Lasso peptides sviceucin and siamycin I exhibit
anti-virulence activity and restore vancomycin
effectiveness in vancomycin-resistant pathogens

Abdelhakim Boudrioua, Benjamin Baëtz, Solenn Desmadril, Christophe Goulard, Anne-Claire Groo, Carine Lombard, Sabrina Gueulle, Marie Marugan, Aurélie Malzert-Fréon, Axel Hartke, et al.

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To cite this version:

Abdelhakim Boudrioua, Benjamin Baëtz, Solenn Desmadril, Christophe Goulard, Anne-Claire Groo, et al. Lasso peptides sviceucin and siamycin I exhibit anti-virulence activity and restore vancomycin effectiveness in vancomycin-resistant pathogens. iScience, 2025, 28 (3), pp.111922. 10.1016/j.isci.2025.111922. hal-04981918

HAL Id: hal-04981918

https://hal.science/hal-04981918v1

Submitted on 7 Mar 2025

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# Lasso peptides sviceucin and siamycin I exhibit anti-virulence activity and restore vancomycin effectiveness in vancomycin-resistant pathogens

## Graphical abstract

<figure><img src="image_3.png" /></figure>

## Authors

Abdelhakim Boudrioua, Benjamin Baëtz, Solenn Desmadril, ..., Axel Hartke, Yanyan Li, Caroline Giraud

## Correspondence

yanyan.li@mnhn.fr (Y.L.),
caroline.giraud@unicaen.fr (C.G.)

## In brief

Drugs; Biochemistry; Peptides; Medical Microbiology

## Highlights

* Lasso peptides sviceucin and siamycin I abolish the virulence of *E. faecalis*

* Sviceucin and siamycin I restore vancomycin susceptibility in vancomycin-resistant bacteria

* Sviceucin and siamycin I target the sensor histidine kinases FsrC and VanS

<figure><img src="image_4.png" /></figure>
Article

# Lasso peptides sviceucin and siamycin I exhibit anti-virulence activity and restore vancomycin effectiveness in vancomycin-resistant pathogens

Abdelhakim Boudrioua,<fnref n="1" />,<fnref n="4" />,<fnref n="5" /> Benjamin Baëtz,<fnref n="1" />,<fnref n="5" /> Solenn Desmadril,<fnref n="1" />,<fnref n="6" /> Christophe Goulard,<fnref n="2" />,<fnref n="6" /> Anne-Claire Groo,<fnref n="3" />
Carine Lombard,<fnref n="2" /> Sabrina Gueulle,<fnref n="1" /> Marie Marugan,<fnref n="2" /> Aurélie Malzert-Fréon,<fnref n="3" /> Axel Hartke,<fnref n="1" /> Yanyan Li,<fnref n="2" />,*
and Caroline Giraud<fnref n="1" />,<fnref n="7" />,*

## SUMMARY

Antibiotic resistance is a major threat to human health and new drugs are urgently needed. Ideally, these drugs should have several cellular targets in pathogens, decreasing the risk of resistance development. We show here that two natural ribosomally synthesized lasso peptides (LPs), sviceucin and siamycin I, (1) abolish bacterial virulence of pathogenic enterococci, (2) restore vancomycin clinical susceptibility of vancomycin-resistant (VR) enterococci *in vitro* and in a surrogate animal model, and (3) re-sensitize VR *Staphylococcus aureus*. Mode of action (MoA) analyses showed that they do so by inhibiting the histidine kinases (HKs) FsrC and VanS controlling these phenotypes. Strains resistant to the vancomycin/LP combination were difficult to obtain, and were still fully susceptible to the anti-virulence effect of the LPs, highlighting the advantage of multiple targets. Together with the highly sought-after MoA as HK inhibitors, such properties make these lasso peptides promising candidates for the development of next generation antibiotics.

## INTRODUCTION

The alarming increase of infections caused by multidrug-resistant (MDR) bacteria is a main threat for human health worldwide.<fnref n="1" />,<fnref n="2" /> The World Health Organization (WHO) published a list of the MDR bacteria for which new antibiotics are urgently needed.<fnref n="3" /> On this list, vancomycin-resistant enterococci (VRE) as well as methicillin-resistant *Staphylococcus aureus* (MRSA) are classified as “high priority”. These organisms are leading causes of hospital-acquired infections in high-income countries, and a high prevalence of MDR isolates among these strains renders clinical treatments difficult. Vancomycin resistant *Staphylococcus aureus* strains (VRSA) were also reported.<fnref n="4" />,<fnref n="5" />

*Enterococcus* sp. and *S. aureus* are commensal gram-positive bacteria.<fnref n="4" />,<fnref n="6" /> *Enterococcus* species are common colonizers of the gastrointestinal tract in humans and other animals.<fnref n="6" /> Pathogenic *Enterococcus* species, mainly *E. faecium* and *E. faecalis*, can cross the intestinal barrier and cause bacteremia, endocarditis, and urinary tract infections in immunocompromised patients.<fnref n="7" /> *S. aureus* is a commensal of human nasal mucosa. This species is a leading cause of bacterial infections in healthcare and com-

munity settings.⁸ Skin infections and bloodstream infections occur when mucosal barriers are damaged or owing to invasive medical devices.⁹

Both pathogenic enterococci and *S. aureus* pose a therapeutic challenge due to their ability to form biofilms at the infection sites in addition to their frequent resistance to antibiotics.⁹,¹⁰ Enterococcal proteases such as the gelatinase GelE and the serine protease SprE are associated with biofilm formation during endocarditis.<fnref n="7" /> In *E. faecalis*, biofilm formation is regulated by the Fsr quorum sensing system. The response regulator FsrA and the histidine kinase (HK) FsrC form a two-component regulatory system (TCS), which is responsible for the quorum sensing-dependent activation of biofilm formation through the activation of gelE and sprE expression.¹¹,¹²

Vancomycin is the antibiotic of choice to treat enterococci and MRSA infections.¹³ Vancomycin inhibits peptidoglycan biosynthesis by binding to N-acetylglycosamine-N-acetylmuramic acid-pentapeptide ending with D-alanyl-D-alanine (D-Ala-D-Ala) dipeptide.¹⁴ Vancomycin resistance occurs following the acquisition of van genes, resulting in the synthesis of modified peptidoglycan precursors. Substitution of the last D-Ala of the

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<footnote n="1">¹Université de Caen Normandie, CBSA UR4312, F-14000 Caen, France</footnote>

<footnote n="2">²Unit Molecules of Communication and Adaptation of Microorganisms (MCAM), UMR 7245 CNRS-Muséum National d'Histoire Naturelle (MNHN), 75005 Paris, France</footnote>

<footnote n="3">³Normandie University, Unicaen, CERMN, 14000 Caen, France</footnote>

<footnote n="4">⁴Present address: Section of Cellular and Molecular Microbiology, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), University of Tübingen, Elfriede-Aulhorn-Str. 6, 72076 Tübingen, Germany.</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>

<footnote n="6">⁶These authors contributed equally</footnote>

<footnote n="7">⁷Lead contact</footnote>

<footnote>*Correspondence: yanyan.li@mnhn.fr (Y.L.), caroline.giraud@unicaen.fr (C.G.)</footnote>

<footnote>https://doi.org/10.1016/j.isci.2025.111922</footnote>
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A

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B

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C

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D

<figure><img src="image_10.png" /><figcaption>**Figure 1. Anti-virulence activity of sviceucin and siamycin I**</figcaption></figure>

(A) Peptide sequences of sviceucin and siamycin I. The macrolactam ring is shown in green. The disulfide bonds are shown in yellow. See also Figure S1.

(B) Effect of sviceucin (10 µM) and siamycin I (2 µM) on the growth of *E. faecalis* V583. "Control" refers to a condition in which the 1:1 water:methanol peptide formulation without peptide was added.

(C) Effect of sviceucin (10 µM) and siamycin I (2 µM) on the expression of *geI*, determined by RT-qPCR. See also Figure S2.

(D) Mortality of *Galleria mellonella* larvae 24 h post-infection (p.i.) with *E. faecalis* V583, pre-treated with either sviceucin (10 µM), siamycin I (2 µM), or formulation. \*\*\*p < 0.0001 (Bonferroni's multiple comparisons test). Data are averages of three biological replicates. Each replicate represents one round of infection on 15 larvae. See also Figure S3.

pentapeptide by D-lactate (D-Lac) greatly lowers the affinity of vancomycin to its target.¹⁵ Several types of van clusters have been described. However, VanA- and VanB-types are the most relevant in the clinic.¹⁶ In VanA-type resistance, the D-Lac dehydrogenase VanH converts pyruvate to D-Lac, whereas the ligase VanA forms the dimer D-Ala-D-Lac and the dipeptidase VanX hydrolyses the endogenous D-Ala-D-Ala dimers. The corresponding enzymes in VanB-type resistance are VanH<sub>B</sub>, VanB, and VanX<sub>B</sub>, respectively.¹⁵ The *van* gene cluster is regulated by a TCS consisting of the HK VanS (named VanS<sub>B</sub> in VanB-type resistance) and the response regulator VanR (named VanR<sub>B</sub> in VanB-type resistance). In the presence of vancomycin, the kinase VanS undergoes auto-phosphorylation, which subsequently leads to the phosphorylation of VanR, triggering its dimerization and increasing its affinity for the promoter located upstream of *vanHAX*. In non-inducing conditions, the phosphatase activity of VanS keeps VanR in a dephosphorylated state, preventing the expression of the *van* operon.¹⁷ Although the genetic organization of the VanA- and VanB-type van clusters is similar, the *vanA* genes are induced by the presence of both vancomycin and teicoplanin, conferring high levels of resistance to both vancomycin and teicoplanin, whereas the *vanB* type genes are only induced by vancomycin, conferring only moderate resistance to vancomycin and no resistance to teicoplanin.¹⁶

The therapeutic efficiency of the few approved antimicrobial agents active against vancomycin-resistant bacteria seems to be temporary, since resistance to these new drugs has already been described.¹⁸ Therefore, as pointed out by the WHO, new antibiotics or alternative approaches are urgently needed to combat these gram-positive pathogens with critical clinical importance. The lack of identification of new drugs with novel molecular scaffolds and uncommon mechanisms of action has renewed interest in the investigation of alternative strategies. Ideally, the next generation antimicrobials would interfere with bacterial virulence without inhibiting bacterial growth, which is

expected to lower the probability of resistance development because of reduced selective pressure.¹⁹,²⁰ Another promising approach consists in interfering with antibiotic resistance mechanisms to restore the original sensitivity.²¹ Such molecules could be used in synergy with conventional antibiotics.²²,²³

TCSs represent the majority of signaling pathways in bacteria and they control a wide range of behaviors crucial for bacterial adaptation, including virulence, biofilm formation, and antibiotic resistance.²⁴,²⁵ Most HKs contain a variable N-terminal sensor domain and a conserved C-terminal cytoplasmic kinase domain.²⁶ The kinase domain comprises a catalytic ATP binding (CA) and a dimerization/histidine phosphorylation domain. The conservation of the kinase domain allows for the potential design of broad-spectrum antimicrobial agents.²⁷,²⁸ The toxicity risk of such molecules for humans is likely low, given the fact that HKs are absent in mammals. Pathogenic bacteria often contain a significant number of HKs. For instance, 17 HKs have been identified in the core genome of *S. aureus* strains.²⁹ This represents multiple possibilities to interfere with bacterial fitness, virulence, and antibiotic resistance, features expected for next generation antibiotics.²⁰

We report herein that the lasso peptides sviceucin³⁰ and siamycin I³¹ display both anti-virulence and resistance-breaking activity in *Enterococcus* sp. and *S. aureus* through the inhibition of corresponding HKs. They are thus very promising candidates for the development of next generation antimicrobials. Lasso peptides belong to the family of ribosomally synthesized and post-translationally modified peptide, natural products produced by prokaryotes. They display a mechanically interlocked structure composed of an N-terminal macrolactam ring and a C-terminal tail that is held firmly within the ring.³²,³³ This compact and rigid scaffold makes them highly stable and excellent protein ligands, leading to enzyme inhibition or receptor antagonism. Sviceucin and siamycin I are among the few representatives of the class I lasso peptides (C1LP), characterized by the presence of two disulfide bonds (Figure 1A; Figure S1). They show distinct growth inhibition activity of gram-positive bacteria: sviceucin is a weak
<table><caption>Table 1. Sviceucin and siamycin I reverse vancomycin resistance</caption><thead><tr><th rowspan="2">Strain</th><th rowspan="2">Resistance type</th><th colspan="5">Vancomycin MIC (µg/mL)</th></tr><tr><th>Control</th><th>Sviceucin (10 µM)</th><th>Siamycin I (2 µM)</th><th>Sviceucin MIC (µM)</th><th>Siamycin I MIC (µM)</th></tr></thead><tbody><tr><td><i>E. faecalis</i> V583</td><td>VanB</td><td>32</td><td>2</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecalis</i> JH2-2 08048</td><td>VanB</td><td>64</td><td>2</td><td>&lt;0.5</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecalis</i> merz96</td><td>VanB</td><td>32</td><td>1</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>S. aureus</i> T-SAR12 VRSA.B</td><td>VanB</td><td>64</td><td>2</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecium</i> 1 231 410</td><td>VanA</td><td>128</td><td>128</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecalis</i> HIP11704</td><td>VanA</td><td>1024</td><td>512</td><td>&lt;0.5</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecium</i> 1 231 502</td><td>VanA</td><td>128</td><td>128</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecium</i> 1 230 933</td><td>VanA</td><td>512</td><td>256</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>S. aureus</i> T-SAR12 VRSA.A</td><td>VanA</td><td>&gt;512</td><td>&gt;512</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>E. faecalis</i> V583 ΔvanB</td><td>van-</td><td>2</td><td>2</td><td>2</td><td>&gt;10</td><td>5</td></tr><tr><td><i>S. aureus</i> 16038 (VISA)</td><td>van-</td><td>4</td><td>4</td><td>4</td><td>&gt;10</td><td>5</td></tr></tbody></table>

MICs of antibiotic alone, in combination with sviceucin, or with siamycin I. "Control" refers to a condition in which the 1:1 water:methanol peptide formulation without peptide was added. See also Table S1.

antibacterial whereas siamycin I displays a good activity, notably toward *Enterococcus* sp. and S. aureus. In S. aureus, siamycin I was shown to bind to lipid II, hence blocking the biosynthesis of peptidoglycan, which is supposed to be the basis of its antibacterial activity.³⁴ Moreover, sviceucin and siamycin I are known to attenuate the TCS FsrAC-mediated quorum sensing in *Enterococcus*,³⁰,³⁵ with direct inhibition of the HK FsrC by siamycin I.³⁶,³⁷ This study extends the activity of these C1LPs to interfere with other TCS-controlled processes, i.e., resistance to vancomycin. Through detailed microbiological, genetic, and biochemical analyses, the molecular target of restoring clinical susceptibility to vancomycin in VRE and VRSA is firmly established to be the HK VanS.

## RESULTS

### Sviceucin and siamycin I attenuate the virulence of *E. faecalis*

It has been shown that siamycin I binds *in vitro* to the HK FsrC of *E. faecalis* and inhibits its ATPase activity.³⁷ We confirmed the direct inhibition of the autophosphorylation activity of FsrC by sviceucin, similarly to siamycin I, using a continuous assay based on the detection of ADP³⁸ (Figure S2). Therefore, we wondered if siamycin I and the related C1LP sviceucin (Figure 1A) decreased virulence of *E. faecalis*. Subinhibitory concentrations of siamycin I (2 µM) and sviceucin (10 µM) were used throughout all experiments of this work. At these concentrations, the peptides decreased the growth rate by about 50% compared to that in the absence of the lasso peptides (Figure 1B). We verified that siamycin I completely inhibited the growth of all tested *E. faecalis* and *E. faecium* strains at 5 µM (Table 1), which is in agreement with the study of Nakayama and collaborators.³⁵ Sviceucin did not completely inhibit the bacterial growth of *E. faecalis* and *E. faecium*, even at concentrations above 10 µM (indicated as MIC >10 µM in Table 1). We first monitored the expression of the Fsr regulated *gelE* gene by RT-qPCR. The

peptides were solubilized in a 1:1 water:methanol formulation, and the formulation was used as the control. Both peptides led to a downregulation of expression of *gelE* (Figure 1C), which is consistent with the previously reported reduction of gelatinase (GelE) activity by sviceucin.³⁰

*Galleria mellonella* larvae were then used as a model of *E. faecalis* infection to assess the anti-virulence activity of siamycin I and sviceucin (Figure 1D). The *E. faecalis* strain V583 was first pre-treated with either C1LP or the formulation and subsequently injected into the larvae. Formulation-treated V583 was highly virulent for the larvae, with an average mortality of 95% at 24 h post-injection. By contrast, bacteria pre-treated with sviceucin or siamycin I were significantly less virulent for the larvae, with mortality rates of 13% and 10%, respectively. Similar effects on virulence have been obtained using sviceucin for *E. faecalis* strains MMH594 and OG1RF (Figure S3).

### Sviceucin and siamycin I restore vancomycin susceptibility

Since sviceucin and siamycin I inhibit signal transduction of the FsrAC TCS *in vivo*, we next wondered if the lasso peptides would also target other TCSs. In *E. faecalis*, the VanRS TCS regulates resistance to vancomycin, an antibiotic of clinical importance. The minimum inhibitory concentrations (MICs) of this antibiotic in combination with the lasso peptides were thus determined for clinically relevant *Enterococcus* and S. aureus strains from our in-house collection. The chosen concentrations of the peptides (2 µM of siamycin I and 10 µM of sviceucin) are the lowest subinhibitory concentrations that allow the maximum synergy with vancomycin, as determined by the checkerboard method on the *E. faecalis* V583 strain (Table S1).

Most interestingly, both lasso peptides re-sensitized VanB-type *E. faecalis* strains to vancomycin whereas only siamycin I was active to restore susceptibility of VanA strains to this glycopeptide (clinical and laboratory standards institute (CLSI) breakpoint >4 µg/mL). Worth of note, the antibacterial activity of the
<figure><img src="image_11.png" /><figcaption>**Figure 2. Siamycin I reverses vancomycin resistance in vivo**</figcaption></figure>

Survival plot of G. mellonella larvae infected with 10⁶ CFU of the *E. faecalis* V583 WT strain, the isogenic Δ*vanB* mutant, or injected with saline solution. Larvae were then treated at t₉₀min and t₂₄h with the formulation, siamycin I, vancomycin or the combination of both. **p** < 0.01 (Log-rank test).

peptides in absence of vancomycin remains the same among the different VanB- or VanA-type strains: 5 µM for siamycin I and >10 µM for sviceucin (Table 1). The MIC of vancomycin in strain V583 decreased from 32 to 2 µg/mL in the presence of either one of the two peptides (Table 1), becoming as sensitive as an isogenic strain with a deleted *vanB* gene (V583 Δ*vanB*). This suggests that the effects of the peptides on vancomycin resistance are *van*-operon type dependent and that the antibacterial activity of siamycin I is independent of the *van*-operon. The same activity spectrum of the lasso peptides related to the *van*-operon type was also observed in VRSA strains. Sviceucin re-sensitized only the VanB-type resistant strain, while siamycin I synergized with vancomycin in both VanA- and VanB-type VRSA strains (Table 1). Additionally, we tested the MIC for vancomycin of a vancomycin-intermediate S. aureus (VISA) strain in combination with the lasso peptides. VISA strains do not harbor a *van* operon. The low level of vancomycin resistance (MICs 4 to 8 µg/mL) is due to spontaneous mutations in genes predominantly involved in cell envelope biosynthesis.³⁹ In the presence of either C1LP, no reduction of the MIC of vancomycin was observed, further suggesting that the action of these peptides is *van* operon-dependent. Considering the relevance of a vancomycin resistance-breaking activity, we subsequently focused our efforts on (1) providing an in-host proof of the efficacy and (2) deciphering the underlying MoA of the lasso peptides.

We used *Galleria mellonella* insect larvae to investigate the efficacy of the vancomycin/C1LP combinatorial treatment (Figure 2). As sviceucin and siamycin I are hydrophobic peptides,³⁰,⁴⁰ they were first solubilized in a nanoemulsion previously shown to improve the solubility and the bioavailability of lipophilic drugs.⁴¹ A nanoemulsion with a sufficient drug payload is necessary for the treatment of infected larvae, and this was only obtained for siamycin I, for which it contained 400 µg/mL of the peptide. Larvae were infected with the wild-type strain *E. faecalis* V583, the isogenic vancomycin-sensitive mutant V583 Δ*vanB*, or an equivalent volume of saline solution. At t₉₀min and t₂₄h post-infection (p.i.), the larvae were treated with the nanoemulsion alone (referred to as formulation), the nanoemulsion loaded with siamycin I (3.92 µg/larvae; about 18 mg/kg), vancomycin (1 µg/larvae; about 4.6 mg/kg)

alone, or a combination of both. Larvae survival was assessed, and statistical analysis was performed at the endpoint t₉₆h. The formulation and siamycin I-loaded nanoemulsions did not exhibit toxicity toward the larvae (Figure 2). 95% of infected larvae with the WT strain

and treated with the formulation were killed 24 h p.i.. Siamycin I treatment significantly delayed the killing of the larvae at the beginning of the experiment (p value = 0.0486 at 48 h p.i.), although most larvae were also dead by the end of the experiment. This attenuation of the killing rate is likely related to the anti-virulence activity of the peptide. Larvae infected with the WT strain and treated with the combined treatment resulted in a significant improvement of survival in comparison to the condition where larvae were treated with either vancomycin or siamycin I alone. Moreover, the combination therapy resulted in a survival rate similar to that of larvae infected with the V583 Δ*vanB* strain and treated with vancomycin. We therefore provide a proof-of-concept for the potential use of siamycin I as an adjunctive therapy for the treatment of vancomycin-resistant *E. faecalis* in an infected host.

## Mutations in VanS<sub>B</sub> lead to resistance to the vancomycin/lasso peptide combinatorial treatment

To gain further insights into the molecular targets of these lasso peptides to reverse vancomycin resistance, resistant clone isolation followed by whole genome sequencing was performed. *E. faecalis* V583, a VanB strain, was subjected to selective pressure in the presence of vancomycin combined with either lasso peptide. Four resistant mutants were isolated when bacteria were plated in the presence of a lethal concentration of combined vancomycin (32 µg/mL) and sviceucin (10 µM), occurring with an overall frequency of resistance of ~10⁻⁸. By contrast, no resistant mutants could be obtained with lethal concentrations of combined vancomycin (32 µg/mL) and siamycin I (2 µM). However, by performing serial passages in the presence of 10 µg/mL vancomycin and increasing sublethal concentrations of siamycin I up to a lethal concentration of 5 µM, we were able to isolate five resistant mutants. The genomes of the mutants and the isogenic parental V583 strain were sequenced and compared. Three mutants identified from the vancomycin/sviceucin combination have mutations in the locus encoding the F1 ATP synthase, including substitutions in the *atpA* and *atpF* genes and an altered RBS upstream the *atpD* gene. Compared to the wild-type, these mutants were more resistant to vancomycin alone and the vancomycin/sviceucin combination, but still susceptible to the vancomycin/siamycin I
<figure><img src="image_12.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Figure 3. Characterization of VanS/VanS<sub>B</sub> as the molecular target of sviceucin and siamycin I</figcaption></figure>

(A–C) Model of the full-length VanS<sub>B</sub> dimer generated using AlphaFold 1. Amino acids D398, S402, and the conserved H233 are mapped on the structure (A). A zoom into the H-ATPase domain (B) and top view of the H-ATPase domain (C), are shown.

(D) Relative expression of *vanY*<sub>B</sub> in presence of vancomycin (1 µg/mL) or vancomycin and sviceucin (10 µM) in *E. faecalis* V583 and its mutant *Svic1*. Data presented are means from three independent biological replicates as indicated by individual data points and error bars represent the standard deviation (SD).

(E) Relative abundance of D-Ala-D-Lac muropeptide in presence of vancomycin (2 µg/mL) or vancomycin and sviceucin in *E. faecalis* V583 and its mutant *Svic1*.
Data presented are means from at least two independent biological replicates as indicated by individual data points and error bars represent the SD.

(F) Differential scanning fluorimetry analysis of VanS-lasso peptides interactions. Changes of the melting temperature (ΔT) of VanS<sup>127-384</sup> were measured in presence of increasing concentrations of sviceucin, siamycin I or microcin J25. Data presented are means from three independent replicates and error bars represent the SD.

(G) Dose-response curve of VanS<sup>127-384</sup> inhibition by sviceucin. Data presented are means from three independent replicates and were analyzed by the nonlinear regression fit function in GraphPad, which gives an IC<sub>50</sub> of 10.8 ± 1.5 µM, with a R<sup>2</sup> = 0.93. Error bars represent the SD. See also Figure S6.

combination (Table S2). Four mutants derived from the vancomycin/siamycin I combination have either point mutations or a frameshift in the *ccpA* gene encoding the catabolite control protein A. These mutants were resistant to the effects of both C1LPs in combination with vancomycin (Table S2). Interestingly, two mutants, named *Svic1* and *SiaA*, isolated respectively from vancomycin/sviceucin and vancomycin/siamycin I combinations, have a direct link to vancomycin resistance. Genome sequencing revealed that both have point mutations in the ATPase CA domain of VanS<sub>B</sub>, leading to a S402I and a D398E substitution, respectively (Figures 3A-3C). Growth of these mutants was very similar to that of the wild-type strain. Both *Svic1* and *SiaA* exhibited a 4-fold increase of the MIC of vancomycin, reaching 128 µg/mL regardless of the presence of sviceucin (Table 2). In the case of the combination of van-

comycin/siamycin I, the *SiaA* mutant showed partial resistance while the *Svic1* mutant remained sensitive. Of note, these mutants are both still fully susceptible to the anti-virulence activity of the C1LPs, as shown by the *G. mellonella* infection assay (Figure S4). To provide evidence of the causal effect of S402I and D398E substitutions in VanS<sub>B</sub> on resistance to the lasso peptides, we introduced both mutations individually into a wild-type V583 background. The resistance profiles of V583 *vanS<sub>B</sub>*<sup>S402I</sup> and V583 *vanS<sub>B</sub>*<sup>D398E</sup> were similar to the mutants *Svic1* and *SiaA*, respectively, indicating that the resistance phenotypes of *Svic1* and *SiaA* are due to the identified mutations in VanS<sub>B</sub>. Additionally, deletion of *vanB* from the resistant mutant *Svic1* led to loss of vancomycin resistance, arguing that the increase of the MIC of vancomycin in *Svic1* and *SiaA* mutants occurs exclusively through *van*
<table><caption>**Table 2.** MICs (µg/mL) of vancomycin of Svic1 and SiaA resistant mutants</caption><thead><tr><th>Strain</th><th>Control</th><th>Sviceucin</th><th>Siamycin I</th></tr></thead><tbody><tr><td>V583</td><td>32</td><td>2</td><td>2</td></tr><tr><td>Svic1</td><td>128</td><td>128</td><td>2</td></tr><tr><td>Svic1 ΔvanB</td><td>2</td><td>2</td><td>2</td></tr><tr><td>V583 vanS<sub>B</sub><sup>S402I</sup></td><td>128</td><td>128</td><td>2</td></tr><tr><td>SiaA</td><td>128</td><td>128</td><td>32</td></tr><tr><td>V583 vanS<sub>B</sub><sup>D398E</sup></td><td>128</td><td>128</td><td>16</td></tr></tbody></table>

“Control” refers to a condition in which the 1:1 water:methanol peptide formulation without peptide was added. See also Figure S4 and Table S2.

resistance genes. Our results strongly support that the histidine kinases VanS and VanS<sub>B</sub> are direct targets of sviceucin and siamycin I.

### Sviceucin leads to a decrease in van operon expression

Based on the previous results, sviceucin and siamycin I are expected to reduce or block the expression of the van operon upon vancomycin induction in the wild-type strain V583 but not in the Svic1 and SiaA mutants. To decipher this part of the mechanism, we chose to focus on sviceucin and the sviceucin-resistant mutant Svic1. The expression of vanY<sub>B</sub>, the first gene of the VanB-type van operon, was monitored by RT-qPCR when bacteria were cultured with vancomycin (1 µg/mL) alone or in combination with sviceucin. As expected, vanY<sub>B</sub> expression was strongly induced by vancomycin alone in both wild-type and Svic1 strains (Figure 3D). In the presence of sviceucin, vanY<sub>B</sub> expression was reduced by a factor of 21 in the wild-type strain upon vancomycin induction, suggesting that sviceucin interferes with the signal transduction of VanR<sub>B</sub>S<sub>B</sub>. By contrast, sviceucin led to only a 4-fold reduction of vanY<sub>B</sub> expression in the Svic1 strain. The vanY<sub>B</sub> expression level in the Svic1 mutant was 6.2 times higher than in the wild-type strain. This difference may explain the sviceucin-resistant phenotype of the Svic1, mutant in which, conversely to the wild-type, a sufficient expression level of van genes is reached to allow synthesis of modified peptidoglycan precursors in order to counter the action of vancomycin.

### Sviceucin leads to a decrease in the percentage of muropeptides ending in D-Ala/D-Lac

To verify the latter hypothesis, we investigated the effect of sviceucin on peptidoglycan biosynthesis in the presence of vancomycin by quantifying the muropeptides ending in D-Ala-D-Ala and D-Ala-D-Lac in the wild-type and Svic1 strains by liquid chromatography coupled to mass spectrometry (LCMS) (Figure 3E). When induced with vancomycin alone (2 µg/mL), the percentage of muropeptides D-Ala-D-Lac reached 48.3% and 62.0% in the wild-type and the Svic1 mutant, respectively, which is consistent with the increased MIC of vancomycin in Svic1. Importantly, the presence of sviceucin resulted in a decrease of the percentage of muropeptides D-Ala-D-Lac (28.5%) in the wild-type strain, while that of the Svic1 mutant remained unchanged. Taken together, our data confirm that sviceucin breaks van operon-mediated vancomycin resistance by affecting the

signal transduction of VanR<sub>B</sub>S<sub>B</sub>, leading to a decreased expression of the van operon and, subsequently, a decreased incorporation of the modified muropeptides D-Ala-D-Lac into the peptidoglycan. This should also hold true for siamycin I.

### Sviceucin and siamycin I directly interact with the Van histidine kinase

Given that the resistant mutants harbor mutations located in the ATPase domain of VanS<sub>B</sub>, this suggests that the peptides directly interact with this HK. This hypothesis was tested via genetic and biochemical experiments. First, a system allowing VanS<sub>B</sub>-independent constitutive expression of the van operon was generated. It has been shown that the conserved aspartate residue D53 at the N-terminal receiver domain of VanR<sub>B</sub> is the phosphate acceptor following phosphorylation by P-VanS<sub>B</sub>,42,43 and that substituting the conserved aspartate by a glutamate residue mimics a phosphorylated response regulator (P-VanR<sub>B</sub>).44 We introduced a copy of a vanR<sub>B</sub> variant gene encoding the phosphomimetic modification D53E into the chromosome of a vancomycin-sensitive V583ΔvanS<sub>B</sub> background strain (V583 ΔvanS<sub>B</sub> malT::vanR<sup>D53E</sup>). In this strain, the MIC of vancomycin (32 µg/mL) was similar to that of the wild-type strain, indicating that the VanS<sub>B</sub>-independent constitutive expression of the van operon was successfully elicited (Table 3). We verified that vanY<sub>B</sub> expression is indeed obtained in this strain even in absence of vancomycin induction (Figure S5). In contrast to the wild-type strain, sviceucin and siamycin I did not suppress the resistance to vancomycin, suggesting that the lasso peptides act upstream of VanR<sub>B</sub>.

To further support this, we investigated the origin of the different activity spectra of sviceucin and siamycin I between VanA- and VanB-type vancomycin resistance. We first replaced vanS<sub>B</sub> by vanS in the VanB-type strain E. faecalis V583. VanS-VanR<sub>B</sub> strain was sensitive to vancomycin (MIC = 2 µg/mL), probably because of an impaired signal transduction between VanS and VanR<sub>B</sub>. To bypass this issue, we replaced the vanR<sub>B</sub>S<sub>B</sub>-P<sub>vanY<sub>B</sub></sub> fragment by the vanRS-P<sub>vanH</sub> fragment from a VanA-type operon, resulting in a VanA-type transduction system fused to VanB-type resistance genes. In this strain, the phenotype of a VanA-type strain was restored, meaning resistance to sviceucin while remaining sensitive to siamycin I (Table 4). Furthermore, the replacement of the vanB gene encoding the D-Ala:D-Lac ligase by its VanA-type homolog vanA did not affect the sensitivity of the strain toward sviceucin, confirming that the activity spectrum of the peptide is linked to the TCS and not the resistance genes. Taken together, these results confirm that sviceucin and siamycin I interfere with the signal transduction of the TCS at the level of the HK VanS<sub>B</sub>/VanS.

To provide evidence for a direct interaction, the cytoplasmic domains of the HKs were recombinantly produced in E. coli. Despite numerous efforts, only the C-terminal His<sub>6</sub>-tagged VanA-type VanS<sup>127-384</sup> could be obtained as a soluble protein. The peptide-protein interactions were analyzed by differential scanning fluorimetry (DSF). The affinities of siamycin I, sviceucin, and an unrelated lasso peptide microcin J25<sup>45</sup> for VanS<sup>127-384</sup> were determined by monitoring the changes of the melting temperature (Tm) of the polypeptide (Figure 3F). While microcin J25 did not affect the Tm of VanS<sup>127-384</sup>, sviceucin and siamycin I
<table><caption>**Table 3.** Effect of the phosphomimetic modification VanR<sup>D53E</sup> on the MICs (µg/mL) of vancomycin</caption><thead><tr><th>Strain</th><th>Control</th><th>Sviceucin</th><th>Siamycin I</th></tr></thead><tbody><tr><td>V583</td><td>32</td><td>2</td><td>2</td></tr><tr><td>V583 ΔvanS<sub>B</sub></td><td>2</td><td>2</td><td>2</td></tr><tr><td>V583 ΔvanS<sub>B</sub> malT::vanR<sup>D53E</sup></td><td>32</td><td>32</td><td>32</td></tr></tbody></table>

"Control" refers to a condition in which the 1:1 water:methanol peptide formulation without peptide was added. See also Figure S5.

caused a dose-dependent reduction of its thermal stability, with an equilibrium dissociation constant ($K_d$) of 52 ± 1.6 µM and 38 ± 1.0 µM, respectively. We therefore conclude that sviceucin and siamycin I specifically interact with the VanA-type HK VanS. Next, we investigated whether this interaction leads to inhibition of the autophosphorylation activity of the HK. Indeed, sviceucin inhibited VanS<sup>127-384</sup> with $IC_{50}$ of 10.9 ± 1.5 µM (Figure 3G). By contrast, microcin J25 had no effect on VanS<sup>127-384</sup> (Figure S6). Unfortunately, we were unable to determine the $IC_{50}$ for siamycin I since the peptide made the enzymes precipitate under our assay conditions. Given that the CA domains in HKs are highly conserved<sup>28</sup> and that sviceucin and siamycin I share the same 3D structure (Figure S1), it is likely that these C1LPs also target the VanB-type kinase VanS<sub>B</sub>.

Collectively, these data confirm that the molecular basis of restoring vancomycin sensitivity by these C1LPs is the direct inhibition of Van HKs. It is worth noting that sviceucin and siamycin I have distinct spectra toward VanA- or VanB-type vancomycin resistance (Table 1), which could, at least in part, be due to the higher affinity of siamycin I for VanS.

## DISCUSSION

Bacteria use TCSs to control a wide variety of physiological responses, including virulence, biofilm formation, competence, and antibiotic resistance, notably in the context of host infection. Interference with these processes likely does not impact bacterial viability, hence reducing the risk of resistance appearance, which is imposed by the selection pressure. Therefore, targeting TCSs represents a promising strategy to combat the antibiotic resistance crisis. To our knowledge, sviceucin and siamycin I are the only natural products known to target both virulence and vancomycin resistance in major gram-positive pathogens via inhibition of related HKs. Previous reports and our study showed that the C1LPs interfere with FsrC in *E. faecalis*, leading to an attenuation of virulence in a *G. mellonella* infection model. Using the biochemical assay established in this study, direct inhibition of the autophosphorylation activity of FsrC by sviceucin, similarly to siamycin I, was confirmed (Figure S2).<sup>36</sup> The anti-virulence effect could be extrapolated to S. aureus because one of its master virulence regulator, AgrC, is a close homolog of FsrC.<sup>46</sup> This work also establishes that C1LPs inhibit directly VanS, which is the molecular basis of reverting vancomycin resistance. The effectiveness of combined vancomycin/C1LP treatments to restore clinical susceptibility of VRE and VRSA to vancomycin has been proved in the *G. mellonella* infection model. Such an elegant dual mechanism of C1LPs to kill two

<table><caption>**Table 4.** Effect of the signal transduction system on the spectrum of activity of sviceucin and siamycin I on vancomycin MIC (µg/mL)</caption><thead><tr><th>Strain</th><th>Control</th><th>Sviceucin</th><th>Siamycin I</th></tr></thead><tbody><tr><td>V583</td><td>32</td><td>2</td><td>2</td></tr><tr><td>V583 ΔvanS<sub>B</sub>-vanR<sub>B</sub>::vanS-vanR</td><td>32</td><td>32</td><td>2</td></tr><tr><td>V583 ΔvanB::vanA</td><td>32</td><td>2</td><td>2</td></tr></tbody></table>

"Control" refers to a condition in which the 1:1 water:methanol peptide formulation without peptide was added.

birds with one stone bears an immense advantage, as having multiple clinically relevant targets further decreases the risk of simultaneous developments of resistance to all actions of these molecules. In this regard, siamycin I seems particularly interesting. Siamycin I has another direct target, lipid II, an essential precursor for cell wall biosynthesis in gram-positive bacteria, and the inhibition of lipid II led to compromised cell growth.<sup>34</sup>

Breaking antibiotic resistance is a validated therapeutic strategy, as exemplified by the clinical use of β-lactam antibiotic and β-lactamase inhibitor combination.<sup>47</sup> Similar approaches should also be successful to potentiate the use of vancomycin. Being the first-line antibiotic for the treatment of MRSA, β-lactam resistant enterococci, and *Clostridioides difficile* infections,<sup>4,5,48,49</sup> vancomycin encounters resistance frequently in enterococci, especially *E. faecium*, but is hitherto rare for MRSA isolates. In *C. difficile*, vancomycin resistance genes are present and have been associated with elevated vancomycin MICs.<sup>50</sup> High-level vancomycin resistance of *C. difficile* would have dramatic clinical consequences. Therefore, drugs that counteract the vancomycin resistance mechanism would be immediately applicable for VRE infections, but could also be used as precautionary drugs for the treatment of other resistant strains, such as *C. difficile*. Previous studies reported several molecules disrupting the TCS VanSR,<sup>18,51</sup> including a synthetic antimicrobial peptide.<sup>23</sup> However, their molecular mechanisms remained elusive as no direct evidence has been provided that they directly inhibit the TCS VanSR.

To understand the molecular details of how the C1LPs suppress vancomycin resistance, *E. faecalis* mutants resistant to vancomycin in the presence of either sviceucin or siamycin I were isolated. The only overlap between the two combinations were point mutations in VanS. Genetic and biochemical characterization showed C1LPs interact directly with VanS and inhibit its autophosphorylation activity. This leads to blockage of van gene expression and subsequent reduction of the incorporation of the modified pentapeptides into the peptidoglycan. Among other spontaneous resistant mutants, mutations have been mapped in the ($F_1F_0$) H+ ATPase (F-ATPase) operon or in the *ccpA* gene, but none of these mutants were shared by the two different combinations. The F-ATPase mutants were specifically resistant to sviceucin, whereas *ccpA* mutants were resistant to both C1LPs. In both cases, introduction of the corresponding mutation in a wild-type background conferred vancomycin resistance in the presence of the C1LPs, although effects on general fitness are expected for such mutants. The F-ATPase functions in lactic acid bacteria, including *E. faecalis*, as a proton pump
to maintain intracellular pH homeostasis.⁵² In *S. aureus*, ATP synthase mutants show altered growth kinetics and biofilm formation, and are more susceptible to antimicrobial peptides.⁵³,⁵⁴ By comparison, CcpA is a global regulator of genes encoding activities for the transport and catabolism of secondary substrates in gram-positive bacteria.⁵⁵ Knock-outs of *ccpA* have pleiotropic effects on the physiology of enterococci and staphylococci, including reduced biofilm formation, antibiotic resistance, and virulence.⁵⁶,⁵⁷ Although not directly linked to the van operon, these mutations could provide insights into the interplay between vancomycin resistance and bacterial physiology, which in turn could inspire novel resistance-breaker strategies.⁴⁷ Remarkably, these mutants are all still fully susceptible to the anti-virulence activity of the C1LPs, as shown by the *G. mellonella* infection assay (Figure S4), supporting strongly that developments of resistance to one action of the C1LPs do not induce resistance to other activities of the C1LPs.

Siamycin I and sviceucin have a high structural homology³⁰ but differ considerably in several aspects. For both anti-virulence and anti-vancomycin resistance activities, sviceucin requires an effective concentration 5-fold higher than that of siamycin I. Moreover, siamycin I acts on VanA- and VanB-type vancomycin resistant strains, whereas sviceucin is only active on VanB-type strains. Surprisingly, *in vitro* characterization using the soluble cytoplasmic domain of VanS showed that siamycin I only has a slight higher affinity (2-fold) for VanS. The discrepancy of *in vitro* assays and the whole-cell phenotyping could be attributed to: (1) a truncated cytoplasmic domain may not reflect the functioning of a full-length HK, which involves the sensor domain and interactions with the response regulator; (2) transport into the cytoplasm and subsequence availability are different for these peptides; (3) binding of siamycin I to lipid II, a membrane-bound precursor for cell wall synthesis, may somehow favor the interaction between this peptide and VanS.

In conclusion, this study identifies the C1LPs as promising HK inhibitors for the development of next generation antibiotics. Their highly stable structure and their capacity to target several HKs, which confers them dual anti-virulence and anti-vancomycin resistance properties, are particularly advantageous. They can be envisioned to be used as an adjuvant in combination with vancomycin to break high-level resistance to this antibiotic. The C1LPs reported here thus represent a blueprint for engineering new HK inhibitors for the development of next generation antibiotics.

## Limitations of the study

We show here that sviceucin and siamycin I restore vancomycin clinical susceptibility of VRE *in vitro* and in the *Galleria mellonella* animal model. Although this model has several advantages (testing at 37°C, innate immune system similar to mammals, ethical considerations), it would be a step forward to verify these conclusions in a mammalian model. Our biochemical analyses showed that the peptides inhibit the ATPase activity of the HKs FsrC and VanS and directly bind VanS. We also obtained genetic evidences that the ATPase CA domain of VanS<sub>B</sub> is involved the activity of C1LPs. However, we lack a complete structural characterization of the HK-C1LP interactions, which will be obtained by solving the X-ray crystal structures of an HK-C1LP complex.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Caroline Giraud (caroline.giraud@unicaen.fr).

### Materials availability

All plasmids bacterial strains generated in this study are available from the lead contact with a completed Materials Transfer Agreement and with reasonable compensation by requestor for its processing and shipping. There are restrictions to the availability of strains T-SAR12 VRSA.B and T-SAR12 VRSA.A because we want to limit the spread of these multi-resistant strains obtained in our laboratory by transfers of conjugative plasmids of VR *E. faecalis*.

### Data and code availability

* Data: all data reported in this article will be shared by the lead contact upon request.

* Code: this article does not report an original code.

* Any additional information required to reanalyze the data reported in this work will be shared by the lead contact upon request.

## ACKNOWLEDGMENTS

The funding supports from the ANR (Agence Nationale de la Recherche) (PRC project no. ANR-19-CE18-0026-01) and the SATT-Lutech are acknowledged. A.B and B.B thank the Normandy region and the French research ministry for the doctoral scholarships. DNA sequencing was performed at the iGE3 genomics platform of the University of Geneva (http://www.ige3.unige.ch/genomics platform.php) and we thank Mylène Docquier for her expertise. We also thank Nicholas J. Harmer for his help in the DSF data analysis process.

## AUTHOR CONTRIBUTIONS

Conceptualization, A.H., Y.L. and C.Giraud; methodology, A.B., B.B. and S.D.; investigation, A.B., B.B., S.D., C.Goulard, A.-C.G., C.L., S.G., M.M., Y.L., and C.Giraud; writing—original draft, A.B., B.B., S.D., A.-C.G., A.H., Y.L., and C.Giraud; writing—review and editing, A.B., B.B., S.D., A.H., Y.L., and C.Giraud; funding acquisition, A.H., Y.L., and C.Giraud; Supervision, A.M.-F., A.H., Y.L., C.Giraud.

## DECLARATION OF INTERESTS

A.B., B.B., S.D., C.Goulard, A.-C.G., A.M.-F., A.H., Y.L., and C.Giraud are listed as inventors in a granted patent (number EP 23306748.7 of the European Patent Office) based on work presented in this paper. All other authors declare they have no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Strains and growth conditions

* *Galleria mellonella* insect model

* METHOD DETAILS

* Plasmids and bacterial genetics methods

* Determination of minimal inhibitory concentration

* Extraction of muropeptides

* Quantification of muropeptides by mass spectrometry

* Analysis of gene expression by qPCR

* Directed evolution and selection of resistant mutants

* Nanoemulsions formulation process

* *Galleria mellonella* infection and C1LP treatments

* Protein purification

* Differential scanning fluorimetry
o Autophosphorylation assays for VanS and FsrC

● QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.111922.

Received: October 25, 2024

Revised: December 6, 2024

Accepted: January 27, 2025

Published: January 30, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Bacterial and virus strains</td></tr><tr><td>Enterococcus faecalis V583</td><td>Sahm et al.<sup>5</sup></td><td>NCBI: txid226185; RRID: WB-STRAIN: WBStrain00042092</td></tr><tr><td>Enterococcus faecalis V583 ΔvanB</td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 Svic1</td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 Svic1 ΔvanB</td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 SiaA</td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 vanS<sub>B</sub><sup>S402I</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 vanS<sub>B</sub><sup>D398E</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 ΔvanS<sub>B</sub>::vanS</td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 ΔvanR<sub>B</sub>S<sub>B</sub>::vanRS-P<sub>vanY</sub></td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 ΔvanB::vanA</td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 ΔvanS<sub>B</sub></td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis V583 ΔvanS<sub>B</sub> malT::vanR<sup>D53E</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>Enterococcus faecalis MMH594</td><td>Shankar et al.<sup>6</sup></td><td>NCBI: 1158653</td></tr><tr><td>Enterococcus faecalis OG1RF</td><td>Dunny et al.<sup>7</sup></td><td>NCBI: txid474186; RRID: WB-STRAIN: WBStrain00041967</td></tr><tr><td>Enterococcus faecalis JH2-2 08048</td><td>Ladjouzi et al.<sup>8</sup></td><td>N/A</td></tr><tr><td>Enterococcus faecalis Merz96</td><td>Harrington et al.<sup>9</sup></td><td>NCBI: txid565645</td></tr><tr><td>Enterococcus faecium 1 231 410</td><td>Broad Institute</td><td>NCBI: txid565660</td></tr><tr><td>Enterococcus faecalis HIP11704</td><td>Broad Institute</td><td>NCBI: txid565646</td></tr><tr><td>Enterococcus faecium 1 231 502</td><td>Broad Institute</td><td>NCBI: txid565657</td></tr><tr><td>Enterococcus faecium 1 230 933</td><td>Broad Institute</td><td>NCBI: txid565656</td></tr><tr><td>Staphylococcus aureus T-SAR12 VRSA.A</td><td>This paper</td><td>N/A</td></tr><tr><td>Staphylococcus aureus T-SAR12 VRSA.B</td><td>This paper.</td><td>N/A</td></tr><tr><td>Staphylococcus aureus 16038</td><td>Caen University Hospital</td><td>N/A</td></tr><tr><td>Escherichia coli Top10F'</td><td>ThermoFisher Scientific</td><td>Cat# C3030-03</td></tr><tr><td>Escherichia coli EC1000</td><td>Leenhouts et al.<sup>10</sup>; Addgene</td><td>Cat# 71852</td></tr><tr><td>Escherichia coli NEB5α™</td><td>New England BioLabs®</td><td>Cat# C2987H</td></tr><tr><td>Escherichia coli BI21DE(3)</td><td>ThermoFisher Scientific</td><td>NCBI: txid469008; Cat# EC0114</td></tr><tr><td>Streptomyces coelicolor M1146</td><td>Li et al.<sup>30</sup></td><td>N/A</td></tr><tr><td>Streptomyces sp. SKH 2344</td><td>Chokekijchai et al.<sup>58</sup></td><td>N/A</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Tris</td><td>Research Organics</td><td>CAS: 77-86-1</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>Merck</td><td>CAS: 7778-77-0</td></tr><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>Merck</td><td>CAS: 7758-11-4</td></tr><tr><td>NaH<sub>2</sub>PO<sub>4</sub></td><td>Honeywell-Fluka</td><td>CAS: 7558-80-7</td></tr><tr><td>Na<sub>2</sub>HPO<sub>4</sub></td><td>VWR</td><td>CAS: 10028-24-7</td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>CAS: 6132-04-3</td></tr><tr><td>HEPES</td><td>Merck</td><td>CAS: 7365-45-9</td></tr><tr><td>PIPES</td><td>Merck</td><td>CAS: 5625-37-6</td></tr><tr><td>MOPS</td><td>Research Organics</td><td>CAS: 1132-61-2</td></tr><tr><td>SYPRO® Orange Protein Gel Stain</td><td>Sigma-Aldrich</td><td>Cat# S5692</td></tr><tr><td>Vancomycin</td><td>Merck</td><td>CAS: 1404-93-9</td></tr><tr><td>Kanamycin</td><td>Merck</td><td>CAS: 25389-94-0</td></tr></tbody></table>

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Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Tetracyclin</td><td>Serva</td><td>CAS: 64-75-5</td></tr><tr><td>Chloramphenicol</td><td>Thermo Fisher Scientific</td><td>CAS: 56-75-7</td></tr><tr><td>Sviceucin</td><td>Li et al.<sup>30</sup></td><td>PDB: 2LS1</td></tr><tr><td>Siamycin I</td><td>Detlefsen et al.<sup>31</sup></td><td>CAS: 164802-68-0</td></tr><tr><td>Nanoemulsion</td><td>Séguy et al.<sup>41</sup></td><td>N/A</td></tr><tr><td>Sviceucin-Nanoemulsion</td><td>This paper.</td><td>N/A</td></tr><tr><td>Siamycin I-Nanoemulsion</td><td>This paper.</td><td>N/A</td></tr><tr><td>VanS<sup>127-384</sup></td><td>This paper.</td><td>N/A</td></tr></tbody></table>

Critical commercial assays

<table><tbody><tr><td>Q5® High-Fidelity DNA Polymerase</td><td>New England BioLabs®</td><td>Cat# M0491L</td></tr><tr><td>NucleoSpin Plasmid kit</td><td>Macherey-Nagel</td><td>Cat# 740588.50</td></tr><tr><td>GoTaq DNA Polymerase</td><td>Promega</td><td>Cat# M7805</td></tr><tr><td>Luna C18(2) HPLC column</td><td>Phenomenex</td><td>Cat# 00G-4252-N0</td></tr><tr><td>Acclaim™ RSLC PolarAdvantage II</td><td>Thermo Fisher Scientific</td><td>Cat# 555-7122</td></tr><tr><td>CytoOne® 96 wells microplate</td><td>Starlab</td><td>Cat# CC7672-7596</td></tr><tr><td>Lysis Matrix B Tube</td><td>MP Biomedicals™</td><td>Cat# 11462410</td></tr><tr><td>Direct-Zol™ RNA Miniprep kit</td><td>Zymo Research</td><td>Cat# R2051</td></tr><tr><td>NucleoSpin® miRNA Miniprep kit</td><td>Macherey-Nagel</td><td>Cat# 740971.50</td></tr><tr><td>QuantiTect® Reverse transcription kit</td><td>QIAGEN</td><td>Cat# 205311</td></tr><tr><td>GoTaq® qPCR Master kit</td><td>Promega</td><td>Cat# A6002</td></tr><tr><td>GenElute™</td><td>Sigma-Aldrich</td><td>Cat# NA2120</td></tr><tr><td>Illumina DNA prep kit</td><td>Illumina</td><td>Cat# 20060059</td></tr><tr><td>HisTrap™ FF Crude 5 mL column</td><td>Cytiva</td><td>Cat# 17528601</td></tr><tr><td>Vivaspin® 6 30kDa MWCO</td><td>Cytiva</td><td>Cat# 28932317</td></tr><tr><td>Pierce™ BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>Cat# 23225</td></tr><tr><td>96 Fast PCR Plates full skirt</td><td>Sarstedt</td><td>Cat# 72.1980</td></tr></tbody></table>

Experimental models: Organisms/strains

<table><tr><td>Galleria mellonella</td><td>Reptilis</td><td>RRID: NCBITaxon_7137</td></tr><tr><td>Oligonucleotides</td><td></td><td></td></tr><tr><td>See Table S5</td><td>Eurofins Scientific</td><td>N/A</td></tr></table>

Recombinant DNA

Plasmid:

<table><tbody><tr><td>P4H7 cosmid</td><td>Li et al.<sup>30</sup></td><td>N/A</td></tr><tr><td>pEBM2</td><td>Lab collection.</td><td>N/A</td></tr><tr><td>pEBM2 ΔvanS<sub>B</sub></td><td>This paper.</td><td>N/A</td></tr><tr><td>pEBM2 ΔvanS<sub>B</sub>::vanS</td><td>This paper.</td><td>N/A</td></tr><tr><td>pEBM2 ΔvanR<sub>B</sub>S<sub>B</sub>::vanRS-P<sub>vanH</sub></td><td>This paper.</td><td>N/A</td></tr><tr><td>pEBM2 malT::P<sub>hup</sub>-vanR<sup>D53E</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>pAS222</td><td>Jönsson et al.<sup>11</sup></td><td>N/A</td></tr><tr><td>PAS222 vanS<sub>B</sub><sup>S402I</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>PAS222 vanS<sub>B</sub><sup>D398E</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>pWS3</td><td>Zhang et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pWS3 ΔvanB</td><td>This paper.</td><td>N/A</td></tr><tr><td>pWS3 ΔvanB::vanA</td><td>This paper.</td><td>N/A</td></tr><tr><td>pET29b(+)</td><td>Novagen</td><td>N/A</td></tr><tr><td>pET29b(+) vanS<sup>127-384</sup></td><td>This paper.</td><td>N/A</td></tr><tr><td>pHisMBPTev</td><td>Lab collection.</td><td>N/A</td></tr><tr><td>pHisMBPTev fsrC<sup>240-447</sup></td><td>This paper.</td><td>N/A</td></tr></tbody></table>

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<figure><img src="image_20.png" /></figure>

Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>DataAnalysis 4.4</td><td>Bruker Daltonics</td><td>https://bruker-compass-dataanalysis.software.informer.com/</td></tr><tr><td>CFX Manager</td><td>BioRad</td><td>https://www.bio-rad.com/fr-fr/sku/1845000-cfx-manager-software?ID=1845000</td></tr><tr><td>Graphpad Prism 10</td><td>GraphPad Software</td><td>https://www.graphpad.com/</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>Tecan Infinite® M Nano</td><td>TECAN</td><td>Cat# 30190086</td></tr><tr><td>Branson 1800 cleaner</td><td>Branson</td><td>N/A</td></tr><tr><td>Gene Pulser™</td><td>BioRad</td><td>N/A</td></tr><tr><td>2002-1BIOBCE</td><td>Bioblock Scientific</td><td>N/A</td></tr><tr><td>Ultimate 3000 RSLC</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td>FastPrep-24™ 5G</td><td>MP Biomedicals</td><td>Cat# 116005500</td></tr><tr><td>KDS100 Syringe pump</td><td>Fisher Scientific</td><td>N/A</td></tr><tr><td>Fisherbrand™ Model 120 sonic dismembrator</td><td>Fisher Scientific</td><td>Cat# 12337338</td></tr><tr><td>ÄKTA start chromatography system</td><td>Cytiva</td><td>Cat# 29022094</td></tr><tr><td>CFX96 Real-Time PCR system</td><td>BioRad</td><td>N/A</td></tr><tr><td>POLARstar OMEGA</td><td>BMG Labtech</td><td>N/A</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Strains and growth conditions

Bacterial strains used in this study are listed in Table S3. *Enterococcus* strains were cultured in M17 medium⁵⁹ supplemented with glucose 0.5% (GM17) at 37°C without shaking. *Staphylococcus* strains were cultured in MH medium⁶⁰ at 37°C with shaking at 120 rpm. *Escherichia coli* strains were cultured in LB medium⁶¹ at 37°C or 30°C with shaking at 120 rpm. Growth was assessed by monitoring the optical density at 600 nm (OD₆₀₀) and performed in 96-well microplates (CytoOne) in a spectrophotometer (Tecan Infinite M Nano.) with agitation for *S. aureus* strains and without agitation for *Enterococcus* strains. Media were supplemented with chloramphenicol at 10 or 20 µg/mL (Thermo Fisher Scientific, USA), kanamycin at 15 µg/mL (Merck, USA) or tetracycline at 10 µg/mL (Serva, Germany) when necessary for mutant constructions.

### Galleria mellonella insect model

*Galleria mellonella* larvae, used as an insect model, are reared in-house and kept in sealed, ventilated boxes in an incubator set at 32°C. The incubator atmosphere is kept moist with water and the larvae are fed pollen and beeswax twice a week. For each new generation, a few large larvae are selected and isolated to form cocoons. The cocoons are then transferred to a rearing cage in the same incubator. In this cage, the moths are able to reproduce and lay eggs on a paper towel, which is used to start a new fresh batch of larvae. The larvae used as models are approximately six weeks old and weigh an average of 218 mg. For each experiment, larvae from the same batch are randomly selected from those that are light-coloured without gray or black markings and then transferred to sterile Petri dishes with 5 larvae per dish.

## METHOD DETAILS

### Plasmids and bacterial genetics methods

All plasmids and primers used in this study are listed in Tables S4 and S5. Plasmids were replicated in *E. coli* strains. DNA fragments were obtained by PCR using Q5 High-Fidelity DNA Polymerase (New England BioLabs, USA) and purified (QIAGEN, Germany). Plasmid extractions were performed using the NucleoSpin Plasmid Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. Recombinant plasmids were constructed by in vivo recombination using DNA fragments with overlapping ends, as previously described.⁶² Inverse PCR was performed on extracted plasmids to linearize them, and the circular matrix was digested by DpnI. Purified DNA fragments and linearized plasmids were then transformed into competent *E. coli* Top10F' cells by heat shock (30 min on ice, 90 s in 42°C water, 2 min on ice). Purified constructed plasmids were then electroporated into competent *E. faecalis* V583 cells using a Gene Pulser (2.25 kV, 200 Ω, 25 µF) (Biorad, USA). Mutations were introduced into the chromosome by double crossing-over of the plasmid as previously described.⁶³ Thermosensitive plasmids were inserted into the genome by
selective pressure with an antibiotic to which the plasmid infers resistance (chloramphenicol in the case of pEBM2) at a non-repli-
cative temperature (37°C in the case of pEBM2). Plasmid excision was then performed by successive subcultures in a minimal growth
medium such as MH at a replicative temperature and without the antibiotic to select clones that lost their resistance. Plasmids and
mutants were verified by amplification of the corresponding loci using GoTaq DNA Polymerase (Promega, USA) and sequencing.
Production and purification of C1LPs.

The heterologous expression strain *Streptomyces coelicolor* M1146 harboring the P4H7 cosmid³⁰ and the native producing strain *Streptomyces* sp. SKH 2344⁶² were used for the production of sviceucin and siamycin I, respectively. Both strains followed the same fermentation procedure. The *Streptomyces* strain was grown from a mycelium stock in 150 mL GYM medium⁶³ for 4 days at 30°C with agitation. This pre-culture was used to inoculate a 3 L fresh GYM culture, which continued growth at 30°C for 7 days. The cell pellets were collected and extracted by 400 mL MeOH at room temperature for 4 h. After solvent evaporation, the dried crude extract was resuspended in 3 mL 80% acetonitrile and purified by semi-preparative HPLC on a Phenomenex C18 column (Luna C18 (2), 5 µm, 100 Å, 250 × 10 mm) at 3 mL/min with UV detection at 280 nm. Eluents used were A (H₂O with 0.1% formic acid) and B (aceto-nitrile). For sviceucin purification, the gradient was a linear increase of 45%-60% B in 10 min. For siamycin I purification, the gradient was a linear increase of 20%-70% B in 30 min. The corresponding peak containing the peptide was collected. For peptide quanti-fication, a predicted extinction coefficient at 280 nm of 11860 M⁻¹ cm⁻¹ for sviceucin and 7450 M⁻¹ cm⁻¹ for siamycin I were used.

Determination of minimal inhibitory concentration

The MIC of vancomycin was determined by the microdilution method described in the guidelines of the European Committee on Anti-
microbial Susceptibility Testing (EUCAST 2024, v14.0 Breakpoint Tables).⁵⁸ For enterococci, GM17 medium was used instead of
CAMH medium since enterococci cannot degrade starch, the main carbohydrate of MH medium. 2-fold serial dilutions of the tested
antibiotic were performed to obtain concentrations ranging from 0.5 to 512 µg/mL. LP were then added to the wells at the desired
concentration. The medium was inoculated with a final inoculum of 5 × 10⁵ CFU/mL. The MIC is the lowest concentration that pre-
vents visible *in vitro* growth.

Extraction of muropeptides

Extraction of muropeptides was performed as previously described⁶⁴ with modifications. Bacteria were cultured overnight in GM17 medium supplemented with 2 µg/mL of vancomycin (Merck, USA), 10 µM of sviceucin, or 2 µM of siamycin I. Bacteria were pelleted by centrifugation for 5 min at 10000 rpm and then resuspended in 1 mL of 0.1 M Tris-HCl (pH 6.8), 0.25% SDS. Samples were boiled for 20 min in a heating block (Biolock Scientific, USA) and then centrifuged for 5 min at 10000 rpm. The pellets were washed twice with 1 mL of cold ultrapure water before sonication for 30 min (Branson 1800 Cleaner, USA). 500 µL of DNase 15 µg/mL and RNase 60 µg/mL (Sigma-Aldrich, USA) were added to each sample then incubated at 37°C for 60 min. Samples were then treated with trypsin at 5 µg/mL (Promega, USA) and incubated at 37°C for 2 h. Enzymes were inactivated by boiling the samples for 3 min and then washed once with ultrapure water. Pellets were resuspended in 12.5 mM sodium phosphate buffer (pH 5.5), then muropeptides concentration was determined and standardized to an OD₅₇₈ of 3. Samples were then treated with mutanolysin (Sigma-Aldrich, USA) at 5000 U/mL and incubated at 37°C, 150 rpm shaking, for 16 h. Samples were boiled for 3 min to inactivate the enzyme and then centrifuged. The supernatants containing the muropeptides were collected and stored for LCMS analysis.

Quantification of muropeptides by mass spectrometry

LCMS analysis was performed on an ultra-high performance LC system (Ultimate 3000 RSLC, Thermo Scientific, USA) coupled to a
high-resolution electrospray ionization-quadrupole-time of flight mass spectrometer (MaXis II ETD, Bruker Daltonics, USA). The mur-
opeptide solution was diluted five times in MeOH before analysis. LC separation was performed on an Acclaim RSLC Polar Advan-
tage II column (2.2 µm, 2.1 × 100 mm, Thermo Scientific, USA) with a flow-rate of 0.3 mL/min using the following gradient (eluent A:
water with 0.1% formic acid (v/v); eluent B: acetonitrile): 2% B for 3 min, followed by a linear increase to 15% B over 7 min and a final
increase to 100% B in 1 min. The mass range of 250–1500 in positive mode was acquired. Mass data were analyzed by Data Analysis
4.4 (Bruker Daltonics, USA). For quantification, the *m/z* corresponding to singly- and doubly-charged species of monomer, as well as
doubly- and triply-charged species of dimers of muropeptides were manually searched. These were the most abundant masses
observed under our experimental conditions. Subsequently, the areas of the peaks corresponding to the same muropeptide in
the extracted ion chromatograms were combined for calculation.

Analysis of gene expression by qPCR

For *gelE* gene expression measurements, bacteria were cultured in GM17 medium supplemented with the 1:1 water:methanol formu-
lation or either sviceucin at 10 µM, or siamycin I at 1 µM. Cultures were incubated at 37°C until reaching OD₆₀₀ of 0.5. For *vanY* gene
expression measurements, bacteria were cultured in GM17 medium supplemented with either vancomycin at 1 µg/mL or sviceucin at
10 µM and vancomycin at 1 µg/mL at 37°C to induce the expression of *van* genes until reaching OD₆₀₀ of 0.5. Bacteria were recovered
by centrifugation at 4200 RCF for 2 min. Once the supernatant was discarded, the pellets were stored at -80°C. Pellets were resus-
pended in TE buffer or ML lysis buffer, depending on kit used, and transferred in Lysis Matrix B Tubes (MP Biomedicals, USA) before
cell lysis with a FastPrep-24 5G (2 × 40 s at 6 m/s, 5 min on ice in-between cycles) (MP Biomedicals, USA). After bead-beating lysis,
RNA extraction was performed using Direct-Zol RNA Miniprep kit (Zymo Research, USA) for *gelE* gene expression measurements or
NucleoSpin® miRNA Miniprep kit (Macherey-Nagel, Germany) for vanY gene expression measurements, according to the manufacturers' instructions. Retrotranscription was performed using QuantiTect Reverse transcription (QIAGEN, Germany). qPCR on cDNA was performed with GoTaq qPCR (Promega, USA). The housekeeping gene gyrA was used as a reference for normalization. Data were analyzed with CFX Manager software (Biorad, USA).

## Directed evolution and selection of resistant mutants

Resistant clones to sviceucin were selected by plating bacteria on GM17 agar medium supplemented with vancomycin at 32 µg/mL and sviceucin at 10 µM. Growth and MIC of vancomycin and sviceucin of the resistant clones were tested. The selection for siamycin I resistance was done by performing serial passages of bacteria in GM17 medium supplemented with vancomycin 10 µg/mL and increasing concentrations of siamycin I from 0.5 to 5 µM. Single nucleotide polymorphism (SNPs) analysis was performed on the sequenced gDNA of each selected resistant mutant.

The genomic DNA of the selected resistant mutants and the WT strain *E. faecalis* V583 were extracted using GenElute (Sigma-Aldrich, USA). Whole genome sequencing was then performed at the iGE3 genomics platform of the University of Geneva. The Illumina DNA prep kit was used for library preparation according to manufacturer's specifications, with 200 ng of genomic DNA as input. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Libraries were sequenced on a NovaSeq 6000 Illumina sequencer with a minimum of 15 M of paired-end 100 reads per sample. The sequencing quality control was done with FastQC. The reads were mapped with the BWA v.0.7.10⁶⁵ software to the NCBI *Entero-*coccus faecalis V583 AE016830.1 reference. After sorting and marking the PCR replicates with samtools,⁶⁶ the alignments were screened for variants with freebayes v.0.9.21, by specifying a haploid genome.

## Nanoemulsions formulation process

The formulation of the nanoemulsions was based on the spontaneous nanoemulsification method previously described by Séguy et al.⁴¹ and transposed to lasso peptides. The present nanoemulsions were developed from anhydrous mixtures composed of excipients already described in the European Pharmacopeia (World Health Organization, 2018) and already proven to be hemocompatible on human whole blood. Monodisperse nanoemulsions could be formulated at a drug payload of 400 µg/mL for siamycin I. At these concentrations, the drug added to the anhydrous mixture during the formulation process appeared to be completely dissolved. After the addition of the aqueous phase, a homogeneous colloidal suspension was observed. The nanoemulsions were characterized in terms of granulometric and physicochemical properties (Table S6). The hydrodynamic diameter, zeta potential, and PDI were not changed relative to the blank nanoemulsions, remaining lower than 0.25, indicating the presence of a monodispersed population.

## Galleria mellonella infection and C1LP treatments

Bacteria were collected from overnight cultures by centrifugation, washed three times with normal saline solution (NSS), then adjusted to an OD₆₀₀ of 0.3 corresponding to ~10⁸ CFU/mL. 10 µL of the pre-treated bacterial suspension were injected following Fuchs et al.⁶⁷ protocol with this modification: larvae were injected subcutaneously into in-house reared *Galleria mellonella* larvae using a syringe pump (KD scientific, USA) in order to inoculate 10⁶ CFU into each larva or the equivalent volume of NSS as a control. To assess the anti-virulence effect of the C1LPs, *E. faecalis* V583 was grown overnight in a medium supplemented with either 10 µM sviceucin or 2 µM siamycin I. 15 larvae were used for each condition. Larvae were recorded as dead in absence of reactivity to stimuli at 24 h p.i.. To assess the C1LPs' efficacy as treatment of *E. faecalis* infection, the strains V583 or V583 Δ*vanB* were grown without previous C1LP exposition. 20 larvae were used for each condition. At 1.5 and 24 h p.i., larvae were injected with 4.76 mg of vancomycin per kilogram and/or 18 mg of siamycin I per kilogram, or the equivalent volume of nanoemulsion as a control. Larvae killing was then monitored between 1.5 and 96 h p.i.. Data and statistical analysis were performed using the Kaplan-Meier R package and the log-rank test, respectively.

## Protein purification

Terrific medium⁶⁸ (1L) equally dispatched in four 1 L baffled Erlenmeyer flasks was inoculated with an overnight culture of *E. coli* BL21(DE3) possessing the plasmid pET29b(+) *vanS*¹²⁷⁻³⁸⁴ to a final OD₆₀₀ of 0.1 (Tables S3 and S4). The culture was incubated at 37°C with agitation at 180 rpm. Induction of protein expression was achieved by adding IPTG to a final concentration of 0.1 mM when the bacterial density reached an OD₆₀₀ of 0.5. The culture was then incubated for 20 h at 25°C with agitation at 180 rpm. The cells were then washed twice in purification buffer (20 mM sodium phosphate buffer, 500 mM NaCl, 5 mM MgCl₂, 10 mM imidazole (pH 7.4)). Cells were treated with lysozyme at 1 mg/mL final concentration for 30 min at 4°C and lysed by sonication with the Fisherbrand Model 120 Sonic Dismembrator (Fisher scientific, USA). The total fraction recovered was centrifuged at 10000 rpm at 4°C for 30 to 45 min and only the supernatant, representing the soluble fraction, was recovered for purification.

Protein purification was carried out on a HisTrap FF Crude 5 mL column (Cytiva, USA) using the ÄKTA start device (Cytiva, USA), following the manufacturer's recommendations. The column was equilibrated with the purification buffer and then loaded with the soluble fraction. The protein of interest was washed and eluted with an imidazole gradient of 10 mM-500 mM in purification buffer. The purified protein was desalted and concentrated on a Vivaspin 6 30 kDa MWCO column (Cytiva, USA) according to the
manufacturer's recommendations. Protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, USA) according to the manufacturer's recommendations and then stored at -80°C.

## Differential scanning fluorimetry

All the experiments were performed in 96 Fast PCR Plates full skirt (Sarstedt, Germany) and in CFX96 Real-Time PCR system (BioRad, USA). Screening was conducted (Table S7) to determine the best buffer and ratios of protein and SYPRO Orange Protein Gel Stain (Sigma-Aldrich, USA). For the experiments, each well contained 25 µL of 10 µM VanS solution, 1/450 X of SYPRO Orange Protein Gel Stain and lasso peptides at various concentrations: siamycin I – 6 to 90 µM; sviceucin – 12 to 180 µM; microcin J25 – 25 to 200 µM. VanS, SYPRO Orange and lasso peptides were diluted in 20 mM potassium phosphate buffer (pH 7.5, Table S7). Each condition was replicated three times per plate and each plate was replicated three times. CFX Manager was used to recover data from runs. The Tm of each well was determined on the Melt Curve panel with a negative peak type. Tm means were calculated for each condition before model analysis. ΔT was calculated from the subtraction of the Tm of the protein with the buffer alone and the Tm with the lasso peptides. All analyses were carried out with Graphpad Prism 10 as previously described.⁶⁹ After setting protein concentration (P) as “Constant equal to” 10 µM in the software, data from sviceucin and siamycin I fitted best with the simple cooperative model from Vivoli et al.⁶⁹

## Autophosphorylation assays for VanS and FsrC

The phosphorylation assay followed reported procedure with some modifications.³⁸ A standard reaction of 200 µL contained 50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 2 µM purified C-His~6~-VanS¹²⁷⁻³⁸⁴ or N-MBP-C-His~6~-FsrC²⁴⁰⁻⁴⁴⁷, 0–100 µM of respective lasso peptide, 5 mM ATP, 10 mM MgCl~2~, 0.5 mM NADH, 2 mM phospho(enol)pyruvic acid (PEP), 18 U of L-lactate dehydrogenase (LDH, Roche, Switzerland) and 9 U of pyruvate kinase from rabbit muscle (PK, Sigma-Aldrich, USA). The peptide was pre-incubated with the kinase at room temperature for 10 min followed by the addition of PEP/LDH/PK and incubation at 37°C for 3 min. The reaction was allowed to start by adding ATP and NADH, and monitored continuously at 340 nm at 37°C by a microplate reader (POLARstar OMEGA, BMG Labtech, Germany). Each measurement was performed in triplicate, and at least three independent experiments were repeated. Nonlinear regression data fitting and IC~50~ calculation were performed with GraphPad Prism 10.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical parameters and significances are reported in the figure legends and figures. The number of independent experimental replicates is specified in each figure legend.