Bacteria used include Salmonella enterica subsp. enterica serovar Typhimurium 14028, Staphylococcus aureus USA300, and Enterobacter cloacae subsp. cloacae (Jordan) Hormaeche and Edwards, subsp. nov. ATCC 13047. All single gene deletion mutants of S. aureus and where indicated, single and multiple gene deletion mutants of S. Typhimurium, were obtained through BEI Resources (NIAID, NIH). See Supplementary Table 1 for details of individual isolates used in this study (Fitzsimmons et al., 2020; Muller et al., 2009; Porwollik et al., 2014).

Individual bacterial colonies were used to inoculate tryptic soy broth ([TSB]; Fisher Cat No. DF0370-07-5) (S. Typhimurium and S. aureus) or Luria Bertani broth ([LB]; Fisher Cat No. BP1426-2) (E. cloacae) for liquid overnight (O/N) cultures grown at 37°C with aeration in a rolling drum.

S. Typhimurium double mutants generated in this study were created using P22HTint transduction. In brief, donor mutants (with either a kanamycin [Kan] or chloramphenicol [Cam] insertion in the first gene of interest) were infected with P22HTint. The donor P22 lysate was then introduced to a recipient mutant (with a different antibiotic cassette insertion in the second gene of interest) in order to create a double gene-deletion mutant. The double mutant was selected for on LB agar with Kan (45 µg/mL) and Cam (20 µg/mL) and then confirmed phage-free on Evan’s blue uridine (EBU) agar.

The conditions used to grow biofilms for each species were previously described in detail (Sandala and Gunn, 2021; Bennett et al., 2023). In brief, single species biofilms were grown in media alone in 96-well plates (Corning Cat No. 7200656) for 24 hours. After the initial incubation, the spent media was removed and replaced with media containing each respective anti-biofilm compound (diluted from 100 mM stock) or equal volume of vehicle (DMSO). Biofilms were then re-incubated for up to 24 hours prior to analysis.

Identification of compounds M4 and JG-1 were previously described (see Supplementary Figures 1A, C for their respective structures) (Sandala et al., 2020; Woolard et al., 2022). HCl-salted samples of M4 and JG-1 were stored as powders, shielded from light, at -20°C. Stock solutions were prepared in DMSO at a concentration of 100 mM and stored at -20°C. All further dilutions were prepared in culture media such that the final DMSO concentration was no greater than 10% (v/v). In experiments where the effects of more than one concentration were tested, the concentration of the vehicle was standardized across all conditions by adding an equal volume of DMSO.

Biofilms of E. cloacae were grown for 24 hours as described above, prior to treatment with 2.5-80 µM JG-1, M4, or vehicle (DMSO) for 1–24 hours. After treatment, biofilms were washed with H₂O prior to fixation and staining with crystal violet as previously described (Sandala and Gunn, 2021). The change in biofilm mass was measured via crystal violet staining and used to compare rate of activity of each compound.

Biofilms of S. Typhimurium and E. cloacae were grown in 96-well plates as described above. The supernatant of each well was removed and replaced with an equal volume of 10 µM (S. Typhimurium) or 80 µM (E. cloacae) JG-1, M4, or DMSO and re-incubated for 2 hours (S. Typhimurium) or 5 hours (E. cloacae). After incubation, the supernatant was removed and replaced with an equal volume of phosphate buffered saline (PBS). Plates were then scraped and/or sonicated for 10 minutes to suspend the biofilm cells. The cells were pelleted at 5000 rpm for 20 minutes at 4°C and the cell pellet frozen at -80°C for at least 30 minutes prior to RNA extraction.

Planktonic cells of S. aureus were grown overnight at 37°C in a rolling drum, prior to being normalized to an optical density at 600 nm (OD₆₀₀) of 0.8 and diluted 1:1000 in 80 µM M4 or DMSO in TSB with 2.5% dextrose (TSBG). Compound treated cells were incubated for 2 hours at 37°C prior to being pelleted as described above, and frozen at -80°C for at least 30 minutes prior to RNA extraction.

RNA was isolated using the hot phenol method. In brief, frozen cells were resuspended in 2M sodium acetate (AE) buffer, 20% sodium dodecyl sulfate (SDS) (w/v), and phenol. The suspension was incubated in a 65°C water bath for 10 minutes and then immediately placed on ice. The cell debris was pelleted and the lysate treated with chloroform to separate the genomic material from the protein fraction. Any remaining protein was removed with isopropanol and cold ethanol washes until a sufficiently pure RNA pellet was obtained. The pellet was resuspended in RNase-free H₂O and stored at -80°C until analysis.

S. Typhimurium library prep and sequencing was performed on the Illumina platform. Quality control and adapter trimming was performed with bcl2fastq (bcl2fastq Conversion Software). Read mapping was performed with HISAT2 (Kim et al., 2019). Read quantification was performed using Subread’s featureCounts (Liao et al., 2014) functionality. Differential gene expression was qualitied by deseq2. Pathway analysis was performed using ShinyGo 0.81 and iDEP 2.01 (Ge et al., 2018; Ge et al., 2020).

Prior to invasion of S. Typhimurium, HeLa cells (gift from the Wang lab, Nationwide Children’s Hospital) were grown in 12-well tissue culture plates (Corning) with Dulbecco’s Modified Eagle Medium (DMEM, Fischer Scientific) with 10% Fetal Bovine Serum (Gibco) in 5% CO₂ at 37°C until confluency was achieved. A single colony of S. Typhimurium ATCC 14028 (JSG210) from an LB agar plate was used to grow an overnight culture in TSB at 37°C. The following day, the overnight culture was diluted 1:33 in TSB or TSB supplemented with 100 µM of either JG-1, M4, or DMSO. The diluted cultures were grown statically at 37°C for 4 hours. After 4 hours, the bacterial cells from the 1:33 diluted cultures were pelleted and washed twice with TSB. The optical density of each culture was read with 1xPBS and normalized to OD₆₀₀ = 0.8. HeLa cells were infected in quadruplicate with 1 mL of normalized cell cultures in DMEM at a multiplicity of infection (MOI) of 100. Infections were performed for 45 minutes in 5% CO₂ at 37°C. Following infection, wells were gently washed once with 1 mL of 1x PBS and treated with 1 mL of 100 µg/mL gentamicin (Corning) in DMEM for 30 minutes in 5% CO₂ at 37°C. The wells were then washed again with 1 mL of 1x PBS to remove any remaining gentamicin. 200 µL of 0.25% Triton X-100 (Fischer Scientific) in 1x PBS was added to each well and scraped. The scraped cells were then serially diluted and plated on LB agar plates for bacterial enumeration. Colony forming units (CFUs) were counted the next day. All invasion experiments were performed in quadruplicate.

Liquid cultures of WT, ΔcheY, and Δtsr isolates of S. Typhimurium were grown in TSB at 37°C, overnight. Following incubation, the overnight cultures were normalized to OD₆₀₀ of 0.8 and diluted 1:100 in 1:20 TSB or 1:20 TSB supplemented with 100 µM of JG-1, M4, or DMSO. The JG-1, M4, or DMSO treated cultures, and untreated controls were incubated statically at 37°C for four hours. After pre-treatment, 5 µL of each condition was inoculated in Sulfur, Indole, Motility (SIM [NEOGEN Cat No. 50-201-5921]) agar plates supplemented with 25 µM JG-1, M4, or DMSO, or plain SIM agar. Agar plates were incubated overnight at 37°C. The diameter of the growth was measured at its widest point using calipers (Fisherbrand™ Cat. No. S90187B), to the nearest one-tenth of a millimeter (mm). Changes in motility were evaluated by comparing the diameter of growth between treatment groups.

To evaluate potential compound targets suggested by the performed assays, single and multiple gene mutants of S. Typhimurium and S. aureus were tested for recalcitrance to compound treatment. See Supplementary Table 1 for a full description of all mutants tested. S. Typhimurium WT mutant biofilms were grown for 24 hours in 96-well plates as described previously. After incubation, the biofilms were treated with 10-160 µM of JG-1, M4, or vehicle control (DMSO) in 1:20 TSB and re-incubated for 24 hours. After treatment, change in biofilm mass was measured via the crystal violet assay. S. aureus WT and mutant cells were incubated in 96-well plates with 0.625-40 µM M4 or DMSO in TSBG and incubated at 37°C for 24 hours. After treatment, remaining planktonic cells were measured by OD₆₀₀ and biofilm mass was measured via the crystal violet assay. Untreated WT biofilms were compared to mutant biofilms by an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. The IC₅₀/EC₅₀ of JG-1 and/or M4 of WT and mutant isolates was calculated with GraphPad Prism 10.3.1 using a nonlinear regression (log(inhibitor) vs. normalized response – Variable slope Least squares fit). To identify any mutants with an altered response to the compound, the mutant IC₅₀/EC₅₀ was compared to the WT value calculated from the same batch of biological replicates with an extra sum-of-squares F test.

We have established that JG-1 and M4 exhibit anti-biofilm activity in vitro against S. Typhimurium, S. Typhi, and members of the ESKAPE pathogens (Sandala et al., 2020; Bennett et al., 2023). To further understand these compounds’ mechanism of action, we wanted to determine if it requires an active response from the bacteria or alternatively, if compound contact with the extracellular matrix alone is sufficient for dispersal. To differentiate these two mechanisms, we sought to induce reversible bacteriostasis in living bacteria within the biofilm, while keeping the extracellular matrix intact. To measure metabolic activity we used S. Typhimurium 14028lux, an isolate of S. Typhimurium previously described to have a chromosomal insertion of a lux operon (Bennett et al., 2024). Bioluminescence is dependent on ATP production, so biofilm relative luminescent units (RLU) were quantified as a proxy for ATP production.

To induce bacteriostasis, we grew 14028lux biofilms in 96-well plates for 24 hours and then incubated them with sodium azide (NaN₃), a bacteriostatic preservative agent that inhibits cytochrome oxidase (Lichstein and Soule, 1944). To determine a minimum time and concentration of NaN₃ necessary to inactivate the bacteria, we treated 14028lux biofilms with varying concentrations of NaN₃ and measured RLUs every 15 minutes for 16 hours ( Figure 1A ). We determined that incubation of a 6% NaN₃ solution for 2 hours is the minimum concentration and time necessary to completely inactivate the bacteria. We also confirmed that the NaN₃ inactivation can persist to at least 4 hours in the presence of compound as indicated by the low RLUs measured after compound + 6% NaN₃ treatment ( Figure 1B ).

To determine if bacteriostasis prevents biofilm disruption, we pre-treated 14028lux biofilms with 6% NaN₃ for 2 hours and then replaced this with media containing 80 µM JG-1, M4, or DMSO with or without 6% NaN₃. These biofilms were incubated for either 2 or 4 hours and then washed and stained with crystal violet to quantify the percent biofilm remaining after treatment. Importantly, 6% NaN₃ treatment leaves the biofilm structure intact as biofilms that were treated with 6% NaN₃ alone for a total 4 or 6 hours do not show any decrease in biofilm mass ( Figure 1C ). Following pre-treatment, biofilms that were treated with JG-1 or M4 in 6% NaN₃ had no change in biofilm mass at 2 or 4 hours ( Figure 1C ). Importantly, pre-treated biofilms that were subsequently treated with JG-1 or M4 in media without the bacteriostatic agent NaN₃ showed a time-dependent decrease in biofilm mass by comparison to vehicle ( Figure 1C ). This indicates that metabolic inhibition with 6% NaN₃ prevents compound induced biofilm dispersal, suggesting that the compounds’ mechanism of action requires metabolically active bacteria able to interact with and respond to the compounds.

Given that the compounds’ antibiofilm activity requires metabolically active cells, we predict that bacterial interaction with the compound would cause transcriptional changes that involve pathways specific to the compounds’ target. We hypothesized that the response to compound would be shared between Salmonella and the other members of the ESKAPE group that are susceptible to compound dispersion. To determine changes in transcription associated with compound treatment, we treated S. Typhimurium (WT) and E. cloacae biofilms with JG-1, M4, or DMSO and planktonic S. aureus cells treated with M4 (bactericidal effect) or DMSO, for the minimum time required to elicit a dispersion response. We have previously reported that S. aureus responds to M4 two hours post treatment (Bennett et al., 2023). Based on the response time indicated in Figure 1C , we estimate that JG-1 and M4 also require two hours of incubation in order to initiate dispersal. For E. cloacae biofilms with JG-1, M4, or DMSO, we determined that the first consistent decrease in biofilm mass occurs at 5 hours for both JG-1 and M4 ( Supplementary Figures 2A, B ), though we observed some rebound with M4 between hours three and four.

For all tested strains with compound, we unexpectedly identified hundreds of differentially expressed genes ( Supplementary Tables 2 - 4 ). Therefore, pathway analysis was performed to narrow our search. The top 15 most enriched pathways for S. Typhimurium treated with JG-1 include a down regulation of chemotaxis, flagella, and fimbriae assembly, and upregulation of antimicrobial and phage responses ( Figure 2A ). As we expected by their different structures, the top 15 most enriched pathways for S. Typhimurium treated with M4 mostly differ from those of JG-1. M4 treatment results in downregulation of processes involved in respiration, amino acid metabolism, and production of the flagellar motor proteins, and upregulation of motility and extracellular polymeric substances (EPS) ( Figure 2C ). E. cloacae biofilms treated with JG-1 down regulate processes involved in organic compound binding, metal ion transport, and transcription broadly, while upregulating processes that are poorly understood, but predicted to be involved in escape from host stressors ( Figure 2B ). Similar to S. Typhimurium, E. cloacae biofilms treated with M4 present a very different transcriptional profile than JG-1 treated biofilms, characterized by downregulation of pathways involved in quorum sensing and membrane transport, and upregulation of pathways involved in chemotaxis, ribosome production, and protein transport ( Figure 2D ). Given the bactericidal effect of M4 against S. aureus, we expected to observe enrichment of stress response pathways, but were surprised to find upregulation of pathways involving cytolysis and metal ion transport ( Figure 2E ). While the pathway analysis greatly improved our understanding of how the bacteria respond to compounds, the diversity in responses suggests that their mechanism of action may be complex and involve multiple processes.

The results of the RNAseq and pathway analysis indicate that JG-1 and M4 induce transcriptional changes related to motility and invasion in S. Typhimurium. Specifically, M4 treatment results in upregulation of genes related to invasion and the SPI-1 type 3-secretion system (T3SS-1) ( Supplementary Table 2 ). It is unclear whether these changes are specific to facilitating biofilm dispersal activity, or represent a phenotypic change independent of dispersal. In order to validate the RNAseq and investigate the suggested relationship between invasion and dispersal, we compared the invasion capacity of S. Typhimurium treated with compound or vehicle control. We infected HeLa cells with untreated WT S. Typhimurium or WT S. Typhimurium pre-treated with JG-1, M4, or vehicle. The percent of invading cells for the JG-1, M4 and DMSO treated groups were normalized to the percent invasion of the untreated bacteria to measure change in invasion induced by compound or DMSO pre-treatment. There was no significant difference in invasion between DMSO and M4 treated groups ( Figure 3A ). Surprisingly, we observed a significant reduction of invasion by JG-1 treated S. Typhimurium in comparison to DMSO treated bacteria ( Figure 3A ). This was unexpected because we did not observe any transcriptional changes obviously related to invasion in JG-1 treated S. Typhimurium.

JG-1 treatment resulted in a significant reduction of genes related to flagella, fimbriae, and chemotaxis ( Supplementary Table 2 ), which are involved in adherence to and motility towards the intestinal epithelium in vivo, independent of the T3SS-1 (Rivera-Chávez et al., 2016). We considered that the decrease in invasion might be a byproduct of loss of motility due to the downregulation of motility-associated genes, and so we evaluated the motility of S. Typhimurium following JG-1/M4 treatment. We pre-treated WT S. Typhimurium and single gene deletion mutants of ΔcheY and Δtsr with JG-1, M4, and DMSO in 1:20 TSB, or untreated 1:20 TSB, and measured their ability to swarm in SIM agar supplemented with the respective compounds/vehicle. Mutants lacking CheY express flagella but are non-motile, and as we expected, all treatment groups did not move from the inoculation point ( Figure 3B ). Tsr enables motility towards the intestinal epithelium, and so mutants lacking Tsr are motile and express flagella, but have an impaired invasion capacity (Rivera-Chávez et al., 2016). The Δtsr mutant, as expected, was motile spreading between 40–50 mm from the inoculation point ( Figure 3B ). Importantly, DMSO treatment did not significantly alter the growth diameter in either the WT S. Typhimurium or the Δtsr mutant, confirming vehicle alone does not alter motility ( Figure 3B ). Neither the WT nor Δtsr isolates demonstrated a change in motility in response to JG-1, by comparison to vehicle ( Figure 3B ), suggesting that JG-1 does not impair motility despite the transcriptional changes observed in the RNAseq ( Supplementary Table 2 ). Based on a lack of transcriptional changes observed in the RNAseq, we did not expect any changes in motility following M4 treatment; however, we observed a significant reduction in motility in the M4 treated WT and Δtsr isolates, with growth diameters similar to the non-motile ΔcheY isolate ( Figure 3B ). The results of the invasion and motility assays together, support that JG-1 inhibits invasion, independent of motility and that M4 dramatically inhibits motility while leaving invasive capacity intact.

To identify potential compound binding targets, we profiled the proteome of treated biofilms with a thermal shift assay. With thermal proteome profiling (TPP), proteins that are stabilized by binding to the compounds will be more resistant to heat denaturation and therefore persist at higher temperatures ( Figure 4A ). Thus, we treated S. Typhimurium and E. cloacae biofilms with compound or vehicle for 2 and 5 hours, respectively, and then heated aliquots of intact cells (S. Typhimurium) or whole cell purified proteins (E. cloacae) to temperatures between 25-90°C, thereby progressively denaturing the proteins as the temperature rises. From the resulting proteome profile, we identified >2500 individual proteins in both species and were able to identify dozens of proteins whose stability was altered in comparison to vehicle ( Supplementary Table 5 ).

To identify protein-protein interactions that may be promoted by compound treatment, we wanted to determine if any of the proteins detected were predicted to interact in situ. Using STRING, we compared the proteins specific to each species tested that were identified in our TPP to those associations reported in the literature. We enriched for connections with significant positive predictive value of forming physical complexes in situ and generated interaction maps based on the results ( Figures 4B–E ; Supplementary Table 5 ). For both S. Typhimurium and E. cloacae treated with JG-1, we identified strong associations between the ribosome associated proteins and translation factors. In addition, there were strong associations with outer membrane proteins (OMP) ( Figures 4B, C ). Owing in part to the much larger list of proteins found in the TPP for S. Typhimurium and E. cloacae treated with M4, there were more numerous and complex predicted interactions than those of JG-1. Of these, there were strong associations between ribosomal structural proteins, OMPs, and membrane-associated dehydrogenases ( Figures 4D, E ). This assay presents a detailed view of how compound treatment changes global protein interactions, in addition to suggesting potential compound-protein interactors.

Performing an additional assay to examine compound-target interactions, we aimed to “fish” for these proteins by performing a pull-down assay with the compounds as “bait.” To do so, the compounds were modified to contain a biotin handle ( Supplementary Figure 1 ) at a predicted neutral site. However, upon testing the activity of a biotin-modified compound (or a “pre-biotinylated” linker compound at the same site), this addition resulted in decreased activity in comparison to the native compound ( Supplementary Figure 3 ). Suspecting that this reduction of activity is due to a reduced ability to penetrate cells, lysates of biofilm or planktonic cells were used to increase the chance of interactions between the biotinylated compound and their target protein(s). The resulting proteins from this pull-down assay were separated using gel electrophoresis and compared to samples incubated with vehicle only ( Supplementary Figure 4 ). Upon identification of unique bands, the proteins were identified using mass spectrometry.

We identified multiple proteins suspected to bind to the biotinylated compounds ( Supplementary Table 6 ). From two unique gel bands, we identified 44 total proteins in S. aureus cell lysates incubated with biotinylated-M4. From 7 and 8 unique gel bands (respectively), for S. Typhimurium and E. cloacae incubated with biotinylated JG-1 and M4, we identified 30 (with biotinylated-JG-1) and 15 (with biotinylated-M4) S. Typhimurium proteins, and 11 (with biotinylated-JG-1) and 31 (with biotinylated-M4) E. cloacae proteins. Because the protein targets of the two compounds are likely different, we chose to focus on proteins that were unique to each condition, resulting in 15 unique to JG-1 and 10 unique to M4 in S. Typhimurium, and 10 unique to JG-1 and 25 unique to M4 in E. cloacae. We hypothesize that S. Typhimurium and E. cloacae share the same or closely related JG-1/M4 target protein(s), and consistent with this, there were multiple orthologous proteins (>90% nucleotide similarity) identified as binding to the same compounds, including OmpA, GapA, and OmpF for JG-1 and AtpA for M4.

The proteins identified in previous experiments are promising potential targets. To identify proteins that are essential to the compounds’ antibiofilm activity, we utilized single gene deletion libraries of S. Typhimurium and S. aureus to screen for resistance, or recalcitrance to JG-1 and/or M4 (a significant increase in the mutant strain’s IC₅₀ or EC₅₀ compared to the WT counterparts). Additionally, we assumed that because these compounds inhibit/disperse biofilm formation, any mutant lacking a true target should demonstrate a deficiency in biofilm formation.

In total, we tested 160 single and multiple gene deletion mutants of S. Typhimurium and 21 single gene deletion mutants of S. aureus ( Supplementary Table 7 ). These mutants included genes encoding proteins identified in the TPP and pull-down, genes identified in the RNAseq, and genes included in the top enriched pathways. We evaluated biofilm-forming ability by measuring the mass of mutant versus WT biofilms grown in plain media for either 24 (S. aureus) or 48 hours (S. Typhimurium). In S. Typhimurium, we identified 41 individual mutations that resulted in significantly less biofilm mass (and 5 individual mutations that resulted in significantly more biofilm mass than WT) ( Supplementary Table 7 ). In S. aureus, we identified four mutations that resulted in significantly less biofilm mass than WT: ΔcodY, Δatl, ΔebpS, and ΔSAUSA300_2097 ( Supplementary Table 7 ).

S. Typhimurium mutants were tested for resistance to JG-1 and M4 dispersal. We first examined genes with altered transcription as evidenced by the RNAseq and pathway analysis ( Figure 2 ; Supplementary Table 2 ). We tested mutants of auto-inducer-2 (AI-2) genes ego (lsrA), luxS, tqsA, ydeV, and ydeW, and the N-acyl homoserine lactone (AHL) sensor sdiA. Of those genes, only the ΔsdiA mutant was recalcitrant to M4 activity ( Table 1 ). We also wanted to evaluate the role of cyclic-di-GMP signaling in compound-triggered dispersion. We tested multiple mutants of genes involved in this process, including yaiC (adrA), typA, yciR, and yegE. The ΔyaiC mutant was more sensitive to JG-1 treatment than WT but had no baseline biofilm deficit ( Table 1 ). Curiously, the ΔyegE mutant was recalcitrant to M4 activity, but produced more biofilm than WT ( Table 1 ). We observed a similar increase in biofilm formation in ΔptsN, ΔrseC, and ΔyhbH mutants ( Supplementary Table 7 ). The TPP results for both S. Typhimurium and E. cloacae treated with M4 suggested an increase in ribosomal protein stability ( Figure 4 ); therefore, we examined S. Typhimurium genes related to ribosome stability and stalling, including: lepA, prfC, relB, relE, rimI, rplI, rsd, STM14_1162, tig, yajC, yciR, and yhbH. Deletion of LepA results in recalcitrance to both JG-1 and M4 ( Table 1 ). Deletion of RimI results in increased susceptibility to JG-1 and M4, while deletion of RelE results in recalcitrance to JG-1 and deletion of RplI, STM14_1162, YciR, or YhbH results in recalcitrance to M4 ( Table 1 ).

In total, we identified 21 S. Typhimurium mutants that exhibit an increased EC₅₀ in response to JG-1 treatment, and 53 that have an increased EC₅₀ in response to M4 treatment ( Table 1 ). In total, 17/53 mutants were both intrinsically deficient in biofilm mass and had an increased EC₅₀ to only M4. Additionally, 4/21 of the mutations were both intrinsically deficient in biofilm mass and had an increased EC₅₀ to only JG-1 (ΔrseP, ΔcsgF, ΔompC, and ΔglnA). Notably, OmpC and OmpF were identified with JG-1 in the pull-down, and OmpC was associated with increased stability at higher temperatures in the TPP. However, OmpF did not have a biofilm deficit or change in EC₅₀ to either compound, in comparison to WT. Interestingly, we also identified 14 mutants that had an unexpected decrease in EC₅₀ (increased susceptibility) in response to JG-1 and/or M4 treatment. Notable among these genes is ompA, whose loss conferred increased sensitivity to JG-1 and increased resistance to M4. OmpA was identified as binding to both JG-1 and M4 in the pull-down ( Supplementary Table 6 ).

Given the interest in ompA and ompC as targets, we back-transduced these mutations from the library derived isolates, JSG 5197 and JSG 5187, respectively, into our WT strain ( Supplementary Table 1 ). This allows us to ensure that the phenotype is a result of the gene deletion and not an unlinked mutation. We compared the new ΔompA and ΔompC mutants, JSG 5575 and JSG 5574, respectively, to WT and identified that JSG 5575 (ΔompC) retains its parental JG-1 resistance phenotype, while JSG 5574 (ΔompA) does not recapitulate the increased sensitivity to JG-1 or recalcitrance to M4 seen in the parent strain. ( Supplementary Table 7 ).

Given the evidence supporting OmpC as an important JG-1 target in vitro, we determined if loss of OmpC affected the in vivo response to JG-1. To evaluate this, we used a murine model of typhoidal chronic carriage previously used to demonstrate JG-1 efficacy in vivo after infection with WT S. Typhimurium (Sandala et al., 2020; Woolard et al., 2022). 129X1/SvJ mice were fed a lithogenic diet to induce gallstone formation prior to being infected with S. Typhimurium ΔompC (JSG 5574). Five days post-infection, mice were assigned to three treatment groups and given daily ip. injections of either 1 mg/kg Cipro (Cipro only), 1 mg/kg Cipro in 5% DMSO (DMSO+Cipro), or 10 mg/kg JG-1 and 1 mg/kg Cipro in 5% DMSO (JG-1+Cipro), in a volume of 100 µL sterile PBS for 10 days. Post-treatment, the mice were euthanized and the bacterial load in the gallbladder, liver, spleen, cecum, and whole blood were compared between groups. There was a significant decrease in bacterial load in the gallbladder of the JG-1+Cipro group by comparison to the Cipro only and DMSO+Cipro groups ( Supplementary Figure 5 ). Therefore, we concluded that OmpC is dispensable for JG-1 efficacy in vivo despite the promising importance in vitro.

S. aureus mutants were evaluated for both M4 antibiofilm activity at low concentrations, and M4 bactericidal activity at higher concentrations. Of the 21 S. aureus mutants tested, 16 had an altered response to M4 treatment in comparison to WT. Specifically, ΔmanA, Δsbi, ΔdeoC, ΔSAUSA300_2097, and ΔhchA mutants were more resistant to M4 killing than WT ( Table 2 ). Additionally, ΔcodY, ΔahpC, and ΔhchA mutants were resistant to M4 antibiofilm activity, as evidenced by their significant increase in IC₅₀ in comparison to WT ( Table 2 ). Of note, only ΔcodY was both resistant to M4 antibiofilm activity and deficient in biofilm formation at baseline. Thus, we propose that while bactericidal activity is facilitated by binding to multiple proteins, M4’s antibiofilm mechanism of action in S. aureus is facilitated by interacting with CodY.