (ANOVA) with multiple comparisons was used to calculate p values. ****, p ≤ 0.0001. (D, E and F) Growth curve analysis of wildtype cells grown in 0 μM (●), 5 μM (■), 10 μM (▲) or 30(▼) μM FeSO4 (D), upon treatment with L-NCR247 (E) and D-NCR247 (F). Data is presented as the mean of three biological replicates ± s.d.

https://doi.org/10.1371/journal.pgen.1011892.g005

We then investigated the mechanism of how the NCR247 peptide’s chirality-dependent interactions, presumably happening in the periplasm, might mediate the cell death caused by periplasmic overaccumulation of NCR247 in a ∆bacA mutant. In the FeuP/FeuQ two-component relay system, FeuN is a periplasmic protein that prevents hyperactive signaling by directly binding and negatively regulating FeuQ [52]. Since our results indicated that hyperactivation of FeuP/FeuQ signaling happens during L-NCR247 treatment on ∆bacA, we hypothesized that abolishing this signaling response might restore the cell viability of this mutant. To test this, a ∆bacA ∆feuP double mutant was created. Consistent with our hypothesis, in a ∆bacA ∆feuP double mutant, the lethality due to the absence of BacA when treated with L-NCR247 was diminished. This is noticed both at a sublethal concentration in a growth curve analysis (Fig 6A and 6B) and at a moderate concentration in a cell viability assay (Fig 6C). This shows that BacA plays a vital role in fine-tuning the amount of peptide available for stimulating signaling in the periplasm at physiologically relevant concentrations. This is also the direct clue for defining the role of BacA during symbiosis, where peptide concentrations are not lethal, yet enough to trigger signaling cascades.

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Fig 6. Deletion of two component signaling through FeuP in a ∆bacA mutant reduces growth inhibition by L-NCR247.

Growth curve analysis of ∆bacA and ∆bacA ∆feuP treated with 4 µM L- (blue) (A) or D -(green) (B) NCR247. C. Cell viability assay of 8 µM L- and D-NCR247 treated ∆bacA and ∆bacA ∆feup mutants. After treatment, cells were serial diluted, spotted and colonies were counted. % survival cells when compared to wildtype untreated cells are plotted. Data is presented as the mean of three biological replicates ± s.d. Two-way analysis of variance (ANOVA) with multiple comparisons was used to calculate p values. ****, p ≤ 0.0001.

https://doi.org/10.1371/journal.pgen.1011892.g006

To test whether the cysteines play a role in the toxicity associated with chirality-dependent interactions of L-NCR247 outside the cytoplasm, we used L and D-NSR247, in which serines replace the cysteines. NSR247 is shown to have no toxicity up to 20 μM [13]. However, at 20 μM, while the WT is sensitive to both L and D-NSR247, the ΔbacA is fully resistant to both the peptides (S5A and S5B Fig). This shows that possible overaccumulation of NSR247 does not cause the lethal phenotype as NCR247, indicating that disulfide bonds are necessary for the lethal phenotype associated with potential NCR247 accumulation outside the cytoplasm. We have previously shown that NSR247 is capable of non-specifically binding to heme with very low affinity [14]. The susceptibility of wildtype to L and D-NSR247 at these high concentrations may be due to weak binding of NSR247 to heme or other proteins in the cytoplasm. To confirm that the phenotypes associated with the ∆bacA mutant are not directly related to the VLCFA defects, we performed growth curve analyses using L- and D-NCR247 on ∆bacA mutant complemented with a wildtype copy of bacA or the R389G mutant version of bacA. No difference was noted between the two strains, confirming the phenotypes are not due to VLCFA modification (S5C Fig).

Conservation across diverse host-associated bacteria

Homologues of BacA are present in many pathogenic bacteria. BacA has been shown to be essential for chronic infection in mice infected with Mycobacterium tuberculosis [53] and Brucella abortus [54], indicating its importance for long-term bacterial survival inside the host. The bacA gene from M. tuberculosis is shown to partially complement the lethality of the S. meliloti ∆bacA mutant upon peptide treatment [55], and a ∆bacA mutant in B. abortus shows modifications in VLCFA [15]. We investigated whether BacA from M. tuberculosis and B. abortus exhibits similar phenotypes as S. meliloti BacA towards L- and D-NCR247. bacA complementation from M. tuberculosis and B. abortus on S. meliloti ∆bacA mutant followed a similar pattern in response to both the peptides, albeit to different extents (Fig 7A,7B and 7C). This suggests that the findings can be translated to the context of host-pathogen interactions and how these pathogens tolerate anti-microbial peptides from their respective hosts. This suggests a conserved, evolutionary role in enabling bacteria to survive hostile, antimicrobial peptide-rich environments.

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Fig 7. Complementation of BacA phenotype with homologs from pathogens.

Growth curve analysis was performed on wildtype or ∆bacA mutant complemented with Sinorhizhobium meliloti BacA (pSmbacA), or BacA homologs of Mycobacterium tuberculosis (pMtbacA) or Brucella abortus (pBabacA). Untreated cells are shown in (A), L-NCR247 treatment in (B) and D-NCR247 treatment in (C). Data is presented as the mean of three biological replicates ± s.d.

https://doi.org/10.1371/journal.pgen.1011892.g007

Discussion

Our study provides new mechanistic insights into how the nodule-specific cysteine-rich peptide NCR247 exerts its effects on S. meliloti. By systematically combining stereoisomeric forms of NCR247 with a ΔbacA background, we were able to discriminate between (i) achiral, charge and hydrophobicity-driven membrane disruption, (ii) stereospecific, protein-dependent interactions within the periplasm and cytoplasm, and (iii) BacA-dependent modulation of subcellular exposure. This integrative approach has clarified several unresolved aspects of NCR peptide biology and of BacA functionality during symbiosis. S1 Table shows the summary of the findings.

At high concentrations, both the L- and D-enantiomers of NCR247 destabilized bacterial membranes, leading to similar bactericidal outcomes. This is consistent with the behavior of conventional cationic AMPs, whose activities are primarily dictated by physicochemical parameters, such as net positive charge, and interaction with negatively charged bacterial envelopes. Importantly, this achiral mode of action explains the antimicrobial capacity of many NCRs when tested in vitro at elevated peptide doses. However, such concentrations are unlikely to be physiologically relevant in the nodule environment, suggesting that peptide-mediated symbiotic regulation involves more subtle, molecularly specific mechanisms.

At sublethal concentrations, only L-NCR247 elicited robust induction of FeuQ/FeuP and ExoS/ChvI two-component systems, while D-NCR247 did not. This demonstrates that NCR247 engages in chirality dependent interactions with periplasmic sensory proteins, triggering signaling cascades that alter envelope physiology, exopolysaccharide production, and stress responses. The stereospecific activation of these systems underscores a protein-targeted mechanism that could allow the host plant to fine-tune bacteroid physiology through evolutionarily selected peptide structures. Our results strongly suggest that in wild-type S. meliloti, BacA-mediated import redirects NCR247 into the cytoplasm, both mitigating excessive periplasmic signaling and enabling cytoplasmic phenotypes such as heme sequestration and translational inhibition. In the ΔbacA mutant, our results suggest that L-NCR247 possibly accumulates in the periplasm, resulting in toxic hyperactivation of signaling pathways and growth arrest, even at nonlethal concentrations. Intriguingly, D-NCR247, which fails to engage in stereospecific protein interactions, did not elicit such detrimental outcomes, emphasizing that toxicity under these conditions is explicitly linked to chirality-dependent periplasmic binding events rather than nonspecific peptide accumulation. The reduction in toxicity during L-NCR247 treatment in a ∆bacA ∆feuP double mutant strongly bolsters this idea. Our findings therefore provide direct evidence to the earlier hypothesis that suggested BacA provides a generic protective function by internalizing toxic peptides [56,57]. Our work further refined the model, showing that BacA appears to play a more sophisticated role in buffering the periplasmic environment against the hyperactivation of regulatory systems, while simultaneously allowing the controlled cytoplasmic engagement of peptides with intracellular targets. This advances a mechanistic framework for how BacA enables long-term symbiotic persistence despite continuous exposure to NCR peptides.

Inside the cytoplasm, both enantiomers were able to sequester heme, leading to an iron starvation response. The equivalence of L- and D-NCR247 in this activity confirms that heme binding is an achiral interaction based on physicochemical complementarity. This strongly supports prior biochemical data showing peptide–heme binding [14]. The reliance on BacA for this phenotype further underscores its essential role in peptide transport across the inner membrane. Translational inhibition, by contrast, represented a mixed phenotype. While both enantiomers reduced protein synthesis, the more pronounced effects of L-NCR247 suggest contributions from stereospecific binding to ribosomal or translational factors. Thus, peptide functionality in the cytoplasm is stratified into chirality-independent (heme sequestration) and partially chirality-dependent (translation inhibition) activities, once again highlighting the mechanistic complexity of NCR peptide action.

Our experiments with NSR247 variants, in which serines replaced cysteines, pointed to an essential role for disulfide bonding in chiral, periplasmic signaling and associated toxicity. The absence of lethality in ΔbacA backgrounds with NSR247 treatment suggests that the structurally constrained, disulfide-stabilized form of NCR247 is required for effective engagement with periplasmic protein partners. This provides molecular insight into why evolution has favored cysteine-rich motifs across the NCR family and related defensin-like peptides, enabling them to adopt precise folds necessary for target recognition [58,59]. Homologs of BacA in pathogens such as M. tuberculosis and B. abortus complement aspects of the S. meliloti ∆bacA phenotype, highlighting a conserved peptide-handling strategy that supports bacterial persistence in diverse hosts [60].

Our findings highlight how plants exploit the chemical versatility of AMPs not only for defense, but also to remodel microbial physiology in prolonged mutualistic associations. Future work should aim to identify the precise periplasmic binding partners responsible for NCR247’s stereospecific effects. These results provide critical insights for the rational design of peptide-based antimicrobials. Identifying the precise balance between achiral, charge-driven membrane disruption and stereospecific, protein-targeted effects could enable the design of peptides that minimize off-target immune responses while enhancing specificity for microbial proteins.

Materials and methods

Growth conditions

All strains of S. meliloti were routinely grown in LB medium supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LBMC) in the presence of 200 μg ml–1 streptomycin at 30°C for 48 h. Strains and plasmid lists are provided in S2 Table.

Growth curves

All growth curve experiments were performed in a Tecan SPARK 10M microplate reader using polystyrene flat-bottomed, non-treated, sterile 96-well plates. Overnight cultures grown in LBMC were washed and were subcultured (1:100 dilution) in MOPS-GS medium. The plates were programmed to continuously shake at 150 rpm. and the temperature was maintained at 30°C. Optical density was measured at 600 nm every 60 min.

Peptides

All chemically synthesized peptides were purchased from Genscript. The purity of all peptides was > 99% and verified by high-performance liquid chromatography. Mixture of all regioisomers were used for all peptides as previously described [12].

Antimicrobial activity assays

NCR247 sensitivity of S. meliloti and E. coli cells was determined as described previously [61] with modifications using early exponential phase cultures in 3-(N-morpholino) propanesulfonic acid (MOPS) buffered minimal medium (50 mM MOPS, 1 mM MgSO4, 0.25 mM CaCl2, 19 mM glutamic acid, 0.004 mM biotin, pH 7.0) Mops-GS supplemented with 1% casaminoacid.

Scanning electron microscopy

SEM analysis was performed as described previously [37] with slight modifications. Untreated or 15 µM L- or D- NCR247 treated cells were fixed with 2% (vol/vol) glutaraldehyde and 3% paraformaldehyde in cacodylate buffer (0.05 M, pH 7.2) overnight. Cells were dehydrated serially in 50%, 75%, 90%, 95% and 3x100% ethanol. Then, the cells were dried in a critical point dryer, mounted on a stub, and sputter-coated with a 10 nm gold coating, and then viewed in a SEM (JOEL JSM-7100F/LV).

ICP-MS

For bacterial samples, 1 ml of sample was spun down, and the pellet was resuspended in 40 μl of 100% HNO3 and heated at 98°C for 1 h. The supernatant of the solution was mixed with metal-free water to make it up to 2 ml and ICP-MS analysis was performed as previously described [14]. The same number of cells were spun down for protein analysis using the BSA method and data were normalized to the amount of protein in each sample. Agilent ICP-MS instrumentation with MassHunter 4.4 was used to collect data.

RNA isolation and RT–qPCR analysis

Cells were grown in LB medium until they reached an OD600 nm of 0.2. Then cells were spun down and suspended in MOPS-GS and synchronized according to previous method. Then cells were then treated with respective peptides and recovered at 30 mins for FeuP/FeuQ, ExoS/ChvI, RirA related genes and 90 mins for CtrA related genes. NCR247 was then added and 5 ml of appropriate cultures were spun down at given time intervals. Total RNA was extracted using the TRIzol (Thermo Fisher Scientific) method. A Qiagen RNeasy kit was used to purify the RNA. On-column DNA removal was carried out using DNase I from NEB. A total of 500 ng of each RNA sample was used to make complementary DNA using an iScript cDNA synthesis kit (Bio-Rad). RT–qPCR assays were performed as previously described [14]. The standard curve method was used for relative quantification. In brief, a standard curve was generated for each gene of interest (including smc00128) by setting up qPCR reactions to amplify increasing amounts of S. meliloti Rm1021 genomic DNA. All the primer sets used resulted in a proportional dose–response curve with R2 > 0.99, which confirmed their efficiency. This curve was then used for extrapolating the relative expression level of each gene of interest in a particular sample to obtain the starting quantities (SQ). This value was then normalized to the SQ values of smc00128 obtained for the respective samples. These normalized values were then expressed as an average of triplicates, with s.d. represented by the error bars.

In vitro translation assay

In vitro translation reactions were performed with the “Pure Express in Vitro Protein Synthesis” (Invitrogen Cat no: K990100) in the presence of different concentrations of NCR247 variants and LL37 as described previously [13]. GFP fluorescence was measured in a Tecan Plate reader (λex = 485 nm, λem = 530 nm) after 30 mins. In the vector used (a gift from the Baker laboratory, Massachusetts Institute of Technology), production of the GFP was controlled by the T7 promoter.

Western blot

Western blot was performed on the reactions from in vitro translation assay. 5 µl of the product was loaded. Anti-GFP antibody (ab6556) was used as primary at a dilution of 1:1000 and Goat anti -rabbit secondary (ab205718) was used at dilution of 1:10000.

Biolayer interferometry

Biolayer interferometry was carried out using ForteBio Octet RED96 biolayer interferometer following the manufacturer’s instructions for a standard kinetic assay. Biotinylated L- or D-NCR247 was loaded on to streptavidin coated biosensor tips. Peptides incubated in 200 μl assay buffer (water (pH 7.4)), each for 60 s. was loaded onto each biosensor tip at the defined concentration until the binding signal reached a value of >1.4. Biosensor tip loading was followed by incubation in assay buffer for 60 s. Association between the ligand and the purified SbmA analyte was observed over a time frame of ~116 s in assay buffer. To stop binding kinetics for dissociation, the biosensor tips were placed back into an assay buffer not containing any analyte for 1200 s.

Pulldown assay: Log phase grown S. meliloti were lysed using French press homogenizer and split into three aliquots. 10 µM of N-terminal Biotin labelled L- or D-NCR247 was added to the extracts and incubated at room temperature for 3 hours. Pull down was performed using Dynabeads MyOne Streptavidin T1s (Invitrogen) on a magnetic rack. After washing 3 X with PBS. Equal amount of SDS loading buffer was added and boiled before loading to 12% SDS PAGE gel. Standard silver staining was then performed.

Supporting information

S1 Fig. Increase in the expression of chvI, a gene target of ExoS/ChvI signaling (A) and smb21440 (B) upon treatment with 4 µM L-NCR247 (blue) when compared to untreated cells (black) as quantified by RT–qPCR analysis.

Treatment with 4 µM D-NCR247 shows very little change in expression when compared to untreated cells (Green). In a ∆bacA mutant, there is a significant increase in expression when treated with L-NCR247 when compared to wildtype treated at the same condition. The data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene smc00128 and are presented as an average of three technical replicates ± s.d, Two-way analysis of variance (ANOVA) with multiple comparisons was used to calculate p values. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

https://doi.org/10.1371/journal.pgen.1011892.s001

(TIF)

S2 Fig. (A) Decrease in the expression of the cell cycle gene gcrA upon L-NCR247 (4 µM) treatment and recovery of expression upon D-NCR247 (4 µM) treatment as quantified by RT-qPCR analysis.

The same treatments resulted in a similar but modest response in a ∆bacA mutant. (B and C) Increase in the expression of the RirA-regulated genes entC and fhuP upon 4 µM L-NCR247 treatment as analyzed by RT-qPCR analysis (blue) as compared to untreated samples (black). Further increase upon D-NCR247 treatment was observed (green). Significant reduction in RirA-regulated gene expression upon both L-NCR247 and D-NCR247 treatment in a ∆bacA mutant when compared to treatments on wildtype is shown. For A, B and C, the data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene smc00128 and are presented as an average of three technical replicates ± s.d. (D) Pulldown experiment using streptavidin beads on S. meliloti extracts treated with biotin labelled L- or D-NCR247. * marks the expected size for NCR247. (E) ∆bacA mutant complemented with empty vector shows similar gene expression change (hmuP) in response to peptide treatments as ∆bacA mutant complemented with peptide transport mutant of BacA (prf-bacA(E207A)) as measured by Rt-qPCR analysis indicating the importance of peptide transport. The ∆bacA mutant complemented with BacA defective in VLCFA modification (prf-bacA(R389G)) shows gene expression change like wildtype BacA complemented ∆bacA mutant (prf-bacA). The data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene smc00128 and are presented as an average of three technical replicates ± s.d. For A, B, C and E, two-way analysis of variance (ANOVA) with multiple comparisons was used to calculate p values. **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

https://doi.org/10.1371/journal.pgen.1011892.s002

(TIF)

S3 Fig. (A) Western blot analysis of products of in vitro translation assay in Fig 4D using anti-GFP antibody.

(B) Ponceau stain of the same blot to show loading controls. (C) In vitro translation assay using LL37 with same molar comparison to L-NCR247. Data is presented as the mean of four technical replicates ± s.d.

https://doi.org/10.1371/journal.pgen.1011892.s003

(TIF)

S4 Fig. Growth curve analysis over a period of 24 hours on wildtype cells treated with increasing concentrations of L-NCR247.

Data is presented as the mean of three biological replicates ± s.d.

https://doi.org/10.1371/journal.pgen.1011892.s004

(TIF)

S5 Fig. A and B. Growth curve analysis over a period of 24 hours on wildtype (A) or ∆bacA (B) mutant cells treated with 20 µM L- (blue) or D- (green) NSR247.

(C) Growth curve analysis over a period of 24 hours on ∆bacA mutant complemented with either wildtype bacA (∆bacA pbacA) or bacA (R389G) ((∆bacA pbacAR389G) upon treatment with 4 µM L –NCR247 (blue) or D-NCR247 (Green). Data is presented as the mean of three biological replicates ± s.d.

https://doi.org/10.1371/journal.pgen.1011892.s005

(TIF)

S1 Table. Summary of phenotypes observed with L and D NCR247 treatment in wildtype and ∆bacA mutant.

https://doi.org/10.1371/journal.pgen.1011892.s006

(DOCX)

S2 Table. Strains and plasmids used in this study.

https://doi.org/10.1371/journal.pgen.1011892.s007

(DOCX)

S1 Data. Raw data files.

https://doi.org/10.1371/journal.pgen.1011892.s008

(ZIP)

Acknowledgments

We thank the Walker lab members for helping with various aspects of preparation of this manuscript. We thank Dr. Joel Griffitts (Brigham Young University) for the plasmid pJG206.

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