{"paper_id":"24eb33c2-7fe7-4c41-8b45-04b2a58e5928","body_text":"Enterococcus faecalis redox metabolism activates the unfolded protein response to impair 1 \nwound healing 2 \n 3 \n 4 \n 5 \n 6 \n 7 \n 8 \nAaron Ming Zhi TAN1,2, Cenk CELIK1,5, Stella Yue Ting LEE1, Mark VELEBA2, Caroline S. 9 \nMANZANO4, Rahim MK ABDUL1, Guillaume THIBAULT1,3,*, Kimberly A. KLINE1,2,4,6,** 10 \n 11 \n 12 \n 13 \n 14 \n 15 \n 16 \n 17 \n 18 \n 19 \n 20 \n 21 \n 22 \n1School of Biological Sciences  and 2Singapore Centre for Environmental Life Science Engineering , 23 \nNanyang Technological University, Singapore 24 \n3Mechanobiology Institute, National University of Singapore, Singapore 25 \n4Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 26 \nSwitzerland 27 \n5Present address: Department of Genetics, Evolution and Environment, Genetics Institute, University 28 \nCollege London, London, United Kingdom 29 \n6Lead contact 30 \n 31 \n 32 \n 33 \n 34 \n 35 \n 36 \n 37 \n 38 \n 39 \n 40 \n 41 \nCorrespondence to: 42 \n*Guillaume Thibault, Tel: +65 6592 1787; email: thibault@ntu.edu.sg 43 \n**Kimberly A. Kline, Tel: +41 23 379 5602; email: kimberly.kline@unige.ch 44 \n 45 \nRunning title: E. faecalis EET drives UPR activation in wound infection.  46 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n1 \nSUMMARY 47 \nEnterococcus faecalis is an opportunistic pathogen that thrives in biofilm -associated infections and 48 \ndelays wound healing, yet how it impairs host tissue responses is unclear . Here, we identif ied 49 \nextracellular electron transport (EET) as a previously unrecognized source of ROS in E. faecalis and 50 \nshow that this activity directly triggers the unfolded protein response (UPR) in epithelial cells and delays 51 \nepithelial cell migration. ROS detoxification with catalase suppressed E. faecalis-induced UPR and 52 \nrescued epithelial cell migration, while exogenous H2O2 was sufficient to restore UPR activation in EET-53 \ndeficient strains. Importantly, UPR disruption by pharmacological inhibition also impaired cell migration, 54 \nhighlighting a critical role for UPR homeostasis in wound repair. Our findings establish EET as a novel 55 \nvirulence mechanism that links bacterial redox metabolism to host cell stress and impaired repair, 56 \noffering new avenues for therapeutic intervention in chronic infections.   57 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n2 \nINTRODUCTION 58 \nEnterococcus faecalis is a gut commensal and opportunistic pathogen that causes difficult -to-treat 59 \nbiofilm-associated infections, including catheter -associated urinary tract infection, infective 60 \nendocarditis, and chronic wound infections1,2. In wound settings, E. faecalis infection is associated with 61 \ndelayed epithelial migration and immune dysregulation 3. The success of E. faecalis in these 62 \nenvironments is often attributed to its metabolic adaptability, including survival under nutrient limitation 63 \nand oxidative stress4. However, the extent to which E. faecalis metabolism actively interferes with host 64 \nrepair mechanisms is poorly understood. 65 \n 66 \nOne way that host cells respond to environmental and infection-induced insults is through the unfolded 67 \nprotein response (UPR), an evolutionarily conserved signalling pathway triggered by endoplasmic 68 \nreticulum (ER) stress. The UPR integrates signals related to protein misfolding, membrane 69 \nperturbations, and redox imbalance to restore homeostasis or induce apoptosis if stress persists5–8. 70 \nPathogens have evolved diverse strategies to manipulate the host UPR, often targeting its three main 71 \npathways (IRE1, PERK, and ATF6) to subvert host cell function, modulate immune responses, or even 72 \nexploit UPR-regulated products as a nutrient source9–14. While some bacterial toxins and effectors can 73 \ninduce the UPR, in most cases, the microbial mechanisms by which the UPR is activated or 74 \ndysregulated are undefined11,15–17. 75 \n 76 \nE. faecalis generates substantial extracellular reactive oxygen species (ROS), including superoxide and 77 \nhydrogen peroxide, in the absence of aerobic respiration or fumarate reduction 18,19. These ROS can 78 \ninflict DNA damage and tissue injury in infection models, as well as modulate host signalling pathways 79 \nincluding redox-sensitive pathways like the UPR20–22. These ROS can inflict DNA damage and tissue 80 \ninjury in infection models, as well as modulate host signalling pathways including redox -sensitive 81 \npathways like the UPR20,21,23. However, the bacterial source of ROS in E. faecalis and its mechanistic 82 \nconsequences for host cell function have not been fully elucidated. 83 \n 84 \nIn this study, we show that E. faecalis activates the host UPR during wound infection. Through a forward 85 \ngenetic screen and functional validation, we identify extracellular electron transport (EET) as a 86 \npreviously unrecognised mechanism by which E. faecalis generates ROS, which in turn activates the 87 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n3 \nUPR in epithelial cells and impedes their migration following wounding . We show that EET and 88 \nassociated demethylmenaquinone (DMK) biosynthesis pathways are required for superoxide and 89 \nhydrogen peroxide generation, mutants of which produce less ROS, fail to activate the UPR, and do 90 \nnot impair epithelial cell migration. These findings not only establish a novel function for EET in ROS 91 \ngeneration but also through its interaction with host UPR, as a novel metabolic virulence mechanism 92 \nby which E. faecalis disrupts epithelial repair, thereby presenting new opportunities for targeting chronic 93 \nE. faecalis-driven pathologies.  94 \n 95 \nRESULTS 96 \nE. faecalis infection activates the UPR in a mouse model 97 \nWe previously show ed that E. faecalis  infection impairs wound healin g3. T o gain insight into the 98 \nmechanisms that influence delayed wound repair , we re-analysed our published single-cell RNA-seq 99 \ndataset of E. faecalis infected mice wounds at 4 days post-infection22 (dpi) (Figure 1A, GSE229257). 100 \nFor each class we calculated enrichment scores for a panel of stress-response signatures24 (Table S1). 101 \nGene-set enrichment for canonical UPR targets revealed that the ER stress-response is not global but 102 \nconcentrated in immune cells (macrophages and neutrophils) and, most strikingly, in an infection -103 \nspecific cluster of keratinocytes ( Figure 1B and 1C). By contrast, oxidative -stress response (OSR) 104 \ngenes were upregulated not only in UPR-elevated keratinocytes and immune cells but also in fibroblasts 105 \n(Figure 1 D, S1A, and S1 B), consistent with high fibroblast redox activity in infected tissue. We 106 \npreviously found that E. faecalis infection interferes with wound closure signatures, drives a partial 107 \nepithelial-to-mesenchymal transition (EMT) in keratinocytes, and skews macrophages toward an anti-108 \ninflammatory phenotype22. The robust UPR response in keratinocytes offers a mechanistic clue where 109 \nexcessive ER stress could amplify the infection -induced EMT shift in these cells, undermining their 110 \nmigratory role and thereby hindering wound repair during E. faecalis infection. 111 \n 112 \nTo corroborate the scRNA-seq findings, we infected full thickness excisional wounds in mice with E. 113 \nfaecalis strain OG1RF and enumerated bacterial colony forming units (CFU) from the wounds at 6 days 114 \npost-infection (6 dpi), chosen to target the proliferation and remodelling phase of healing, where we 115 \nobserved a 2-log reduction in E. faecalis bacteria burden in wounds compared to the starting inoculum, 116 \nwith no significant animal weight loss or attrition, consistent with our previous studies3 (Figure S1C and 117 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n4 \nS1D). We quantified UPR-associated mRNA levels of spliced Xbp1 (Xbp1s), Chop, and Herpud1 as 118 \nmarkers of Ire1, Perk1, and Atf6 activity, respectively, in whole wound tissue  (Figure 1 E). Xbp1s 119 \ntranscripts were significantly higher in uninfected wound s at 6 d pi compared to unwounded skin, 120 \nindicating that the UPR is activated in wounds  regardless of infection state  (Figure 1F). A similar 121 \nobservation was reported in wounded transgenic mice expressing a XBP1-Luc fluorescence marker 8-122 \n10 days post-wounding25. By contrast, both Xbp1s and Chop transcript levels were significantly higher 123 \nin E. faecalis-infected wounds at 6 dpi compared to unwounded skin samples, while Herpud1 levels 124 \nremained unchanged, indicating a lack of ATF6 activation  (Figure 1F). Taken together, t hese data 125 \nsuggest that E. faecalis  infection results in UPR dysregulation, which could impact normal wound 126 \nhealing26. 127 \n 128 \nIRE1 activation by E. faecalis impedes keratinocyte migration in vitro 129 \nTo corroborate our in vivo and in silico findings, we examined UPR activation in keratinocytes (HaCaT) 130 \nand fibroblasts (NIH-3T3) which are the dominant cell types in healthy skin that contribute to wound 131 \nhealing27. E. faecalis  infection significantly increased mRNA expression of  all three UPR pathway 132 \nmarkers in NIH-3T3 cells (Figure 2A), whereas in HaCaT cells only XBP1s and CHOP were significantly 133 \nupregulated (Figure 2B). As a positive control, we treated both cell lines with tunicamycin (Tm), which 134 \ninduces the UPR by inhibiting protein glycosylation in the ER  leading to an accumulation of unfolded 135 \nproteins28. Tm treatment significantly upregulated all three UPR pathway markers in both cell lines to a 136 \ngreater extent compared to E. faecalis infection. Since IRE1 is the most evolutionarily conserved branch 137 \nof the UPR, we further examined its activation by E. faecalis by assessing the expression of XBP1s 138 \ntarget genes29,30. These included the ER chaperone  BiP (encoded by HSPA5) and EDEM1, which 139 \npromote ER homeostasis and are hallmarks of IRE1 activation31,32. Infected cells exhibited a significant 140 \nincrease in EDEM1 transcripts along with elevated levels of XBP1s and BiP proteins (Figure 2C and 141 \n2D), confirming that E. faecalis activates the conserved IRE1 pathway in vivo and in vitro. 142 \n 143 \nWe next investigated the impact of infection-induced UPR on wound closure using an in vitro HaCaT 144 \nscratch wound assay. By 15 hpi, both E. faecalis-infected and Tm-treated cells exhibited significantly 145 \nslower migration compared to uninfected cells, with the difference becoming more pronounced by 27 146 \nhpi (Figure 2E and S2A; Video S1 ). Notably, neither infected nor Tm-treated cells displayed 147 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n5 \nappreciable migration throughout the assay  period, and infected cells even show ed signs of wound 148 \nedge retraction as early as 6 hpi, consistent with cell shrinkage or detachment which can be early 149 \nindicators of apoptosis. To determine whether UPR induction  via IRE1 was responsible for impaired 150 \nmigration, we treated both uninfected and infected cells with IRE1 inhibitor (4µ8c), which blocks the 151 \nRNase activity of IRE133 (IRE1i). Treatment with 50 µM IRE1i was sufficient to block E. faecalis-induced 152 \nIRE1 in HaCaT cells (Figure 2F). IRE1i also slowed migration in uninfected wells, with the delay evident 153 \nby 18 hpi and more pronounced at 27 hpi. (Figure 2G and S2B; Video S2). In infected cells, migration 154 \nwas minimal regardless of IRE1i treatment, showing no significant migration at 27 hpi relative to the 3 155 \nhpi baseline. 156 \n 157 \nWe did not use proliferation inhibitors, such as mitomycin C, which are typically used to differentiate 158 \nbetween proliferation and migration in these assays, because the compound caused widespread cell 159 \ndetachment in infected cells during preliminary experiments. Nonetheless, we attribute the observed 160 \nwound closure primarily to cell migration rather than proliferation for several reasons: (i) the short 24-h 161 \nduration of the assay, (ii) the long doubling time of confluent HaCaT cells (approx. 32 -36 h), and (iii) 162 \nprevious studies have established that migration is the dominant factor in similar short -term scratch 163 \nassays26. Thus, our findings suggest that neither UPR hyperactivation (during infection) nor 164 \nhypoactivation (after IRE1 inhibition) alone fully explains keratinocyte migration; rather, both extremes 165 \nof UPR activity are implicated.  166 \n 167 \nExtracellular electron transport drives E. faecalis UPR activation and migration arrest 168 \nTo dissect how E. faecalis induces the UPR, we designed a high-throughput assay for UPR induction: 169 \na NIH-3T3 reporter line (3T3R) that fluoresces when IRE1 splices a 26-nt intron from a truncated human 170 \nXBP1 fused to mApple (Figure 3A and 3B). While IRE1 is quiescent, a premature stop codon between 171 \nXBP1 and mApple blocks translation, causing cells to remain non-fluorescent. When IRE1 is activated, 172 \nintron excision shifts the reading frame, removes the stop codon, and allows production of the full 173 \nXBP1s-mApple fusion, generating a red signal quantifiable by wide -field microscopy. Using this 174 \nreporter, we screened a defined transposon (Tn) library of 14,976 E. faecalis OG1RF mutants (Figure 175 \n3A) to identify mutants that failed to trigger UPR activation. 176 \n 177 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n6 \nApplying an upper threshold of 30% XBP1s-mApple positive (XBP1s+) cells in a given population, we 178 \nidentified 457 UPR defective mutants, corresponding to  369 unique genetic loci (Figure S3A). We 179 \nfiltered out mutants with multiple transposon insertions, insertions outside of coding regions, and those 180 \nwith significant growth defects after overnight culture in BHI  media. Pathway analysis did not show 181 \nconsistent enrichment patterns; however, w e notice d that a substantial number of mutants had 182 \ninsertions in genes associated with carbohydrate catabolism and respiration, key components of redox 183 \nmetabolism. This  observation prompted us to focus on pathways involved in the  synthesis of 184 \ncomponents and co -factors that facilitate electron flow  from carbohydrate catabolism to terminal 185 \nelectron acceptors. Transposon insertions mapped to three respiratory pathways of E. faecalis: aerobic 186 \nrespiration (ndh2, cydD), fumarate reduction  (frd), and extracellular electron transport (EET)  (ndh3, 187 \neetB) (Figure 3C). Additional insertions were identified in genes involved in  the biosynthesis of the 188 \nquinone electron carrier demethylmenaquinone (DMK) (menF, menE), as well as upstream precursors 189 \nsuch as chorismate and geranyl pyrophosphate, generated either by the shikimate pathway (aroD) or 190 \nthe mevalonate pathway (ispA). DMK is an integral part of all three respiratory pathways by mediating 191 \nelectron transfer between their membrane-associated components. 192 \n 193 \nWe validated mutants of these respiratory pathways identified in the primary screen and found that only 194 \nmutants disrupted in quinone electron carrier synthesis (menE, menF, aroD), and EET ( ndh3, eetB) 195 \ndisplayed significantly reduced UPR induction compared to wild-type E. faecalis (WT) (Figure 3D). 196 \nSince these genes are involved in central metabolic processes, we quantified the growth of each mutant 197 \nto ensure that reduced  UPR induction was not simply due to impaired bacterial replication during 198 \ninfection. However, all Tn mutants grew similarly to WT in cell culture media (Figure S3B and S3C), 199 \neliminating growth defects as a confounding factor . Given the strong association between  EET and 200 \nUPR induction, we also tested a deletion mutant lacking entire EET operon (ΔEET) (Figure 3C and 201 \n3D). As expected, the ΔEET mutant was defective in UPR induction, yet retained growth and antibiotic 202 \nsusceptibility profiles similar to the parental  OG1RF WT strain (Figure S3B-S3D). To rule out the 203 \npossibility that lower UPR induction by ΔEET  could be due to higher cytotoxicity causing cell loss 204 \nresulting in weaker XBP1s-mApple fluorescent signals, we assessed cytotoxicity in HaCaT cells at 3 205 \nand 24 hpi following infection with WT and ΔEET. There was no difference in cytotoxicity between 206 \nuninfected, WT- or ΔEET-infected cells at 3 hpi (Figure S3E). However, WT but not ΔEET-infected cells 207 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n7 \nhad significantly higher cytotoxicity compared to uninfected cells at 24 hpi.  The significantly higher 208 \ncytotoxicity in WT-infected cells at a delayed timepoint of 24 hpi but not immediately after infection at 3 209 \nhpi suggests that WT infection was not directly causing cell death but rather indirectly via UPR 210 \nhyperactivation. T he lack of difference at 24 hpi between uninfected and ΔEET -infected cells also 211 \nconfirms the UPR defective nature of ΔEET, as delayed cell death is a hallmark characteristic of chronic 212 \nUPR hyperactivation34,35. To further confirm that the loss of UPR induction was specific to the EET 213 \npathway and not to secreted toxins, as is the case for Group A Streptococcus14,36, we tested a deletion 214 \nmutant of the Type 7 secretion system (ΔT7SS), encoding predicted secreted toxins 37. The ΔT7SS 215 \nmutant induced the UPR to the same extent as WT (Figure S3F), supporting the conclusion that the 216 \nphenotype of the ΔEET mutant is a direct consequence of its function in redox metabolism. 217 \n 218 \nBased on these characteristics, ΔEET was selected as the model UPR defective mutant for downstream 219 \nstudies. We used this mutant to assess  whether E. faecalis  UPR induction  via the EET  pathway 220 \ncontributes to the inhibition of keratinocyte cell migration. Unlike WT infection, ΔEET did not significantly 221 \nimpair migration, instead showing similar migration, comparable to uninfected controls (Figure 3E and 222 \nS3G; Video S3 ). However, treating ΔEET-infected cells with IRE1i resulted in significantly slower 223 \nmigration at the 27 hpi endpoint  (Figure 3 F and S3H). The se findings suggest that  lack of UPR 224 \ninduction by ΔEET allows for physiological levels of UPR induction that support normal cell migration. 225 \nFurthermore, IRE1i treatment induces UPR hypoactivation in ΔEET-infected cells (Figure 3F and S3H; 226 \nVideo S4), dysregulating UPR homeostasis and impairing cell migration, similar to that observed in 227 \nuninfected cells treated with IRE1i (Figure 2G, Figure 3G). The recovery of cell migration with ΔEET 228 \ninfection and its reversal by IRE1i treatment demonstrate that E. faecalis EET is associated with UPR 229 \ninduction and impaired cell migration. 230 \n 231 \nEET-derived ROS is sufficient to activate the UPR, disrupting epithelial migration 232 \nDisruption of DMK synthesis in E. faecalis impairs both EET function and extracellular superoxide 233 \ngeneration18,38. OG1RF mutants disrupted in genes involved in quinone electron carrier synthesis (aroE, 234 \naroC, aroA, menB, menD, menE ) produce less  superoxide (O2•–) than WT19. Superoxide  radicals 235 \nundergo pH-dependent spontaneous dismutation to generate hydrogen peroxide (H2O2) which can then 236 \nparticipate in  Fenton chemistry to generate hydroxyl radicals and other ROS in the presence of 237 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n8 \ntransition metals (Figure 4A). H2O2 has been shown to induce the UPR in myotubule and epithelial 238 \ncells at 50 µM and 200 µM , respectively20,23. However, this may not be a universal response among 239 \nhost cells as another group reported no significant increase in XBP1s expression for fibroblast when 240 \ntreated with 1 mM H2O239. We hypothesised that superoxide generation via EET drives UPR induction 241 \nin epithelial cells via H2O2. To test whether H2O2 alone is sufficient to induce the UPR in our models, we 242 \ntreated 3T3R cells with increasing concentrations of H2O2 for 3 hours followed by a 21 -hour recovery 243 \nperiod. XBP1s fluorescence increased significantly at 250 and 500 µM (Figure 4B). This finding 244 \nconfirms that NIH-3T3 cells can mount a UPR in response to H2O2, supporting the idea that ROS 245 \ngenerated via EET contributes to UPR induction during infection. 246 \n 247 \nNext, to test whether EET indeed generates ROS , we quantified superoxide generation by validated 248 \nUPR-defective mutants (ΔEET, and transposon insertion mutants in menE, menF, arodD, ndh3, eetB). 249 \nAt the same time, we generated in-frame deletion mutants for each of these genes. All exhibited 250 \nsignificantly lower superoxide generation compared to WT and non-UPR defective mutants (ispA, frd, 251 \nndh2, cydD), and the in-frame deletion mutants were similar to their respective transposon mutants  252 \n(Figure 4C and S4A), supporting a link between EET -dependent superoxide production and UPR 253 \nactivation. 254 \n 255 \nSuperoxide dismutase (SOD) and catalase are antioxidants that catalyse the dismutation of superoxide 256 \nradicals into H2O2 or H2O2 into water and oxygen, respectively (Figure 4A). To determine whether E. 257 \nfaecalis-derived ROS drives UPR induction, we treated infected 3T3R cells with exogenous SOD and 258 \ncatalase. In control experiments, neither enzyme altered XBP1s expression in uninfected cells, whereas 259 \ntreatment with 250 µM H 2O2 to simulate ROS -induced stress robustly increased XBP1s expression  260 \n(Figure 4D). In cells stimulated with H 2O2, the addition of catalase significantly reduced XBP1s 261 \nexpression, whereas SOD had no effect. This result confirms that H2O2 is the specific reactive oxygen 262 \nspecies driving UPR induction in this assay. 263 \n 264 \nIn WT-infected cells, catalase again significantly reduced XBP1s expression, while SOD had no effect 265 \n(Figure 4E). Adding H2O2 to WT-infected cells did not further increase UPR activation, suggesting that 266 \nE. faecalis-generated ROS levels are already saturating whereas co-treatment with catalase reversed 267 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n9 \nthis effect. By contrast, catalase had no impact on ΔEET-infected cells, consistent with their low ROS 268 \ngeneration (Figure 4F). However, functional complementation of the ΔEET with H2O2 partially restored 269 \nUPR induction, which was again reversed by catalase, confirming that H2O2 is sufficient to rescue the 270 \nUPR defective phenotype when EET is disrupted . These findings were replicated in  HaCaT cells 271 \n(Figure 4G-4I). Since lipid peroxidation is a known cause of UPR activation40. We next measured it in 272 \ninfected cells using a BODIPY 581/591 C11 fluorescent probe. WT infected cells exhibited significantly 273 \nmore lipid peroxidation than uninfected controls, while cells infected with the ΔEET mutant showed no 274 \nsignificant change (Figure 4J). These data support a model where EET-derived ROS from E. faecalis 275 \ninduces the UPR via lipid peroxidation. 276 \n 277 \nTo determine whether E. faecalis generated ROS impairs wound healing, we assessed in vitro wound 278 \nclosure after treatment with H 2O2 or/and catalase. H2O2 treatment of uninfected cells significantly 279 \nslowed cell migration at 27 hpi, although no wound edge retraction was observed, unlike in WT-infected 280 \ncells (Figure 4K and S4B; Video S5 ). Co-treatment with catalase  restored cell migration  to 281 \nphysiological levels . Similarly, catalase  treatment of WT-infected cells significantly improved cell 282 \nmigration, which approached that of uninfected controls and without retraction at 27 hpi (Figure 4L and 283 \nS4C; Video S6). Adding H2O2 to untreated WT-infected cells had no further effect, consistent with UPR 284 \nsaturation seen earlier  (Figure 4E and 4H ). Finally, co-treatment with H2O2 and catalase mirrored 285 \ncatalase-treatment alone, reinforcing the role of E. faecalis-derived H2O2 in cell migration arrest (Figure 286 \n4L and S4C; Video S6). Altogether, these findings demonstrate that the EET pathway of E. faecalis 287 \ngenerates ROS which oxidises lipids in epithelial cells, hyperactivating the UPR , and inhibiting cell 288 \nmigration. 289 \n 290 \nDISCUSSION 291 \nOur findings identify E. faecalis extracellular electron transport (EET) as a novel virulence mechanism 292 \nthat links bacterial redox metabolism to host stress responses and impaired tissue repair. We show that 293 \nEET-dependent production of reactive oxygen species (ROS), particularly H2O2, activates the unfolded 294 \nprotein response (UPR) in epithelial cells. UPR dysregulation by E. faecalis disrupts normal epithelial 295 \nfunction and cell migration , revealing a direct mechanistic connection between bacterial energy 296 \nmetabolism and host healing processes. 297 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n10 \n 298 \nE. faecalis is known to produce ROS, which have been previously linked to host cellular injury and even 299 \ncarcinogenesis21. In this study, we refine the genetic basis of this activity in terms of a dedicated electron 300 \ntransport system. Previous studies showed that E. faecalis generates extracellular superoxide through 301 \na process that requires demethylmenaquinone (DMK) , which is diminished when terminal 302 \nquinol/cytochrome oxidases are functional, suggesting that respiratory disruption changes the dynamics 303 \nof electron flow to one that favours the reduction of oxygen to superoxide, resulting in ROS production19. 304 \nIn this model, NADH incompletely reduces DMK, producing semiquinone intermediates which, in the 305 \nabsence of classical aerobic or anaerobic respiration, donate electrons univalently to molecular oxygen, 306 \ngenerating superoxide which spontaneously dismutate into hydrogen peroxide which can contribute to 307 \nhost oxidative stress 19. This movement of electrons may be mediated by soluble shuttles, such as 308 \nflavins41,42. In this study, we refine the mechanistic basis of E. faecalis ROS generation by identifying a 309 \ncritical role for extracellular electron transport (EET) in this process. 310 \n 311 \nWhile disrupting aerobic respiration is required for ROS generation and EET function19,38, our data show 312 \nthat the disruption of aerobic respiration alone is not sufficient and that ROS generation also depends 313 \non an intact EET system. However, the mechanistic contribution of specific EET components involved 314 \nin ROS generation remains to be determined. Whether these components overlap with those required 315 \nfor extracellular metal reduction or electrode respiration or represent a distinct branch of EET machinery 316 \nactivated under redox stress, remains an important question for future work. 317 \n 318 \nThe requirement for EET in E. faecalis ROS production aligns with growing evidence that extracellular 319 \nelectron transfer in Gram -positive bacteria extends beyond classical anaerobic respiration. While 320 \ncanonical diderm systems in general like Shewanella and Geobacter use cytochromes and conductive 321 \npili to transfer electrons to external acceptors 43,44, re cent studies show that monoderm bacteria, 322 \nincluding Listeria monocytogenes and Lactobacillus plantarum, use flavin-based EET to support energy 323 \nconservation, redox homeostasis, and virulence under host-relevant conditions41,42,45. In particular, E. 324 \nfaecalis and L. monocytogenes rely on EET for fitness in the mouse gastrointestinal tract 41,46, and L. 325 \nplantarum employs EET to enhance ATP yield via substrate-level phosphorylation in the absence of a 326 \nclassical respiratory chain47. Our findings position E. faecalis within this emerging framework and reveal 327 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n11 \na distinct facet: in the presence of oxygen within a heme-free environment, EET contributes to univalent 328 \nelectron transfer from reduced DMK to oxygen, generating superoxide and hydrogen peroxide. Unlike 329 \nenvironmental microbial EET systems that avoid oxygen to prevent radical formation, E. faecalis  330 \nappears to exploit this interaction, linking EET to oxidative stress at the host-pathogen interface. 331 \n 332 \nThe UPR is increasingly recognized as a central node in host stress responses during infection, 333 \nparticularly in epithelial and immune cells. Multiple pathogens including Salmonella enterica , 334 \nHelicobacter pylori, Pseudomonas aeruginosa, Streptococcus pyogenes, and Brucella melitensis have 335 \nbeen shown to manipulate the UPR through secreted toxins or effector proteins, often to promote 336 \nintracellular survival or dampen immune response s9–11,14,48. Here w e demonstrate that E. faecalis  337 \nselectively activates the IRE1 and PERK arms of the UPR both in vivo and in vitro, manifesting in 338 \nimpaired epithelial cell migration. Unlike previously described examples, E. faecalis induces the UPR 339 \nindependent of dedicated virulence factors, instead leveraging metabolic ROS production via EET. 340 \nSimilar redox-based virulence strategies have been described in other pathogens. S. pneumoniae 341 \nproduces hydrogen peroxide via SpxB, contributing to epithelial damage49,50. P. aeruginosa phenazines 342 \ngenerate intracellular ROS in airway cells 51,52. Yet E. faecalis is the first example, to our knowledge, 343 \nwhere a defined EET system is shown to drive ROS production that directly alters host stress signalling 344 \nand function. 345 \n 346 \nA compelling hypothesis arising from our data is that the observed UPR hyperactivation and impaired 347 \ncell migration are consequences of ferroptosis. Ferroptosis is a regulated, iron-dependent form of cell 348 \ndeath distinct from apoptosis, driven by the catastrophic accumulation of lipid peroxides. This process 349 \nis typically restrained by the antioxidant enzyme GPX4, and its failure leads to membrane damage53,54. 350 \nFerroptosis is increasingly recognized as a critical factor in diverse pathologies and host -pathogen 351 \ninteractions55–57. Importantly, t he accumulation of lipid peroxides, the biochemical hallmark of 352 \nferroptosis is a known trigger of severe endoplasmic reticulum stress, providing a direct mechanistic 353 \nlink to UPR activation40,58,59. Our findings align remarkably well with this framework. The lipid 354 \nperoxidation we observed in infected keratinocytes is the defining feature of ferroptosis, and the 355 \nsubsequent cell retraction is a classic morphological correlate of this death pathway53,54,60. This model 356 \nmechanistically connects E. faecalis  EET-driven ROS production to the downstream cellular 357 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n12 \npathologies of lipid stress, UPR activation, and inhibition of cell migration. While our experiments with 358 \ncatalase confirm ROS as the primary trigger, they do not exclude ferroptosis as the ultimate executioner 359 \npathway. We therefore propose that the E. faecalis-host interaction may represent a novel model of 360 \ninfection-induced ferroptosis. Testing this hypothesis, for which our study provides the foundational 361 \nrationale, will be a critical next step and could be directly addressed by employing specific inhibitors like 362 \nferrostatin-1. 363 \n 364 \nOur data further show that UPR activation must be tightly regulated for effective wound healing. These 365 \nfindings are consistent with previous studies in aged keratinocytes and fibroblasts displaying higher 366 \nbaseline levels of UPR markers and slower in vitro wound cell migration, which could be reversed upon 367 \ntreatment with 4-phenylbutyrate, a broad-acting UPR inhibitor26. Similarly, we show that pharmacologic 368 \ninhibition of IRE1, the key UPR sensor, led to hypoactivation and impaired cell migration even in 369 \nuninfected cells, underscoring the importance of physiological UPR signalling during repair. Regulated 370 \nUPR induction is also necessary for other wound healing processes like the differentiation of dermal 371 \nfibroblasts to myofibroblasts which promote wound contracture and collagen deposition61. Furthermore, 372 \nUPR inhibitors especially PERK inhibitors have demonstrated significant cytotoxicity to pancreatic islet 373 \ncells which depend on mild UPR induction to perform their secretory function62. Even if UPR induction 374 \nis not lowered to below baseline levels, UPR inhibition will still be counterproductive in restoring  375 \nhomeostasis in pathologies where host cells are already dependent on some level of UPR Induction to 376 \nperform physiological processes. Therefore, targeting the source of UPR dysregulation i.e. bacterial 377 \nROS production, rather than host UPR, may constitute a more effective therapeutic strategy. 378 \n 379 \nIn summary, this work reveals that E. faecalis leverages its respiratory machinery not only for metabolic 380 \nflexibility but also to perturb host cell physiology. While EET has been linked to efficient infection of the 381 \ngastrointestinal tract, this work presents molecular details that may contribute to its role in pathogenesis. 382 \nFuture studies should examine the role of EET in vivo , its regulation and contribution  within 383 \npolymicrobial settings, and the potential for targeting redox metabolism to mitigate E. faecalis infections 384 \nthat are increasingly recalcitrant to antibiotic therapy. 385 \n 386 \n 387 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n13 \nRESOURCES AVAILABILITY 388 \n 389 \nLead contact 390 \nFurther information and requests for resources and reagents should be directed to and will be fulfilled 391 \nby the lead contact, Kimberly Kline (kimberly.kline@unige.ch). 392 \n 393 \nMaterials availability 394 \nThis study did not generate new unique reagents. 395 \n 396 \nData and code availability 397 \nThis paper did not generate any new original datasets nor codes. 398 \nAny additional information required to reanalyse the data reported in this paper is available from the 399 \nlead contact upon request. 400 \n 401 \nACKNOWLEDGEMENTS 402 \nWe are grateful to Thibault and Kline lab members  for helpful discussions and critical reading of the 403 \nmanuscript. We thank Gary Dunny and Jennifer Dale for providing the E. faecalis transposon mutants. 404 \nWe would also like to thank the Centre for Biomedical Informatics (Drs. James A. Miller and Bernett 405 \nTeck Kwong Lee) and the NTU Optical Bio-imaging Centre (NOBIC) for their support. This work was 406 \nsupported by funds from the National Medical Research Council Open Fund (MOH-000566 to GT and 407 \nKAK), the Singapore Ministry of Education Academic Research Fund Tier 1 (RG31/24 to GT), NTU 408 \nResearch Scholarship to SYTL  and RMKA (predoctoral fellowship), and the Swiss National Science 409 \nFoundation (SNSF grant 310030_212262 to KAK) . Parts of this work were also supported by the 410 \nNational Research Foundation and Ministry of Education Singapore under its Research Centre of 411 \nExcellence Program (SCELSE). 412 \n 413 \nAUTHOR CONTRIBUTIONS 414 \nConceptualization, A.M.Z.T., G.T. and K.A.K.; Resources A.M.Z.T., M.V., C.S.M., and R.M.K.A.; Data 415 \ncuration, C.C. and R.M.K.A.; Formal analysis, A.M.Z.T., C.C., S.Y.T.L., and R.M.K.A.; Validation, 416 \nA.M.Z.T.; Investigation, A.M.Z.T., C.C., S.Y.T.L.; Visualization, A.M.Z.T., C.C., S.Y.T.L. and G.T. ; 417 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n14 \nMethodology, A.M.Z.T., C.C., S.Y.T.L., G.T. and K.A.K.;  Writing – original draft, A.M.Z.T., G.T. and 418 \nK.A.K.; Writing – review and editing, A.M.Z.T., C.C., G.T. and K.A.K.; Project administration, G.T. and 419 \nK.A.K. 420 \n 421 \nDECLARATION OF INTERESTS 422 \nThe authors declare no competing interests. 423 \n 424 \nMATERIALS AND METHODS 425 \n 426 \nBacterial strains and growth conditions  427 \nAll bacterial strains used in this study are listed in Table S2. E. faecalis strains were routinely cultured 428 \non brain-heart infusion (BHI; Neogen #NCM0016A) agar plates and grown in BHI broth. E. coli strains, 429 \nused for DNA isolation and plasmid manipulation, were cultured in Luria -Bertani (LB) broth or on LB 430 \nagar plates at 37°C. When required, antibiotics were added at the following final concentrations: 431 \nerythromycin (Em), 500 µg/ml for E. coli and 25 µg/ml for E. faecalis; rifampin (Rif), 25 µg/ml; and 432 \nchloramphenicol (Cm), 10 µg/ml.  To prepare overnight cultures, a single E. faecalis  colony was 433 \ninoculated into a 14 ml tube containing 4 ml of BHI broth, with the lid tightly sealed. Cultures were grown 434 \nstatically for 18-24 hours at 37°C. The following day, the overnight culture was centrifuged (4,000 x g, 435 \n10 min), and the bacterial pellet was washed once with PBS before being resuspended in 1 ml of 436 \ncomplete DMEM (see Cell Culture section). The bacterial suspension was then normalized by optical 437 \ndensity (OD 600) to a concentration equivalent to 8x10⁸ CFU/ml (OD 600 = 1) and further adjusted 438 \ndepending on the specific experimental application. 439 \n 440 \nMouse wound excisional model 441 \nAll in vivo procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at 442 \nNanyang Technological University, Singapore (Protocol #ARF SBS/NIEA -0314), in accordance with 443 \nnational guidelines. Male C57BL/6J mice (6–7 weeks old, 22–25 g; InVivos, Singapore) were housed 444 \nunder specific-pathogen-free conditions. The wound infection model was adapted from a previous  445 \nstudy3. Briefly, mice were anesthetized with 3% isoflurane, and dorsal hair was removed using clippers 446 \nand a depilatory cream (Nair). The skin was disinfected with 70% ethanol, and a 6 mm full -thickness 447 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n15 \nexcisional wound was created using a sterile biopsy punch (Integra Miltex #33 -36). The wound was 448 \nimmediately inoculated with 10 µl of an E. faecalis OG1RF suspension containing 2x10⁶ CFU. The 449 \nwound was then sealed with a transparent dressing (Tegaderm, 3M  #7100252702) to prevent 450 \ncontamination. Post-procedure, mice were housed individually to prevent wound disruption.  At the 451 \nexperimental endpoint, mice were euthanized, and a 1 cm2 piece of skin tissue centered on the wound 452 \nwas excised. For bacterial enumeration, tissues were collected in sterile PBS, homogenized, and plated 453 \non BHI agar supplemented with rifampin to confirm infection by the inoculated strain. For molecular 454 \nanalysis, tissues were homogenized in either TRIzol reagent (Thermo Fisher  #15596026) for RNA 455 \nextraction or ice-cold RIPA buffer (Thermo Scientific #89900) supplemented with a protease inhibitor 456 \ncocktail (Roche #11697498001) for protein extraction. 457 \n 458 \nscRNA-seq integration and downstream analysis 459 \nSingle-cell datasets (GSE229257) were re-processed in R 4.3.263 following the workflow established in 460 \nour original study22. Raw matrices were imported with Seurat 5.1.064–67. Cells expressing < 200 genes, 461 \n> 6,000 genes or > 12 % mitochondrial reads were removed; genes detected in < 5 cells were discarded. 462 \nLibrary size variation was normalised with SCTransform (method = “glmGamPoi”, vst.flavour = “v2”). 463 \nBatch effects between biological replicates (uninfected vs. E. faecalis-infected wounds) were corrected 464 \nwith Seurat’s reciprocal PCA integration (30 PCs). Principal components ( n = 30) were used for 465 \nFindNeighbors/FindClusters (resolution = 0.4) and RunUMAP (dims = 1–30). Broad cell classes were 466 \nassigned on canonical markers as previously reported22, sub-clustering of keratinocytes and fibroblasts 467 \nemployed a second round of PCA/UMAP at resolution = 0.6. All UMAPs use colour-blind-safe palettes 468 \ngenerated with RColorBrewer 1.1-368 (brewer.pal, palette = “Set2”). 469 \n 470 \nDifferential expression between infected and uninfected cells within each cluster was ranked by the 471 \nWilcoxon area-under-curve statistic using wilcoxauc69 (presto 1.0.0). Ranked lists served as input for 472 \ngene-set enrichment analysis with fgseaMultilevel (fgsea 1.28.0, minSize = 15, maxSize = 5,000, eps 473 \n= 0)70,71 Gene sets for unfolded  protein response (UPR), oxidative -stress response (OSR) and heat  474 \nshock response (HSR) were curated from MSigDB (v2023.1). Enrichment was considered significant at 475 \nBenjamini–Hochberg FDR < 0. 0572. For each cluster, the positive, significant normalised enrichment 476 \nscores (NES) for infected cells were projected onto the UMAPs. 477 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n16 \nRNA extraction, reverse transcription, and quantitative real-time PCR 478 \nTotal RNA was extracted from cell lines and homogenised mice wound samples using the EZ-10 479 \nDNAaway RNA Miniprep Kit (Biobasic #BS88136) following the manufacturer’s protocol. RNA 480 \nconcentrations were quantified using Qubit Broad Range RNA Quantification Assay (Thermo Fisher 481 \n#Q10210) together with a Qubit 3 Fluorometer following manufacturer’s protocol. Complementary DNA 482 \n(cDNA) was synthesised from extracted RNA normalised to 1 µg per sample using RevertAid Reverse 483 \nTranscriptase (Thermo Fisher #EP0441) following manufacturer’s protocol. qPCR was performed using 484 \nLuna Universal qPCR Master Mix (New England Biolabs #M3003E) together with a CFX-96 (Bio-Rad) 485 \nor a QuantStudio3 (Thermo Fisher) Real-Time PCR system following manufacturer’s protocol. Each 20 486 \nµl reaction has a final concentration of 2.5 ng/µl of cDNA and 0.25 µM of primer pairs (Table S3) for 487 \ntarget genes. Relative mRNA was normalized to the housekeeping gene Gapdh/GAPDH using the 2-488 \n∆∆Ct method73. 489 \n 490 \nCell culture 491 \nMurine embryonic fibroblasts (NIH -3T3) and human keratinocytes ( HaCaT) were cultured in DMEM  492 \n(Gibco # 11995065) supplemented with 10% heat -inactivated foetal bovine serum (FBS, Cytiva 493 \n#SV30160.03) and 4 mM GlutaMAX (Gibco #35050061) . This medium is referred to as \"complete 494 \nDMEM. The lentivirus packaging line, 293FT, was cultured in complete DMEM further supplemented 495 \nwith 0.1 mM MEM Non -Essential Amino Acids (Gibco  #11140050), 6 mM L -glutamine (Gibco  496 \n#25030081), 1 mM Sodium Pyruvate (Cytiva  #SH30239.01), and 500 μg/ml Geneticin (Gibco  Gibco 497 \n#10131027). All cells were maintained at 37°C in a humidified 5% CO2 incubator. Cells were washed 498 \nonce with PBS (Gibco #14190144) and detached using 0.25% Trypsin-EDTA. Trypsinization times were 499 \n15 min for HaCaT, 5 min for 293FT, 4 min for 3T3R, and 3 min for NIH-3T3 cells. The reaction was 500 \nneutralized with an equal volume of complete DMEM. Cells were pelleted by centrifugation , 501 \nresuspended in fresh medium, and counted using a Countess 3 Automated Cell Counter.  For 502 \nexperiments, cells were seeded in 12-well plates at densities of 2.85x104 cells/cm2 (NIH-3T3), 1.15x104 503 \ncells/cm2 (3T3R) or 1.15x105 cells/cm2 (HaCaT) and in cubated for 24 hours prior to use.  For the 504 \ntransposon scree, 3T3R cells were seeded in 96 -well plates at 2.85x104 cells/cm2. Unless otherwise 505 \nspecified, the following final concentrations of reagents were used: tunicamycin at 0.2 µg/ml (for qPCR), 506 \n2.5 µg/ml (for scratch wound assays), or 5 µg/ml (for immunoblot/microscopy); the IRE1 inhibitor 4µ8c 507 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n17 \nat 50 µM (MedChemExpress #HY-19707), added 1 hour prior to infection; H2O2 at 250 µM; catalase at 508 \n100 units/ml; and superoxide dismutase (SOD, Sigma-Aldrich #S5395) at 100 units/ml.  509 \n 510 \nOptimisation of in vitro infection 511 \nAssay conditions were optimised across (i) multiple timepoints and (ii) multiplicities of infection (MOI), 512 \nto identify those that maximise in vitro XBP1s expression (data not shown). 513 \n 514 \nIn vitro infection 515 \nConfluent NIH-3T3 and HaCaT cells were infected with E. faecalis at a MOI of 800 (800 CFU per host 516 \ncell) and 600 (600 CFU per host cell), respectively. Following a 3-hour infection period, the medium was 517 \nremoved, and cells were washed three times with PBS. To eliminate extracellular bacteria, fresh 518 \ncomplete medium supplemented with a gentamicin-penicillin antibiotic cocktail (50 µg/ml) was added, 519 \nand the cells were incubated for an additional 21 hours74. 520 \n 521 \nImmunoblotting 522 \nCells were lysed with RIPA buffer (Thermo Fisher #89901) supplemented with reconstituted cOmplete 523 \nprotease inhibitor cocktail ( Roche # 11697498001) by gentle agitation on ice for 5 min before 524 \ncentrifugation for 15 min at 12,000 x g at 4°C. A mixture of 15 µg of total proteins was separated on 10% 525 \nSDS-PAGE and transferred on nitrocellulose membranes. Immunoblotting was performed with 526 \nappropriate primary antibodies and IRDye-conjugated secondary antibodies (Table S4). Proteins were 527 \nvisualized using the NIR fluorescence system (Odyssey CLx Imaging System). 528 \n 529 \nIn vitro scratch wound assay model 530 \nScratch assays were performed in 12-well plates, adapting a previously published protocol to facilitate 531 \nautomated microscopy75. Confluent HaCaT cell monolayers were scratched with a sterile P200 pipette 532 \ntip and subsequently infected as described in the In vitro infection section. Following the post-infection 533 \nwash, fresh complete medium supplemented with antibiotics and 25 mM HEPES (Gibco #15630080) 534 \nwas added to each well. Wound closure was monitored on a Zeiss Axio Observer 7 microscope (10x 535 \nmagnification), acquiring brightfield images every 30 min for 45 h in a controlled environment (37°C, 5% 536 \nCO2). The resulting time -lapse images were analyzed using a customi sed CellProfiler pipeline to 537 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n18 \nquantify the scratch area76. To ensure accuracy, images where the wound area was misidentified by 538 \nthe automated pipeline were manually measured in ImageJ using the Wound Healing Size Tool plugin77. 539 \n 540 \nXBP1s reporter cell line 541 \npLVX-XBP1-mNeonGreen-NLS plasmid was a gift from David Andrews ( Table S5). Codon-optimized 542 \nmApple cDNA was synthesized as a gBLOCK fragment (IDT)  and inserted in pLVX -XBP1-543 \nmNeonGreen-NLS to replace mNeonGreen using Gibson Assembly (New England Biolabs #E2611L) 544 \naccording to manufacturer protocol. The resulting plasmid, pLVX-XBP1-mApple-NLS, was sequence-545 \nverified and then co -transfected into 293FT cells with the pLP1, pLP2, and pLP/VSVG packaging 546 \nplasmids (Table S5). Supernatants containing lentiviral particles were harvested at 36 - and 60-hour 547 \npost-transfection, pooled, and filtered (0.45 μm). NIH-3T3 cells were then transduced with the filtered 548 \nvirus for 24 h ours in the presence of 8 µg/ml of polybrene (Sigma -Aldrich #H9268). After 24  hours 549 \nrecovery, infected cells were selected with 2 µM puromycin. Clonal cell lines were established by 550 \nseeding single cells into 96-well plates and expanding them for two weeks. Finally, positive clones were 551 \nvalidated by assessing homogenous fluorescent signal upon tunicamycin treatment. One validated 552 \nclone, designated the 3T3 reporter (3T3R) line, was selected for this study. 553 \n 554 \nTransposon screen 555 \nA high-throughput screen was performed using an established E. faecalis OG1RF mariner transposon 556 \nlibrary containing 14,976 mutants arrayed in 96-well plates78. Following overnight growth, the optical 557 \ndensity (OD 600) of each mutant culture was measured with a Tecan M200 microplate reader . For 558 \ninfection, 5 µl of each culture was added  to 3T3R cells s eeded in 96 -well plates, and plates were 559 \ncentrifuged at 300 x g for 5 minutes to synchronize contact. Following infection, cells were stained with 560 \n2.5 µg/ml Hoechest 33342 (Thermo fisher #H21492) for 15 minutes, and the medium was then replaced 561 \nwith phenol-red free DMEM (Gibco #31053028) supplemented with 25 mM HEPES , 1 mM sodium 562 \npyruvate, and 4 mM GlutaMAX . Plates were imaged on a Zeiss CellDiscoverer 7 microscope (10x 563 \nmagnification) using two fluorescence channels to detect the XBP1s-mApple reporter (Ex/Em: 570/594 564 \nnm) and Hoechst-stained nuclei (Ex/Em: 348/455 nm). The images were subsequently analysed with 565 \nCellProfiler 4.2.1 to quantify the percentage of UPR-positive (mApple-expressing) cells in each well. 566 \n 567 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n19 \nConstruction of in-frame deletion mutants in E. faecalis 568 \nGeneral molecular biology reagents were sourced as follows: genomic DNA from E. faecalis  was 569 \nisolated using the Wizard Genomic DNA Purification Kit (Promega  #A1120), while plasmid DNA was 570 \nisolated from E. coli using the PureLink Plasmid Miniprep Kit (Invitrogen #K210011). All primers used 571 \nin this study (Table S6) were designed based on the E. faecalis OG1RF genome (NC_017316). Gene 572 \nfragments were amplified with Phusion High-Fidelity DNA Polymerase (Thermo Scientific #F530), and 573 \nroutine screening was performed with Taq DNA Polymerase (NEB  #M0273). T4 DNA ligase and all 574 \nrestriction enzymes used according to the manufacturer's protocols  (NEB). In-frame deletion mutants 575 \nwere generated using the temperature -sensitive shuttle vector pGCP213  (Table S5 ), following a 576 \npreviously described protocol79. Deletion constructs were created using two main strategies. For most 577 \nsingle genes and smaller operons, regions of approximately 450 bp flanking the target were amplified 578 \nfrom OG1RF gDNA; the upstream region was amplified with primer pair P1/P2 and the downstream 579 \nregion with P3/P4. These fragments were then fused by overlap extension PCR using the outer primers 580 \nP1 and P4 and subsequently cloned into the PstI site of pGCP213. For the larger ΔEET and ΔT7SS 581 \noperons, a gBlock Gene Fragment (IDT) containing the fused upstream and downstream flanking 582 \nregions was synthesized and cloned into the vector. The resulting deletion constructs were transformed 583 \ninto the appropriate E. faecalis parent strain by electroporation. Transformants were first selected on 584 \nBHI-erythromycin agar at the permissive temperature of 30°C. To promote chromosomal integration, 585 \ncolonies were then passaged at the non-permissive temperature of 42°C with erythromycin selection. 586 \nCuring of the integrated plasmid was achieved by passaging the bacteria at 30°C in antibiotic-free BHI. 587 \nFinally, the successful deletion of the target gene or operon was verified by colony PCR using external 588 \n(Screen F/R) and internal (Intern F/R or Intern R) primer pairs (Table S6). 589 \n 590 \nXBP1s fluorescent reporter assay 591 \nFollowing infection or treatment, 3T3R cells were stained with Hoechst 33342 (2.5 µg/ml) and 592 \ntransferred to a phenol-red-free imaging medium. For each well, a 3x3 grid of images was acquired on 593 \na Zeiss CellDiscoverer 7 microscope. The intensity of the XBP1s -mApple signal within each nucleus 594 \n(identified by Hoechst staining) was quantified using a CellProfiler pipeline, and the average intensity 595 \nper well was used to gauge the level of UPR induction. 596 \n 597 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n20 \nGrowth curve assay 598 \nTo assess bacterial growth, overnight bacterial cultures were processed as described in bacterial strains 599 \nand growth conditions. After pelleting, they were normalised to a starting OD600 of 0.05 in phenol red 600 \nfree complete media  on a 96 -well plate which was sealed with a Breathe -Easy sealing membrane 601 \n(Sigma-Aldrich #Z380059) following manufacturer’s protocols . OD600 readings were taken at 10-min 602 \nintervals over 20 h. The rate of change of the OD600 readings was calculated at 50 -min intervals and 603 \nthe highest rate of change was used to calculate the doubling time.  604 \n 605 \nAntibiotic time-kill assay 606 \nTo assess antibiotic killing, the supernatant of infected HaCaT cells were collected at 4, 5, 6, and 24 607 \nhpi. These were serially diluted on 96-well plates and 5 µl of the dilutions were spotted onto BHI-Agar 608 \nplates which were incubated at 37°C for 24 hours. Plates were imaged using a ProtoCOL3 Plus system 609 \n(Don Whitely Scientific) and bacterial colonies were manually enumerated on ImageJ using the multi -610 \npoint tool. 611 \n 612 \nCytotoxicity assay 613 \nTo quantify total cytotoxicity, both detached (floating in the medium) and attached cells were collected 614 \nand analyzed from infected HaCaT cell cultures. First, to collect the detached cell fraction, the culture 615 \nmedium was harvested, and each well was washed once with 1 ml of PBS. This wash was pooled with 616 \nthe collected medium, and the mixture was centrifuged (300 x g, 5 min). The resulting cell pellet was 617 \ncarefully resuspended in 20 µl of complete medium.  Next, to collect the attached cell fraction, the 618 \nremaining cells in the well were trypsinised for 15 min, neutralized with complete medium, pelleted by 619 \ncentrifugation, and resuspended in 500 µl of fresh complete medium. The viability of both the detached 620 \nand attached cell suspensions was determined separately using a Countess 3 Automated Cell Counter 621 \nwith trypan blue staining. Total cytotoxicity was then calculated by summing the number of non-viable 622 \ncells from both fractions and dividing by the total number of cells (viable and non -viable) from both 623 \nfractions (Equation 1). 624 \nCytotoxicity = \nNo. of dead cells (detached + adhered)\nNo. of cells (detached + adhered)  × 100% Eq (1) 625 \n 626 \n 627 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n21 \nSuperoxide assay 628 \nExtracellular superoxide generation was measured by adapting a previously published cytochrome C 629 \nreduction assay for a 96-well plate format18. Briefly, E. faecalis cultures were normalized to an an OD600 630 \nof 0.005 in 200 µl of phenol red free complete media containing 20 µM of cytochrome C (Sigma-Aldrich 631 \n#C3131). The reduction of cytochrome C was measured as the change in absorbance at 550 nm (with 632 \n650 nm as the reference wavelength) every 2 minutes for 90 minutes at 37°C using a Tecan M200 633 \nmicroplate reader. The rate of superoxide generation was calculated from the maximal rate of change 634 \nin absorbance, after correcting for pathlength. This rate was determined using Beer's Law with an 635 \nextinction coefficient of 21.5 mM ⁻¹cm⁻¹ for reduced cytochrome C  (Equation 2). To determine the 636 \namount of superoxide specifically, the rate measured in a parallel reaction containing 100 units/ml of 637 \nsuperoxide dismutase (SOD; Sigma -Aldrich #S5395) was subtracted from the rate measured in its 638 \nabsence. 639 \nRate of superoxide generation (nmol per minute per\t109CFU) =\t\n∆MAXOD550\n21.5 × 1 ×\n1×109 CFU\n2×107 CFU\n×\n60\n600 ×\n109\n106 Eq (2) 640 \n 641 \nLipid peroxidation assay 642 \nLipid peroxidation was assessed  in HaCaT cells  using the Image-iT Lipid Peroxidation Kit (Thermo 643 \nFisher #C10445) at 24 hpi following manufactu rer’s protocols. Imaging was performed on a Zeiss 644 \nCellDiscoverer 7 microscope using two fluorescence channels to detect fluorescence from reduced 645 \n(Ex/Em: 592/614 nm) and oxidised (Ex/Em: 495/519 nm) BODIPY 581/591 C11. The mean intensity of 646 \nthe reduced and oxidised fluorescent reporters was quantified using a CellProfiler pipeline , with lipid 647 \nperoxidation calculated based on the ratio of reduced:oxidised signals for each condition. 648 \n 649 \nStatistics 650 \nStatistical analyses were performed using GraphPad Prism 9 and 10. In bar-dot plots, dots represent 651 \nindividual replicates, and the bar height indicates the median. Statistical significance was determined 652 \nusing either a one-way ANOVA with Dunnett’s multiple comparisons test or, for scratch wound assays, 653 \na two-way ANOVA with Tukey’s multiple comparisons test. An adjusted p-value < 0.05 was considered 654 \nsignificant. Unless otherwise stated in the figure legends, all experiments were performed with a 655 \nminimum of three independent replicates. 656 \n 657 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n22 \nSUPPLEMENTAL INFORMATION 658 \nDocument S1. Figures S1-S4, Tables S2-S6 659 \nTable S1. Stress Response Gene Set. Related to Figure 1. Excel Spreadsheet. 660 \nVideo S1. E. faecalis infection and tunicamycin treatment impair HaCaT cell migration. Related 661 \nto Figure 2E. 662 \nVideo S2.  IRE1 inhibition alters HaCaT cell migration in uninfected and E. faecalis -infected 663 \nconditions. Related to Figure 2G. 664 \nVideo S3. The ΔEET mutant does not impair HaCaT cell migration. Related to Figure 3E. 665 \nVideo S4. The effect of IRE1 inhibition on HaCaT cell migration during infection with WT or ΔEET 666 \nE. faecalis. Related to Figure 3F. 667 \nVideo S5. Catalase rescues H2O2-induced migration defects in uninfected HaCaT cells. Related 668 \nto Figure 4K. 669 \nVideo S6. Catalase restores migration in E. faecalis-infected HaCaT cells. Related to Figure 4L. 670 \n 671 \nFIGURES 672 \n 673 \nFigure 1. E. faecalis infection activates the UPR in a mouse model 674 \n(A) Uniform manifold approximation and projection (UMAP) of ~23,000 single-cell transcriptomes from 675 \nuninfected and E. faecalis-infected wounds22, recoloured here into the six broad cell classes used for 676 \ndownstream stress-response analyses. (B) Per-cell enrichment score for a curated unfolded protein 677 \nresponse (UPR) gene set projected onto the UMAP in ( A). (C) Same UPR enrichment as in ( B) but 678 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n23 \ndisplayed only for keratinocyte clusters.  (D) Enrichment of an oxidative  stress response (OSR) gene 679 \nset plotted for fibroblast clusters. (E) Schematic representation of the UPR in mice, where spliced Xbp1 680 \n(Xbp1s), Chop, and Herpud1 serve as downstream markers of the IRE1, PERK, and ATF6 pathways, 681 \nrespectively. (F) Gene expression of UPR markers (Xbp1s, Chop, and Herpud1) at 6 dpi for uninfected 682 \nand E. faecalis -infected 6 –7 week-old C57BL/6J mouse skin wounds, normalized to intact skin . 683 \nSignificance was determined using one-way ANOVA, Dunnett’s test (unwounded skin, n = 16; wounded, 684 \nuninfected, n = 20; wounded, infected n = 33; ***p < 0 .001, ****p < 0.0001). 685 \n 686 \nFigure 2. IRE1 activation by E. faecalis impedes keratinocyte migration in vitro 687 \n(A-B) Gene expression of UPR markers (Xbp1s, Chop, and Herpud1) in infected with E. faecalis at MOI 688 \nof 800 (A) or 600 (B)  or tunicamycin (Tm) treated NIH-3T3 mouse fibroblasts (A) or HaCaT human 689 \nkeratinocytes (B) (n = 3). See Methods for MOI optimization description. (C) Gene expression of IRE1 690 \ndownstream gene ( EDEM1) in cells treated as in (A) ( n = 3). (D) Quantitative analysis and 691 \nrepresentative immunoblots showing levels of XBP1 s and BiP in HaCaT cells treated as in (A). (E) 692 \nScratch wound assay quantification for uninfected, infected and Tm-treated cells (positive control) (n = 693 \n4 biological replicates). (F) Gene expression of XBP1s from HaCaT cells treated with the DMSO control 694 \n(open circles) or the IRE1 inhibitor (IRE1i) 4µ8c (closed circles) (n = 3, one-way ANOVA, Dunnett’s test) 695 \n(G) Scratch wound assay quantification for uninfected and infected cells treated  with 0.5% DMSO 696 \ncontrol (open circles) or IRE1i (close circles) (n = 4 biological replicates). Significance was determined 697 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n24 \nusing one-way ANOVA Dunnett’s test (A, B, and F) or two-way ANOVA Tukey’s test (E and G) (*p < 698 \n0.05, **p < 0.01, ***p < 0 .001, ****p < 0.0001). 699 \n 700 \nFigure 3. Extracellular electron transport drives E. faecalis UPR activation and migration arrest 701 \n(A) E. faecalis OG1RF transposon screen in which a library of 14976 mutants was screened against a 702 \nNIH-3T3 cell line expressing the Xbp1 -mApple reporter system (3T3R ). (B) Representative 703 \nepifluorescnece microscopy images of 3T3R under different conditions (uninfected,  WT, and Tm-704 \ntreated) at 21 hpi. Scale bar, 100 μ m. (C) Diagram showing the pathways in which a subset of UPR 705 \ndefective mutants (genes/proteins with red font) were identified. (D) Validation of UPR defective mutants 706 \nwith 3T3R (n = 3). (E-F) Scratch wound assay quantification for HaCaT infected with (E) WT and ΔEET, 707 \nwhich were also (F) treated with either 0.5% DMSO control (open circles) or IRE1i (close circles).  708 \nUninfected and WT data are identical to Figure 2E and 2F and replicated here for ease of comparison 709 \n(n = 4 biological replicates). (G) UPR induction at 24 hpi in HaCaT after treatment with 0.5% DMSO 710 \ncontrol (open circles) or IRE1i (closed circles) under uninfected, WT -infected, and ΔEET -infected 711 \nconditions. Uninfected and WT-infected findings are identical to Figure 2F and replicated here for ease 712 \nof comparison (n = 3). Significance was determined using one-way ANOVA Dunnett’s test (D and G) or 713 \ntwo-way ANOVA Tukey’s test (E and F) (***p < 0 .001, ****p < 0.0001). 714 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n25 \n 715 \nFigure 4. EET-derived ROS is sufficient to activate the UPR, disrupting epithelial migration 716 \n(A) Diagram showing the dismutation of superoxide radical (O2•–) into hydrogen peroxide (H2O2) via the 717 \ncatalytic activity of superoxide dismutase (SOD), or spontaneously via a pH-dependent process. H2O2 718 \ncan be converted into a highly reactive hydroxyl radical in the presence of transition metals like ferrous 719 \nions (Fe2+) via the Fenton reaction or neutralised into water and oxygen if catalase is present. (B) Dose-720 \ndependent H2O2 mediated UPR induction in 3T3R (n = 3). (C) In-frame deletion mutants corresponding 721 \nto the Tn mutants of the EET and DMK synthesis pathways which were identified as UPR defective hits 722 \nduring the transposon screen. The ΔMEN mutant had its entire operon deleted, which contained the 723 \nmenF/D/C/E/B genes (n = 3). (D-F) UPR induction in 3T3(R) for (D) uninfected (E) WT infection, and 724 \n(F) ΔEET infection, in the presence (closed circles) or absence (open circles) of  H2O2, SOD, and 725 \ncatalase (n = 4). (G-I) UPR induction in uninfected (G), WT infected (H), and ΔEET infected (I) HaCaT 726 \ncells in the presence (closed circles) or absence (open circles) of H 2O2 and catalase (n ≥ 3). (J) Lipid 727 \noxidation in uninfected, WT infected, and ΔEET infected HaCaT cells using BODIPY 581/591 C11 (n = 728 \n3). (K-L) Scratch wound assay quantification in uninfected (K) or WT infected (L) HaCaT cells treated 729 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n26 \nwith catalase and/or H2O2 (n = 3 biological replicates). Significance was determined using one -way 730 \nANOVA Dunnett’s test (B-J) or two-way ordinary ANOVA, Tukey’s multiple comparisons test (K-L) (*p 731 \n< 0.05, **p < 0.01, ***p < 0 .001, ****p < 0.0001).  732 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 8, 2025. ; https://doi.org/10.1101/2025.08.07.669218doi: bioRxiv preprint \n\n27 \nREFERENCES 733 \n1. Chng, J.-H., L Chong, K.K., Ning Lam, L., Jie Wong, J., and Kline, K.A. Biofilm-associated infection 734 \nby enterococci. https://doi.org/10.1038/s41579-018-0107-z. 735 \n2. 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