Absence of fsrA and gelE enhances intracellular survival of E. faecalis in RAW264.7 macrophages

To investigate how E. faecalis regulates intracellular survival, we screened a panel of E. faecalis mutants of genes associated with intracellular persistence or overall virulence for their ability to survive intracellularly within RAW264.7 macrophages. These included transposon insertion mutants from the OG1RF EfaMarTn library [39] and in-frame deletion mutants (S1 Table). Macrophages were infected at an MOI of 10 for 1 h, followed by antibiotic treatment to rapidly eliminate extracellular bacteria in the absence of significant host cell cytotoxicity (S1A–S1B Fig), and viable intracellular bacteria were quantified at 2, 6, and 20 hpi (S1C–S1D Fig). We did not observe significant differences in intracellular colony forming units (CFU) for any mutant at 2 hpi (S1E Fig). However, at 6 hpi when intracellular CFU peaked (S1C–S1D Fig), ΔmprF2 and OG1RF 12401::Tn (encoding enzyme IIC in PTS8) showed significantly fewer intracellular CFU compared to WT (Fig 1A). At 20 hpi, both ΔmprF2 and OG1RF_12401::Tn continued to show significantly fewer intracellular CFU, as did msrB::Tn and ΔmptD (Fig 1B), corroborating previous studies implicating each of these genes in survival within macrophages [15,16,18,20]. Surprisingly, we also found that virulence mutants fsrA::Tn and gelE::Tn accumulated to significantly higher intracellular CFU compared to WT (Fig 1B). Because FsrA regulates the transcription of gelE, sprE, and entV [33,34] and GelE proteolytically activates downstream targets including Ace, AtlA, and EntV [40–42], we examined whether loss of these downstream effectors might account for the increased intracellular survival. In contrast to gelE::Tn, transposon insertion mutants of ace, atlA, and entV displayed similar intracellular CFU as WT at 20 hpi. These data suggest that FsrA and GelE, rather than other downstream effectors of the Fsr regulon, mediate the modulation of intracellular survival.

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Fig 1. Absence of fsrA and gelE enhances intracellular survival of E. faecalis in RAW264.7 macrophages at 20 hpi.

(A-B) Intracellular CFU in RAW264.7 macrophages infected with OG1RF WT and derived mutants of genes implicated in intracellular persistence (blue), virulence (orange), or GelE proteolytic targets (pink). (n = 18 for WT and n = 5-6 for mutants) (C) Schematic diagram of the genomic locus comprising fsrABDC/gelE/sprE, represented in different shaded arrows for each transcriptional unit. Promoters are represented by bent arrows (grey: constitutive promoter of fsrA, black: fsrA-regulated promoter). The respective gene size in base pairs (bp) is indicated below each gene. (D-E) Intracellular CFU in RAW264.7 macrophages infected with genetic deletion mutants of fsrABDC and gelE (n = 9), as well as a transposon insertion mutant of sprE (n = 5). (F-J) Analysis of gelatinase activity phenotype. (F, I) Gelatinase activity of E. faecalis strains on Todd-Hewitt agar + 3% gelatin at 24 h. Halo formation (white dashed line) indicates gelatinase activity. Representative images of n = 3 are shown. (G, J) Detection of GelE secretion in E. faecalis culture supernatants at 4, 12 and 24 h by Western blotting. CTD = C-terminal domain. Representative images of n = 2 are shown. (H) Schematic diagram of GelE domains: a putative signal peptide (S.P., 1-30 a.a.), a propeptide (30-192 a.a.), the mature GelE protease (192-495 a.a.) and a C-terminal domain (CTD, 495-509 a.a.). (K-L) Intracellular CFU in RAW264.7 macrophages infected with WT, ΔgelE and ΔgelE-complemented OG1RF strains. (n = 4); All bar graphs shown represent mean ± SD of biological replicates. Statistical significance of each strain against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. Only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.1371/journal.ppat.1013738.g001

To validate these findings, we generated in-frame deletion mutants for individual transcriptional units (ΔfsrA, ΔfsrBDC, ΔgelE and a combined transcriptional unit ΔfsrABDC; Fig 1C) and quantified their intracellular CFU. We additionally tested a transposon insertion mutant in sprE, a serine protease encoded downstream of gelE that is also regulated by FsrA, for its contribution to intracellular survival. All mutants had similar intracellular CFU at 2 hpi (S1F Fig). However, ΔgelE and ΔfsrABDC displayed increased intracellular CFU than WT at both 6 and 20 hpi, and ΔfsrA displayed increased intracellular CFU at 20 hpi (Fig 1D–1E). These phenotypes correlated with loss of GelE secretion as confirmed by agar-based activity assays and Western blot of culture supernatants collected at different growth timepoints (Figs 1F–H and S2A). WT initially secreted a 34.5 kDa isoform of GelE with lower activity at 4 h that was progressively cleaved to a 33 kDa fully-active form by 24 h, consistent with previous observations [43]. By contrast, ΔfsrBDC retained weak gelatinase activity and had intracellular CFU comparable to WT, consistent with low-level gelE expression, likely driven by constitutive fsrA transcription from its own promoter. sprE::Tn, which was gelatinase-positive, showed comparable intracellular CFU to WT at all timepoints, further supporting a specific role of GelE in modulating intracellular survival (Figs 1D–1E and S1G).

To determine whether augmented intracellular CFU depends on GelE proteolytic activity, we chromosomally complemented ΔgelE with either the WT allele (ΔgelE::gelE) or two catalytically inactive variants carrying E329 or E352 substitutions (S3 Fig). The WT complement restored active GelE secretion, whereas the E329A and E352A mutants produced enzymatically inactive isoforms with impaired secretion particularly for E329A, suggesting differential roles for these residues in GelE proteolytic maturation and secretion (Figs 1H–1J and S2B). Accordingly, ΔgelE::gelE restored intracellular CFU to WT levels, whereas the E329A and E352A mutants lacking gelatinase activity retained elevated intracellular CFU (Figs 1K–1L and S1H). These findings demonstrate that the ability of GelE to regulate intracellular CFU depends on its proteolytic activity.

Increased intramacrophage survival in the absence of fsrA and gelE is conserved across wound isolates of E. faecalis

The fsr QS locus varies among E. faecalis clinical isolates from the gut, infective endocarditis, bloodstream, and urinary tract infections [44–47]. To determine whether elevated intracellular CFU observed for OG1RF ΔfsrA, ΔfsrABDC, and ΔgelE mutants extends to clinical strains, we examined 49 E. faecalis wound isolates [48,49]. Whole genome sequence analysis of gelE, fsrA and fsrBDC revealed three genotypes: 22/49 strains (44.9%) had the full fsrABDC and gelE locus (fsrABDC⁺gelE⁺), 10/49 (20.4%) lacked both fsrABDC and gelE (fsrABDC^-gelE^-), and 17/49 (34.7%) had a complete gelE gene sequence but lacked fsrA and had a truncated fsrBDC (fsrABDC^-gelE⁺). Phenotypically, fsrABDC^-gelE^- and fsrABDC^-gelE⁺ strains lacked gelatinase activity on gelatin agar (GelE^-), whereas fsrABDC⁺gelE⁺ strains retained it to varying degrees (GelE⁺) (Figs 2A and S4B). All fsrABDC^-gelE⁺ strains shared a conserved 23.9 kb deletion spanning the 5’ end of fsrC (junction at position 261 in fsrC, V583 coordinates: 1,765,811) to the 3’ end of yqeK (EF1841, V583 coordinates: 1,789,714) (S4A–S4B Fig), consistent with previous reports [50,51]. Genomic analysis of fsrABDC^-gelE^- strains showed a different 27.8 kb conserved deletion spanning the 5’ end of EF1814 (V583 coordinates: 1,759,390) to the 3’ end of EF1838 (V583 coordinates: 1,787,212), while fsrABDC⁺gelE⁺ strains displayed diversity in the genomic region upstream of fsrA, including a previously reported 14.8 kb insertion in 11 out of 22 fsrABDC⁺gelE⁺ wound isolates as well as in OG1RF [51].

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Fig 2. Increased intramacrophage survival in the absence of fsrA and gelE is conserved across wound isolates of E. faecalis.

(A) Characterization of 49 E. faecalis wound isolates for fsrABDC/gelE genotypes and gelatinase activity phenotypes. (B-D) Intracellular CFU in RAW264.7 macrophages infected with four selected gentamicin-susceptible fsrABDC⁺gelE⁺ (grey bars), fsrABDC^-gelE⁺ (green bars) and fsrABDC^-gelE^- (orange bars) E. faecalis wound isolates and laboratory strains (JH2-2, OG1RF WT, ΔfsrABDC and ΔgelE) at (B) 2 hpi, (C) 6 hpi and (D) 20 hpi. Bars represent mean ± SD from n = 4. Statistical significance of each strain against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. Only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.1371/journal.ppat.1013738.g002

We next infected RAW264.7 macrophages with four gentamicin-susceptible strains from each genotype group (highlighted in red in Figs 2A, S4B, and S5A–S5B), as well as JH2–2, a fsrABDC^-gelE⁺ laboratory strain [52]. Except for one fsrABDC^-gelE^- isolate (20_EF), all clinical strains exhibited comparable intracellular CFU at 2 hpi (Fig 2B). At 6 and 20 hpi, all fsrABDC^-gelE^- strains, as well as the ΔfsrABDC and ΔgelE controls, showed significantly higher intracellular CFU compared to WT OG1RF (Fig 2C–2D). By 20 hpi, three of the fsrABDC^-gelE⁺ strains (04_EF, 12_EF, JH2–2) also exhibited increased intracellular CFU relative to WT, whereas fsrABDC⁺gelE⁺ strains maintained WT levels at all time points (Fig 2B–2D). GelE secretion was nearly undetectable in culture supernatants of most of the fsrABDC^-gelE⁺ clinical strains tested, suggesting that the variability in their intracellular CFU at 20 hpi is likely due to other genomic factors and not a leaky Fsr-independent GelE expression (S6 Fig). Therefore, we conclude that enhanced intramacrophage CFU in the absence of FsrABDC/GelE is conserved among wound isolates and links naturally occurring fsrABDC/gelE variants with increased intracellular persistence.

Lack of GelE promotes the accumulation of replicating intracellular bacterial reservoirs

The elevated intracellular CFU observed in GelE^- strains at 20 hpi was not due to differences in initial uptake or infection efficiency (S1E–S1F, S1H, and S7 Figs). To determine if this phenotype reflected increased intracellular replication, E. faecalis was pre-stained with the proliferation dye eFluor 670, which is diluted with each division (S8A Fig). Infected macrophages were lysed at 2, 6, and 20 hpi and intracellular bacteria were analyzed by flow cytometry following immunolabeling of lysates with the anti-Group D antigen (AgD⁺ , labelling all E. faecalis cells) (S8B Fig). At 2 hpi, approximately 80–90% of intracellular E. faecalis were eFluor⁺ (Fig 3A–3B, red histograms), indicating limited replication within macrophages regardless of their GelE phenotype. By 6 and 20 hpi, an eFluor^- peak of lower fluorescence intensity emerged, indicating intracellular replication (Fig 3A–3B, blue and orange histograms). This eFluor^- peak was absent in PFA-fixed non-replicative controls at all timepoints, validating the specificity of this assay in identifying replicating bacteria (Fig 3A). Strikingly, GelE^- strains (ΔfsrA, ΔfsrABDC, ΔgelE) displayed significantly higher proportions of replicating eFluor^- bacteria (~80–90%) compared to GelE⁺ strains (WT and ΔfsrBDC), which reached only ~40–60% at 20 hpi. (Fig 3A-3B). Confocal microscopy supported these findings: At 20 hpi, we observed macrophages infected with GelE⁻ strains (ΔfsrA, ΔfsrABDC, ΔgelE) containing large intracellular clusters of eFluor^- bacteria, consistent with active replication, whereas infection with GelE⁺ strains (WT, ΔfsrBDC) primarily contained eFluor⁺ non-replicating bacteria (Fig 3C). The higher proportion of replicating intracellular GelE⁻ strains was not attributable to increased growth rate, as all fsrABDC/gelE deletion mutants exhibited growth kinetics comparable to WT OG1RF when cultured in macrophage culture media (S9 Fig). Collectively, these results demonstrate that the elevated intracellular CFU observed in GelE^- E. faecalis results from a higher proportion of replicating bacterial reservoirs within macrophages rather than altered uptake or cytotoxicity.

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Fig 3. Lack of GelE promotes the accumulation of replicating intracellular bacterial reservoirs.

(A) Quantification of intracellular replicating (eFluor^-) and non-replicating (eFluor⁺) E. faecalis strains from macrophage lysates at 2, 6, and 20 hpi using flow cytometry. WT OG1RF fixed with 4% PFA prior to infection (Fixed WT) was included as non-proliferating controls. Representative histograms from n = 2-4 are shown. (B) Proportion (%) of intracellular replicating E. faecalis (eFluor^-) population from WT and mutant E. faecalis-infected macrophage lysates. Bars represent mean ± SD of n = 4, except fixed WT (n = 2). Statistical significance of each strain against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. Only comparisons with p < 0.05 are annotated. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. (C) Representative confocal microscopy images of RAW264.7 macrophages infected by E. faecalis strains pre-stained with eFluor 670 (magenta) at 20 hpi from n = 3. Samples were fixed and post-stained for Enterococcus-specific Group D antigen (green), double-stranded DNA (dsDNA; blue) and F-actin (white).

https://doi.org/10.1371/journal.ppat.1013738.g003

Intracellularly replicating WT OG1RF induce Fsr quorum sensing at 6 hpi

Since FsrA and GelE negatively regulate intracellular persistence and replication, we next examined whether these genes are differentially expressed in intracellularly replicating GelE⁺ WT OG1RF. To distinguish macrophages containing replicating versus non-replicating intracellular E. faecalis at 6 and 20 hpi, we adapted a two-color fluorescence dilution assay combined with fluorescence-assisted cell sorting (FACS) [53,54]. Constitutive Dasher GFP-expressing WT OG1RF were pre-stained with the proliferation dye eFluor 670 to identify macrophages containing replicating bacteria. As intracellular bacteria replicate, the macrophage GFP intensity increases while the eFluor signal remains constant, enabling separation of macrophages containing replicating E. faecalis (eFluor⁺GFP^hi) and non-replicating E. faecalis (eFluor⁺GFP^lo) (Fig 4A). At 2 hpi, < 0.5% of infected macrophages contained replicating bacteria (GFP^hi macrophages), compared to 1–3% and 3–5% at 6 and 20 hpi, respectively (Fig 4A–4B). Confocal microscopy confirmed that GFP^hi macrophages contained clusters of replicating E. faecalis with a diluted eFluor signal, while GFP^lo macrophages contained lower numbers of non-replicating eFluor⁺ E. faecalis (Fig 4C). Quantitative PCR (qPCR) showed that the shift from the extracellular to intracellular environment led to an early decrease in Fsr QS-regulated genes compared to extracellular E. faecalis, likely due to a shift into a microenvironment with little to no GBAP autoinducer peptide. At 6 hpi, replicating E. faecalis within GFP^hi macrophages significantly upregulated the fsrABDC locus genes and its known regulon compared to non-replicating intracellular bacteria within GFP^lo macrophages at the same timepoint, approaching extracellular expression levels (Figs 4D and S10). By 20 hpi, expression levels were similar between replicating (GFP^hi population) and non-replicating (GFP^lo population) intracellular E. faecalis (Fig 4E). These results suggest that intracellular E. faecalis activates the Fsr QS system and its regulon following early replication within macrophages, likely in response to increased intracellular density, initiating a regulatory program that ultimately promotes the intracellular-to-extracellular transition.

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Fig 4. Intracellularly replicating WT OG1RF induce Fsr quorum sensing at 6 hpi.

(A) Gating of eFluor⁺GFP^hi (GFP^hi) and eFluor⁺GFP^lo (GFP^lo) infected macrophages used for cell sorting at 2, 6 and 20 hpi. Representative dotplots from n = 4 are shown. (B) Relative quantification (%) of GFP^hi and GFP^lo macrophages as gated in (A). Bars represent mean ± SD of n = 4. Statistical analysis was assessed using one-way ANOVA with Tukey’s multiple comparisons test. (C) Confocal fluorescence images of sorted GFP^hi and GFP^lo macrophages, with intracellular GFP⁺eFluor670^lo bacteria indicating proliferating E. faecalis (n = 1). Scale bar = 10 μm. (D-E) Relative gene expression of fsrABDC and its associated regulon in intracellular replicating (GFP^hi) and non-replicating (GFP^lo) E. faecalis populations at (D) 6 hpi and (E) 20 hpi, normalised by the -ΔΔCt method to the housekeeping gene recA and to the gene expression of the baseline 2 hpi GFP^lo population. Bars represent mean ± SEM of n = 4. Statistical significance between GFP^hi and GFP^lo populations at each timepoint was assessed using unpaired T-test. For all graphs, only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.1371/journal.ppat.1013738.g004

Loss of GelE proteolytic activity delays egress from macrophages and promotes intracellular accumulation of E. faecalis in macrophages

We hypothesized that GelE secreted by replicating intracellular bacteria promotes host cell lysis and bacterial release, thereby reducing the intracellular bacterial population. To test this hypothesis, we quantified the lytic release of intracellular lactate dehydrogenase (LDH) from macrophages. At 6 hpi, infection with GelE⁺ strains caused ~5% macrophage death, whereas GelE^- strains caused significantly lower cytotoxicity comparable to uninfected controls (Fig 5A). By 20 hpi, comparable cytotoxicity was seen in both GelE⁺ and GelE^- strain infections. These data suggest that intracellular GelE accelerates macrophage lysis and bacterial release into the antibiotic-containing media, eventually reducing overall intracellular GelE⁺ populations by 20 hpi.

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Fig 5. Loss of GelE proteolytic activity delays egress from macrophages and promotes intracellular accumulation of E. faecalis in macrophages.

(A) LDH cytotoxicity quantification of culture supernatants from E. faecalis-infected macrophages at 2, 6 and 20 hpi using the antibiotic protection assay. Bars represent mean ± SD from n = 5. Statistical significance against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. A subset of this data comparing macrophage cytotoxicity from WT infection at 2, 6 and 20 hpi is shown in S1B Fig. (B-C) Quantification of extracellular egressed bacteria from culture supernatants of RAW264.7 macrophages infected with (B) fsrABDC/gelE deletion mutants and (C) gelE-complemented OG1RF strains at 6 to 12 hpi. Each data point represents mean ± SD of n = 3-4. At each timepoint, statistical significance against WT was assessed using two-way ANOVA with Dunnett’s multiple comparisons test. LOD = limit of detection. (D) Representative Z-projections of WT- and ΔgelE-infected RAW264.7 macrophages at 6, 9 and 12 hpi from (B), captured with confocal microscopy and stained for Enterococcus specific Group D antigen (yellow), dsDNA (cyan), and F-actin (magenta) (n = 2). Orthogonal Z-axis projections along the blue and red lines are shown in the adjacent colored boxes. White and yellow arrows indicate matched top view and Z-axis projections of selected macrophages with dense intracellular E. faecalis. For all graphs, only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.1371/journal.ppat.1013738.g005

To investigate the role of GelE in promoting egress, we performed a modified antibiotic protection assay up to 6 hpi, following which the bactericidal cocktail was replaced with vancomycin at a bacteriostatic concentration to E. faecalis, allowing intracellular bacteria to exit without extracellular replication or death [55] (S11A–S11C Fig). Supernatants were sampled hourly from 6-12 hpi for CFU enumeration. WT E. faecalis egressed rapidly, plateauing by 7 hpi, whereas ΔgelE egressed more slowly (Fig 5B). Complementation restored ΔgelE to WT egress kinetics, while protease-inactive E329A and E352A mutants remained defective (Fig 5C). The weakly GelE⁺ ΔfsrBDC mutant displayed an intermediate phenotype, supporting a correlation between gelatinase activity and egress efficiency. Both ΔfsrA and ΔfsrABDC surpassed WT egress levels at later time points, suggesting that additional FsrA-regulated factors may restrict intracellular replication or release. Consistent with the extracellular CFU recovered, confocal microscopy of infected ΔgelE-infected macrophages between 6–12 hpi showed large, highly dense intracellular bacteria encased within F-actin (Fig 5D, white and yellow arrows), which were largely absent in WT-infected macrophages, suggesting a population of continuously replicating ΔgelE that is egress-defective. By contrast, WT-infected macrophages harbored smaller and more dispersed bacterial clusters, consistent with active egress and host cell lysis. To investigate whether intracellular replication and the formation of dense intracellular clusters occurred in the host cytosol, we applied a differential permeabilization method for staining cytosolic but not phagosomal bacteria in infected host cells [56–58]. Although we found large replicating intracellular clusters within the host cytosol by 6 hpi, several smaller clusters of replicating E. faecalis were observed to be non-cytosolic, and these were observed in both WT- and ΔgelE-infected macrophages (S12A Fig). These results suggest that E. faecalis intracellular replication is likely a heterogenous process, in which large replicating intracellular E. faecalis clusters eventually localize to the cytosol regardless of gelE presence. Transmission electron microscopy further supported the heterogenous subcellular localization of both WT and ΔgelE at 6 hpi in both host membrane-bound compartments and the cytosol (S12B Fig). Taken together, these data show that GelE proteolytically regulates E. faecalis egress from RAW264.7 macrophages, without which replicating bacteria remain as dense cytosolic populations due to delayed egress.

Absence of GelE promotes intracellular survival of E. faecalis in murine wounds

Intracellular bacterial populations of traditionally extracellular pathogens can contribute to persistent and/or recurrent infections, including wound infection, bacteremia, and lung infection [59–63]. To evaluate whether GelE deficiency affects E. faecalis persistence in vivo, we compared WT and ΔgelE in a mouse wound infection model. Both strains established infection and persisted at similar levels at 1, 3, and 5 dpi in mono-species (Fig 6A) and competitive infections (Figs 6B and S13), showing that GelE is dispensable for wound infection at these timepoints.

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Fig 6. Absence of GelE promotes intracellular survival of E. faecalis in murine wounds.

(A-B) Total CFU from wound homogenates of (A) mono-infection or (B) competitive infection at 1, 3, and 5 dpi. Median of 3-5 animals per group from one independent experiment is shown. Dotted lines show inoculum CFU for mono-infection. (C) Flow cytometry analysis of intracellular E. faecalis (stained for Group D antigen) in various immune cells at 5 dpi. Median from 6-9 animals per group from 2 independent experiments is shown. (D-E) Quantification of (D) intracellular CFU at 1, 3 and 5 dpi or (E) total CFU at 5 dpi from enzymatically dissociated wounds. Median from 4-5 animals per group from one independent experiment is shown. For A-D, only comparisons with p < 0.05 are annotated. At each timepoint, statistical significance between infection groups was assessed using Mann-Whitney test, except for C, which was assessed using Kruskal-Wallis test with Dunn's multiple comparisons test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n.s. = not significant.

https://doi.org/10.1371/journal.ppat.1013738.g006

We next investigated whether ΔgelE contributes to intracellular persistence during wound infection. At 5 dpi, dissociated wound tissues were analyzed by flow cytometry for relevant immune subpopulations [64] (S14 Fig). Both WT and ΔgelE infections elicited comparable innate immune infiltration at 5 dpi, with greater populations of neutrophils (CD45⁺ Ly6G⁺) and monocytes (CD45⁺ Ly6G^- Ly6C⁺) compared to mock-infected wounds (S15A–S15B Fig). Intracellular E. faecalis were found in approximately 0.1-5% of all the cell types investigated for both WT and ΔgelE infection (Fig 6C). To quantify intracellular CFU, excised wound tissues were enzymatically dissociated ex vivo in antibiotics-containing medium (500 μg/mL each of gentamicin and penicillin G) to eliminate extracellular bacteria. The single cell suspension was then washed and lysed to release intracellular bacteria. While intracellular CFU were similar between WT and ΔgelE at 1 and 3 dpi, significantly more intracellular ΔgelE CFU were present at 5 dpi as compared to intracellular WT (Fig 6D). Parallel experiments without ex vivo antibiotic treatment to capture both intracellular and extracellular bacteria yielded no significant differences in total CFU between WT and ΔgelE (Fig 6E). Thus, although GelE does not contribute to overall fitness during wound infection, its absence promotes E. faecalis intracellular survival during wound infection.

Ethics statement

All animal infection procedures were approved and performed in accordance to the requirements of Institutional Animal Care and Use Committee in Nanyang Technological University (ARF SBS/NIEA0198Z). Murine wound infections were performed according to the approved animal use protocol (AUP) “Mechanisms of Polymicrobial Wound Infection” (AUP codes: A19061, A24066). Anonymized bacterial isolates used in this study did not require IRB approval, as described previously [49].

Bacterial strains and culture

Enterococcus faecalis strain OG1RF [80] and its derivative transposon insertion mutants [39], genetic deletion mutants and other isogenic strains (S1 Table), as well as strain JH2–2 [52] and clinical wound isolates of E. faecalis previously collected by Timothy Mark Sebastian Barkham from patients in Tan Tock Seng Hospital, Singapore (S2 Table) were all grown statically in Brain Heart Infusion (BHI) broth (Neogen, Singapore) and BHI + 1.5% Bacto-Agar (Becton-Dickinson, USA). For all experiments, bacterial suspensions were normalized to an optical density at 600 nm (OD₆₀₀) of 0.5, corresponding to approximately 3.0 x 10⁸ colony forming units (CFU)/mL, prior to further bacterial count normalisations. Where appropriate, we added chloramphenicol (Cm) at 10 μg/mL, rifampicin (Rif) at 25 μg/mL, spectinomycin (Spec) at 60 μg/mL (OG1RFS) or 120 μg/mL (plasmid selection), and erythromycin (Erm) at 25 μg/mL (E. faecalis) or 500 μg/mL (E. coli).

Mammalian cell culture

RAW264.7 cells (ATCC, USA) or RAW-Blue cells (Invivogen, Singapore), a derivative of RAW264.7 cells containing a SEAP reporter plasmid, were cultured in complete Dulbecco’s Modified Eagle Medium (complete DMEM; Gibco, Singapore) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, Singapore) at 37°C,5% CO₂. 200 μg/mL Zeocin (Gibco, Singapore) was added for RAW-Blue cell line maintenance as per manufacturer’s protocol.

Antibiotic protection assay

1 x 10⁶ or 3 x 10⁵ RAW-Blue cells were seeded per well of a 6-well plate or 24-well plate respectively and left to attach overnight at 37°C, 5% CO₂, and overnight cultures of E. faecalis were incubated statically at 37°C. The following day, overnight cultures were subcultured 1:10 in BHI broth to log-phase growth at 37°C, washed, and normalised to OD₆₀₀ of 0.5 in complete DMEM. Macrophages were infected with E. faecalis at MOI 10, centrifuged at 500 x g for 5 min to enhance macrophage-bacteria contact, and incubated for 1 h at 37°C, 5% CO₂. When indicated, the bacterial inoculum was pre-stained with eFluor 670 proliferation dye prior to normalization in DMEM, as described for flow cytometry. At 1 hpi, media was changed to complete DMEM with 10 μg/mL vancomycin (Sigma, USA) and 150 μg/mL gentamicin (MP Biomedicals, USA) to kill extracellular bacteria until indicated timepoints. Then, infected macrophages were washed thrice with Dulbecco’s phosphate buffered saline (DPBS; Gibco, Singapore) and lysed with 1 mL 0.1% Triton X-100 (Sigma, USA) in DPBS with vigorous pipetting. Lysates were serially diluted, spotted on BHI agar, and incubated at 37°C overnight for intracellular CFU enumeration. The antibiotic susceptibility of OG1RF, JH2–2 and selected clinical wound isolates tested in this antibiotic protection assay is verified in S5B Fig.

Molecular cloning

All plasmids were transformed and propagated in Stellar E. coli competent cells (TakaraBio, USA), which were cultured aerobically in LB with the appropriate antibiotics at 37°C, except for pFR213-transformed E. coli strains which were cultured aerobically in BHI with 100 μg/mL erythromycin at 30°C. Plasmids were extracted using Monarch Plasmid Miniprep Kit (New England BioLabs, Singapore) and verified by Sanger sequencing prior to transformation into E. faecalis. The plasmids and primers used in this study are detailed in S3 and S4 Tables respectively. All engineered strains were confirmed by whole genome sequencing to have the intended insertions/deletions with minimal off-target mutations.

Chromosomal insertions or deletions were performed by allelic exchange using the temperature sensitive shuttle plasmid pGCP213 [81] or its derivatives, pFR212 and pFR213, which were generated in this study as follows: DasherGFP was first PCR amplified from pBSU101::DasherGFP [82] using Q5 High-Fidelity DNA Polymerase (New England BioLabs, Singapore), and its ends were double-digested with SacI/SphI (New England Biolabs, USA). The insert was then ligated via T4 ligase (New England Biolabs, USA) to SacI/SphI-digested pSD15 [83], which contains full OG1RF_11778 (abbreviated to 11778) and OG1RF_11779 (abbreviated to 11779) sequences flanking the multiple cloning site for genomic insertion site for expression (GISE) [83], generating pSD15::DasherGFP. The constitutive DasherGFP promoter was then replaced via InFusion Cloning (TakaraBio, USA) with gelE promoter region (P_gelE; -86 bp to +121 bp, relative to +1 transcriptional start site), which was PCR amplified from WT OG1RF genomic DNA, generating pSD15::P_gelE-DasherGFP. The entire GISE cassette, which is the 11779-P_gelE-DasherGFP-11778 sequence from pSD15::P_gelE-DasherGFP, was then PCR amplified and ligated to pGCP213 via InFusion cloning, generating pFR212. Finally, a P-pheS* negative counterselection cassette was PCR-amplified from pRV1 [83,84] and ligated to pFR212 upstream of the GISE cassette via InFusion cloning to generate pFR213.

Chromosomal deletion strains ΔfsrA and ΔfsrBDC were generated as described previously [65]. Briefly, upstream (US) and downstream (DS) flanking regions were PCR-amplified from OG1RF genomic DNA and ligated by overlap extension PCR. The pGCP213 vector was linearized by inverse PCR, vector-insert ligation was performed via InFusion Cloning, and transformed into Stellar E. coli with selection on LB agar + Erm. Sequence-verified plasmids were extracted and transformed into WT OG1RF by electroporation, which were then serially passaged for allelic exchange to obtain plasmid-free chromosomal deletion strains. For the ΔgelE deletion mutant of OG1RFC, the chromosomal deletion was performed similarly as above, except using pFR213 instead of pGCP213, in which the full 11779-P_gelE-DasherGFP-11778 cassette in pFR213 was replaced with the US_gelE-DS_gelE insert via InFusion cloning, generating pFR213::gelE_del. This allowed for a counterselection for plasmid-free clones by plating serial BHI cultures on MM9YEG agar supplemented with 10 mM p-chloro-phenylalanine (MM9YEG + p-Cl-Phe) [84]. Colonies on MM9YEG + p-Cl-Phe agar were then verified for Erm susceptibility, indicating loss of shuttle plasmid, prior to colony PCR. All deletion strains were verified by colony PCR and Sanger sequencing of the deletion locus using the respective sequencing primers indicated in S4 Table.

The generation of chromosomal complementation strains of ΔgelE (ΔgelE::gelE, E329A, E352A) have also been described previously [65]. Briefly, WT gelE allele including homologous flanking regions were PCR-amplified with OG1RF genomic DNA and subjected to overlap extension PCR, introducing a silent A29A (GCA > GCG) mutation to distinguish complementation gelE sequences from WT gelE allele. This complementation gelE insert was then ligated to pGCP213 via InFusion cloning, generating pGCP213::gelE. Site directed mutagenesis (SDM) by PCR was then performed on pGCP213::gelE using mutagenic primer pairs oFR80/oFR81 (E329A mutation) or oFR82/oFR83 (E352A mutation). The PCR products were treated with DpnI (New England Biolabs, USA), purified using the Wizard SV Gel and PCR Clean-Up Kit (Promega, USA) and re-ligated by InFusion cloning, generating pGCP213::gelE^E329A and pGCP213::gelE^E352A respectively. These plasmids (including pGCP213::gelE) were then separately electroporated into ΔgelE OG1RF and subjected to serial passaging for allelic exchange as described above. Correct gelE complementation was verified by colony PCR and Sanger sequencing of the gelE locus, as well as susceptibility of the complemented gelE locus to SacII digestion (New England Biolabs, USA).

To generate OG1RF-derived strains OG1RFC and OG1RFS for competitive infection, the P_gelE-DasherGFP cassette from pFR212 was replaced via InFusion cloning with PCR-amplified chloramphenicol resistance gene cat from the EfaMarTn cassette [39] or spectinomycin resistance gene spc from pBSU101::DasherGFP [82], generating pFR212::cat and pFR212::spc respectively. Sequence-verified pFR212::cat and pFR212::spc plasmids were extracted and transformed into WT OG1RF by electroporation, and transformants were subjected to serial passaging for allelic exchange, similar to pGCP213 constructs. OG1RFC and OG1RFS clones were verified by colony PCR and Sanger sequencing, as well as for their susceptibility to erythromycin (indicating pFR212 plasmid loss) and their differential resistance against chloramphenicol and spectinomycin respectively (S13A Fig).

Agar-based gelatinase assay

Up to 5 μL of overnight OG1RF cultures were spotted onto Todd-Hewitt agar (Sigma-Aldrich, USA) supplemented with 3% gelatin (Sigma-Aldrich, USA), and incubated at 37°C overnight. The following day, the plates were incubated at 4°C for 1 h, allowing any degraded gelatin around the colony to form a halo, before imaging with ProtoCOL 3 system (Synbiosis, USA). Halo area, defined as the area between the halo edge and the colony edge, was measured using Fiji 2.9.0, normalized to the known area (in mm²) of the agar plate.

Western blot

Overnight cultures of WT and mutant OG1RF strains were subcultured 1:40 in 20 mL BHI broth and incubated for up to 24 h at 37°C. At indicated timepoints, 1 mL bacterial culture was pelleted at 12,000 x g for 5 min, for which all cultures were at similar bacterial densities and growth phases (S2A Fig). Supernatants were filtered through a 0.2 μm syringe filter (Pall, USA) to remove residual bacteria, and pellets (normalized to OD₆₀₀ = 0.5 in 1 mL DPBS) were lysed in 10 mg/mL lysozyme for 1 h at 37°C. Both supernatants and cell lysates were denatured in 4X LDS Sample Buffer (Invitrogen, USA) at 100°C for 20 min, and 10 μL was loaded onto NuPAGE 4–12% Bis-Tris gel (Invitrogen, USA) and separated at 120 V, 100 min in ice-cold MOPS SDS Running Buffer (Invitrogen, USA). The gel was then transferred to a PVDF membrane using iBlot2 Gel Transfer Device (Thermo Scientific, USA), and blocked with 5% BSA (Sigma, USA) in Tris-buffered saline (Thermo Scientific, USA) + Tween-20 (Sigma, USA) (TBST) for 1 h at room temperature with shaking. The membrane was probed with 1:1000 rabbit anti-GelE polyclonal antibody (Invitrogen, Cat# PA5–117682, USA) or 1:3000 rabbit anti-SecA polyclonal antibody [85] in TBST + 1% BSA overnight at 4°C with shaking, followed by 1:5000 HRP-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Cat# 32460, USA) in TBST + 1% BSA for 1 h at room temperature with shaking. Membranes were washed thrice with TBST after incubation with each antibody. Immunoblotted proteins were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Invitrogen, USA) in an Amersham IQ800 Imager (Cytiva, USA).

Whole genome sequencing analysis of E. faecalis wound isolates

Genomic DNA from 49 wound isolates of E. faecalis were isolated using DNEasy Blood And Tissue Kit (Qiagen, Germany) and whole genome sequencing reads were acquired using MiSeq V3 (300 bp paired-end) (Illumina, USA) to an average coverage of 18X per genome. The reads were quality- and adapter-trimmed using bbduk (bbtools v39.17) [86] to a minimum phred score of 20 and a minimum read length of 20. Draft genomes were then de novo assembled using SPAdes v4.1.0 [87] with the “--careful” setting, and the assembled contigs were analyzed by blastn v2.16.0 against OG1RF nucleotide sequences of fsrA, fsrB, fsrC and gelE to identify the presence or absence of these genes. The blastn results also indicated the start and end residues of any sequence match to pinpoint the deletion junction for partial matches. To identify sequence type, trimmed reads were directly analyzed by SRST2 v0.2.0 [88] with E. faecalis PubMLST database [89].

To compare the genomic locus surrounding fsrABDC/gelE, contigs from SPAdes were scaffolded to the V583 reference genome (Accession ID: NC_004668.1) using the scaffold function of RagTag v2.1.0 [90], and were passed as queries to blastn v2.16.0 against the genomic region corresponding to EF1814-EF1844 from the V583 reference genome. Matching query start and end positions were used to extract the genomic locus from the scaffolded contig of each strain, as well as published OG1RF reference genome (Accession ID: NC_017316.1) and JH2–2 genomic scaffold (Accession ID: KI518257.1), using samtools faidx v1.21 [91], which was subsequently annotated by prokka v1.14.6 [92] using V583 GenBank annotation (Accession ID: NC_004668.1) as reference via the “--proteins” option, and clustered and visualized for gene cluster similarity by clinker v0.0.31 [93].

Flow cytometry of replicating intracellular bacteria in macrophage lysates

Detection of intracellular bacteria replication using eFluor 670 amine-reactive proliferation dye was adapted from Flannagan and Heinrichs [94]. Log-phase E. faecalis cultures in BHI were pelleted at 12,000 x g for 5 min, washed thrice with DPBS, and stained with 10 μM eFluor 670 (eBioscience, Singapore) in DPBS, at room temperature for 30 min with shaking (200 rpm). eFluor 670-stained bacteria were then washed once with BHI broth to quench unreacted eFluor 670, and once with DPBS to wash off excess media. Bacteria were then normalized to MOI 10 in complete DMEM and used for infecting macrophages as described for the antibiotic protection assay.

At indicated infection timepoints, 600 μL of intracellular lysates were aliquoted, kept on ice, and blocked for 15 min in DPBS with 2% BSA. Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature. Washing with staining buffer was done after every immunolabelling step. Samples were then resuspended in 200 μL staining buffer and kept on ice until analysis using BD C6 Accuri flow cytometer (Becton Dickinson, USA).

Cell sorting of infected macrophages

Prior to infection, WT OG1RF was transformed with pBSU101::DasherGFP (S3 Table, hereby referred to as pDasher OG1RF) for the constitutive expression of GFP. pDasher OG1RF was maintained in BHI agar supplemented with 120 μg/mL spectinomycin and was freshly transformed for every independent replicate. Macrophages were infected using the antibiotic protection assay with proliferation dye staining, in which RAW-Blue cells were seeded at 1 x 10⁶ cells/well in four to eight 6-well plates per timepoint and pDasher OG1RF were inoculated and subcultured in BHI broth + 120 μg/mL spectinomycin. eFluor-stained pDasher OG1RF were normalised to MOI 10 in DMEM + 10% FBS with no spectinomycin, and macrophages were infected as described in the antibiotic protection assay. At 2, 6 and 20 hpi, the antibiotic media was aspirated, and the infected macrophages were gently scraped and collected in ice-cold 2% BSA in DPBS. Collected macrophages were pelleted at 500 x g, 4°C for 5 min, resuspended in 1–3 mL ice-cold 2% BSA in DPBS, and sorted into eFluor⁺GFP^hi and eFluor⁺GFP^lo populations in a cooled FACSFusion Cell Sorter (Becton-Dickinson, USA). Sorted cell suspensions were pelleted and resuspended in 800 μL ice-cold TRIZol reagent (Invitrogen, USA) to be transferred to Lysing Matrix B tubes (MP Biomedicals, USA). In parallel, E. faecalis (10⁷ CFU inoculum) was separately grown without macrophages in DMEM + 10% FBS for 6 h, and were harvested and resuspended in Trizol without cell sorting. All Lysing Matrix B tubes were homogenised in a FastPrep-24 Bead Beating System (MP Biomedicals, USA) for one round at 6.0 m/s, 40 s and stored at -80°C prior to RNA extraction. To image sorted cell populations, approximately 10,000 eFluor⁺GFP^hi and eFluor⁺GFP^lo cells were each sorted, fixed in 4% paraformaldehyde (PFA; Sigma, USA) in DPBS for 15 min at room temperature, and resuspended in 1:1000 Hoechst 33342 (Invitrogen, Cat# H1399, USA) in PBS for 20 min at room temperature. Cells were then resuspended in 5 μL ProLong Glass Antifade Mountant (Invitrogen, Cat# P36980, USA) and spotted onto glass slides. Microscopy images were acquired at NTU Optical Bio-Imaging Centre (NOBIC) imaging facility at SCELSE using the LSM 780 confocal laser scanning microscope (Carl Zeiss, Germany) equipped with 405 nm, 488 nm, 561 nm, and 633 nm lasers.

Total RNA extraction and quantitative PCR (qPCR)

Frozen TRIZol samples were thawed on ice, and RNA was extracted by phenol-chloroform extraction followed by purification of the aqueous phase using RNeasy Mini Kit with on-column DNAse I digestion (Qiagen, USA) following manufacturer protocols. The quantity and quality of extracted RNA were assessed by Qubit RNA Broad Range and/or High Sensitivity kits (Invitrogen, USA) and RNA ScreenTape (Agilent, USA) respectively. Approximately 250 ng of RNA from each sample was reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, USA) with random hexamers as primers to capture both host and bacterial RNA, and the resulting cDNA was diluted 1:10 for use as template in gene expression analysis by qPCR. qPCR primer efficiency was determined by performing qPCRs on 10-fold serial dilutions of pooled cDNA (1:2–1:2000) and calculating efficiency (in %) using StepOne software v2.3 (Applied Biosystems, USA). Primer pairs with efficiency 90–100% were selected for subsequent qPCR experiments. All qPCR reactions were performed in three technical replicates, including no-template controls, using KAPA SYBR FAST qPCR Kit (Roche, USA) and StepOnePlus Real-Time PCR System (Applied Biosystems, USA), according to manufacturer protocols. Data were analyzed using the -ΔΔCt method and presented as log₂ fold change (log₂FC) relative to the control group (2 hpi GFP^lo) and to the recA housekeeping gene. The primers used for qPCR are listed in S4 Table.

Lactate dehydrogenase cytotoxicity assay

To assess host cell cytotoxicity, supernatants from infected and uninfected macrophages were collected in 4 technical replicates at 2, 6 and 20 hpi following antibiotic protection assay, and subjected to the Cytotoxicity Detection Kit (LDH) (Roche, USA) following manufacturer instructions. Media-only controls and 100% dead controls (uninfected cells that were fully lysed using 0.1% Triton X-100, 30 min before each timepoint) were included as 0% and 100% cytotoxicity controls. Cytotoxicity was calculated as follows:

in which A_S represents the average absorbance at 490 nm (A₄₉₀) of 4 technical replicates, A₀ represents the average A₄₉₀ from media-only controls (0% cytotoxicity), and A_max represents average A₄₉₀ of 100% dead controls (100% cytotoxicity).

Modified antibiotic protection assay for egress measurements

To determine the minimum inhibitory concentration of vancomycin for OG1RF, 2 μL of overnight cultures of OG1RF strains were inoculated to 200 μL of colorless DMEM (Gibco, Singapore) supplemented with 10% HI-FBS and indicated concentrations of vancomycin in a 96-well plate and incubated overnight at 37°C, 5% CO₂. Bacterial growth after 24 h of incubation was measured as absorbance at 600 nm (Abs₆₀₀) using a Tecan Infinite M200 microplate reader (Tecan, Switzerland).

For egress measurements, RAW-Blue macrophages were infected at MOI 10 in an antibiotic protection assay. At 6 hpi, the media was sampled for CFU enumeration to ensure that no extracellular bacteria were present at the start of the measurements. The cells were then washed thrice with DPBS, the media was replaced with complete DMEM supplemented with 5 μg/mL vancomycin, and then re-incubated at 37°C, 5% CO₂ for up to 12 hpi. 100 μL of the media was sampled hourly for serial dilution and CFU quantification on BHI agar, and replaced with 100 μL of fresh complete DMEM + 5 μg/mL vancomycin.

For E. faecalis-only bacteriostatic controls, 1 μL of MOI 10/mL-normalised E. faecalis cultures (~1 x 10⁴ CFU) were inoculated onto 2 mL complete DMEM + 5 μg/mL vancomycin in 6-well plates, and incubated at 37°C, 5% CO₂ for up to 6 h. These cultures were sampled hourly as described above to determine viable bacteria CFU in the bacteriostatic media.

Confocal microscopy of infected macrophages

To visualise infected macrophages, RAW-Blue cells were seeded on glass coverslips in 6-well plates (5 x 10⁵ cells/well), and antibiotic protection assay at an MOI of 10 was performed. For visualisation of replicating bacteria, macrophages were infected with E. faecalis pre-stained with eFluor 670 as described for flow cytometry. At 20 hpi, infected macrophages were washed thrice with DPBS and fixed with 4% PFA for 15 min at room temperature. For egress experiments, at 6, 9 and 12 hpi, infected macrophages were spun down at 500 x g for 5 min to sediment any extracellular bacteria. Then, half of the culture media was carefully removed from the top and replaced with an equivalent volume of 8% PFA (final concentration = 4%) for 15 min at room temperature to fix the cells and extracellular bacteria. Subsequently, for all experiments, fixed cells were washed thrice with DPBS and permeabilized with 0.1% Triton X-100 for 20 min at room temperature. Samples were then blocked 2% BSA in DPBS (staining buffer) and immunolabelled with 1:500 rabbit anti-AgD in staining buffer overnight at 4°C, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG for 1 h at room temperature. All samples were then stained with 1:1000 phalloidin-Alexa Fluor 568 (Invitrogen, Cat# A12380, USA) and 1 μg/mL Hoechst 33342 for 20 min at room temperature before mounting on glass slides using ProLong Glass Antifade Mountant. DPBS washes were done between every staining step. Confocal microscopy was performed at NTU Optical Bio-Imaging Centre (NOBIC) imaging facility at SCELSE using an LSM780 laser scanning confocal microscope (Carl Zeiss, Germany). Representative images are shown as maximum intensity projections of Z-stack acquisitions (0.32 μm sections) with orthologous Z-stack projections shown in blue or red adjacent boxes.

Cytosolic staining of intracellular E. faecalis was performed using a differential permeabilization method previously described for cytosolic S. pyogenes and Salmonella [57,56]. Briefly, following antibiotic protection assay at 2, 4 and 6 hpi, cells were washed and equilibrated ice-cold KHM buffer, before being permeabilized with 50 μg/mL digitonin for exactly 5 min on ice. Subsequently, cells were washed once with ice-cold KHM buffer and once with DPBS, and then incubated with 1:500 rabbit anti-AgD in DPBS + 10% FBS for 30 min at room temperature. Cells were then fixed with 4% PFA and stained with 1:1000 Alexa Fluor 488-conjugated anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 1 h at room temperature. To stain for total bacteria, these cells were permeabilized with 0.1% saponin (Sigma, USA) + 10% FBS in DPBS (hereby called permeabilization buffer) for 15 min, then stained with 1:500 anti-AgD in permeabilization buffer overnight at 4°C, and subsequently with 1:1000 Alexa Fluor 568-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11011, USA) in permeabilization buffer for 1 h at room temperature. Cells were then counterstained with 1 μg/mL Hoechst 33342 in permeabilization buffer for 20 min at room temperature before mounting onto glass slides. Cells were washed with DPBS (cytosolic bacteria staining) or permeabilization buffer (total bacteria staining) between staining steps. Confocal microscopy and Z-stack acquisition was performed as described previously. RAW-Blue cells infected with PFA-fixed E. faecalis were used as non-cytosolic controls.

Transmission electron microscopy

RAW264.7 cells were seeded on glass coverslips in 6-well plates (1 x 10⁶ cells/well), and antibiotic protection assay at an MOI of 10 was performed. At 6 hpi, infected macrophages were washed thrice with PBS and fixed with 2.5% glutaraldehyde and 2% PFA for 1 hour at room temperature. Cells were scraped, pelleted, and washed with PBS. Pelleted cells were then post-fixed, stained and dehydrated as described previously [95], except that uranyl acetate staining was done at 4°C overnight and dehydration by the graded ethanol series was done as follows: 2 x 50%, 70%, 90%, 2 x 100% ethanol for 10 min each, then followed by 100% ethanol for 30 min and 2 x propylene oxide for 10 min. Cells were infiltrated with Epon:propylene oxide (1:1) for 1 h, followed by infiltration with fresh 100% Epon overnight, and then embedded in resin-filled gelatin capsules and polymerized in oven at 60°C for 48 h. 50 nm thin sections were cut with Leica EMFC7 ultramicrotome and diamond knife (DiATOME) and collected onto Cu 100-hexagonal EM grid coated with Formvar plastic support film. Sections were imaged with Morgagni TEM (FEI, Netherlands) operating at 80 kV acceleration voltage equipped with Veleta side-mounted CCD camera controlled by iTEM camera software (Olympus-Soft Imaging Solutions) at the Electron Microscopy Facility (PFMU), Faculty of Medicine, University of Geneva.

Murine excisional wound infection model

Murine wound infection was performed as described previously [48,96]. Briefly, overnight cultures of OG1RF, OG1RFC or OG1RFS strains were washed thrice with PBS and normalised to ~2.6 x 10⁸ CFU/mL in PBS. For mixed species infection, the strains were mixed 1:1 (equivalent to ~2 x 10⁸ CFU/mL per strain) prior to infection. 7–10-week-old C57BL/6J male mice were anaesthetised, their dorsal hair trimmed by shaving and Nair depilatory cream application (Church & Dwight Co. Inc., USA), and their skin disinfected. Then, wounds were created using a 6-mm biopsy punch (Integra Miltex, USA), and 10 μL of the bacteria suspension (approximately 2.6-4 x 10⁶ CFU) was applied and sealed with a clear wound dressing (Tegaderm 3M, USA). Mice were then euthanized at 1, 3 or 5 dpi, and wounds were excised into sterile DPBS in Lysing Matrix M (MP Biomedicals, USA). Wound were homogenized using FastPrep-24 Bead Beating System (MP Biomedicals, USA) in 5 rounds of 4.0 m/s, 20 s each. Wound homogenates were serially diluted in DPBS and plated onto BHI agar with 25 μg/mL rifampicin (OGIRF and OG1RFC + OG1RFS total counts), 60 μg/mL spectinomycin (OG1RFS) and 10 μg/mL chloramphenicol (OG1RFC) for output CFU enumeration. The competitive index (CI) of mixed-species infection was calculated as .

To quantify intracellular bacteria in infected mice wounds, wound sections were excised at 1, 3 or 5 dpi and transferred into 24-well plates containing FBS-free DMEM supplemented with 500 μg/mL gentamicin and 500 μg/mL penicillin G. 250 μg/mL Liberase TL (Merck, USA) was added to this antibiotic-containing DMEM and incubated for 1 h at 37°C, 5% CO₂ with occasional shaking to dissociate wound cells and kill extracellular bacteria. Complete DMEM was added to stop tissue dissociation and wounds were flushed to collect the wound cell suspension. Cells were pelleted at 1000 x g, 5 min, 4°C and resuspended in 1 mL 10% FBS in DPBS for washing. Cells were pelleted once more and then lysed with 0.1% Triton X-100 in DPBS for 30 min to release the intracellular bacteria. Lysates were serially diluted and plated on BHI agar + 25 μg/mL rifampicin for CFU enumeration. For total CFU quantification using this enzymatic dissociation method, antibiotics were excluded during the tissue harvest and dissociation steps. 20 μL of the cell suspension was then transferred into 180 μL of 0.1% Triton X-100 in DPBS (10X dilution) for 30 min to release any intracellular bacteria, followed by serial dilution in PBS and plating on BHI agar + 25 μg/mL rifampicin for CFU enumeration.

To analyze the host cell profile of infected mice wounds at 5 dpi, wound cells dissociated with Liberase TL in antibiotic-containing DMEM were resuspended in 200 μL staining buffer (2% BSA in DPBS). 50 μL of each cell suspension was aliquoted and blocked with 1:50 TruStain FcX PLUS anti-mouse CD16/32 antibody (BioLegend, Cat# 156604, USA) for 30 min on ice, followed by staining with 50 μL antibody cocktail of BV510 anti-mouse CD45 (BioLegend, Cat# 103138, USA), APC anti-mouse Ly6G (BioLegend, Cat# 127614, USA), BV421 anti-mouse Ly6C (BioLegend, Cat# 128031, USA), PE anti-mouse/human CD11b (BioLegend, Cat# 101208, USA), and BV711 anti-mouse F4/80 (BioLegend, Cat# 123147, USA), each diluted to a final concentration of 1:100 in staining buffer, for 1 h on ice. The cells were then washed once with cold staining buffer, fixed with 4% PFA for 20 min at room temperature, and then stored in staining buffer overnight at 4°C. The following day, cells were permeabilized for intracellular staining by washing thrice with Intracellular Staining Permeabilization Wash Buffer (BioLegend, Cat# 421002, USA; hereby called permeabilization buffer). Then, cells were stained with 1:500 rabbit anti-AgD diluted in permeabilization buffer for 30 min at room temperature, washed with permeabilization buffer once, and stained with 1:1000 Alexa Fluor 488-conjugated goat anti-rabbit IgG diluted in permeabilization buffer for 30 min at room temperature. Cells were then resuspended in 500 μL staining buffer, and 400 μL of the cell suspension was aliquoted and mixed with 100 μL AccuCheck Counting Beads (Invitrogen, Cat# PCB100, USA) for analysis using BD LSRFortessa X-20 flow cytometer (Becton Dickinson, USA). Flow cytometry compensation was achieved using the AbC Total Antibody Compensation Bead Kit (Invitrogen, USA) following manufacturer protocols, and fluorescence-minus-one (FMO) controls were included to set gating thresholds. AgD⁺ events were gated from the histogram using AgD-stained, mock-infected wound cells as AgD^- gating threshold (S14 Fig).

Data and statistical analysis

Graphs and statistical analyses were performed in GraphPad Prism 9.01 (GraphPad, USA). For flow cytometry data, visualization and quantification of populations were done using FlowJo v10.8.0 (Becton Dickinson, USA). Error bars were represented as means ± standard deviation (SD) or means ± standard error of mean (SEM), as indicated in the respective figure captions. Statistical tests were indicated in individual figure captions, with statistical significance represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.