LPxTG Proteins Mediate Microbial Interactions to Modulate Lactiplantibacillus plantarum C8 in Alleviating Antibiotic-Associated Diarrhea

LPxTG Proteins Mediate Microbial Interactions to Modulate Lactiplantibacillus plantarum C8 in Alleviating Antibiotic-Associated Diarrhea | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article LPxTG Proteins Mediate Microbial Interactions to Modulate Lactiplantibacillus plantarum C8 in Alleviating Antibiotic-Associated Diarrhea Jiaying Liang, Jiang Liu, Ao Zhang, Shuolei Zheng, Ting Jiang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8618174/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract To address the challenges of intestinal dysbiosis and antibiotic-associated diarrhea (AAD) caused by antibiotic treatment, this study investigated the potential of an LPxTG surface protein overexpression strategy for probiotic modulation. Specifically, we established a co-culture model comprising a recombinant Lactiplantibacillus plantarum C8 strain (overexpressing LPxTG proteins) and the wild-type strain, and evaluated its efficacy in alleviating clindamycin-induced AAD in mice. Our findings indicate that the overexpression of LPxTG proteins enhanced the adhesive colonization capabilities and anti-inflammatory properties of the strain. Crucially, this co-culture model demonstrated significantly superior efficacy in regulating the intestinal microecology compared to single-strain models. Experimental data revealed that the co-culture system not only effectively restored the abundance and diversity of the compromised gut microbiota ( p < 0.05) but also exerted protective effects via a dual mechanism: it significantly improved the immune microenvironment (reducing IL-6 and TNF-α while elevating IL-10) and repaired the intestinal physical barrier by upregulating Occludin and Claudin-1. These results confirm that constructing a co-culture microecological system utilizing LPxTG-overexpressing recombinant strains represents a highly efficient and superior probiotic therapeutic strategy for managing antibiotic-induced intestinal dysbiosis. antibiotic-associated diarrhea (AAD) Lactiplantibacillus plantarum C8 LPxTG protein gut microbiome co-culture system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Antibiotic-associated diarrhea (AAD) is a common adverse effect during antimicrobial therapy [ 13 ]. Its occurrence is not only related to the abnormal overgrowth of pathogenic microorganisms but is also closely associated with disruption of gut microbiota composition and imbalance of mucosal immune homeostasis [ 40 ]. Recent studies demonstrate that antibiotic-induced gut microbiota alterations correlate with immune dysfunction and various diseases, including diabetes, obesity, and cardiovascular disorders. Notably, antibiotic-mediated disruption of the gut microbiota may persist for months or even years after treatment, thereby increasing susceptibility to diseases[ 10 ]. Therefore, it is necessary to explore better approaches to prevent or mitigate the adverse effects of antibiotics on the gut microbiota. In recent years, LAB have attracted increasing research interest due to their long history of safe use and demonstrated therapeutic benefits for human health[ 3 ]. L. plantarum possesses multiple probiotic characteristics, including the ability to ferment a wide spectrum of plant-derived carbohydrates, achieve high-density growth, tolerate bile salts and low pH conditions, and exert antagonistic effects against intestinal pathogens [ 36 ]. As live microorganisms, probiotics can modulate gut microbiota composition and correct aberrant mucosal immune responses associated with chronic intestinal inflammation. Furthermore, they enhance intestinal barrier function through multiple mechanisms: regulating cytokine production, stimulating regulatory T cell (Treg) release, and promoting intestinal epithelial cell survival [ 42 ]. However, the efficacy of probiotics in AAD intervention varies markedly among individuals, largely due to insufficient intestinal adhesion and unstable functional performance of probiotic strains in vivo. Proteins containing the LPxTG motif are a class of adhesion-related surface proteins predominantly anchored to Lactiplantibacillus plantarum [ 4 ]. These proteins enhance interactions with intestinal epithelial cells, thereby promoting stable gut colonization, and their expression is regulated by quorum sensing, contributing to colonization capacity and functional persistence in the intestinal environment [ 18 ] [ 21 ]. LPxTG-containing surface proteins have been reported to improve resistance to gastrointestinal stresses, enhancing bacterial survival and colonization stability [ 19 ]. Moreover, studies in Listeria monocytogenes indicate that LPxTG-anchored proteins are involved in environmental stress adaptation and host cell adhesion, supporting the functional conservation of this protein family in microbe–host interactions [ 29 ]. In the context of antibiotic-induced dysbiosis, ecological niches within the gut microbiota become highly unstable, and single-strain interventions often fail to achieve sustained colonization and functional recovery. Emerging evidence suggests that probiotics derived from co-cultivation strategies may exhibit improved adaptability and activity in the gastrointestinal environment compared with those obtained from monoculture [ 20 ]. We therefore hypothesize that cooperative colonization between wild-type and engineered strains may confer survival and functional advantages in alleviating antibiotic-associated diarrhea and promoting gut microbiota restoration. LPxTG-anchored surface proteins may act as structural or adhesive factors that facilitate such inter-bacterial cooperative interactions, thereby enhancing collective persistence and functional stability within the intestinal ecosystem. Materials and methods Preparation of Strains The L. plantarum C8 (CGMCC NO.30504) strain used in this study was independently isolated by our laboratory and is currently preserved at the China General Microbiological Culture Collection Center. In this study, signal peptides were identified from the proteome of L. plantarum C8 using the NCBI and UniProt databases. Subsequently, the LPxTG motif protein gene and the signal peptide gene were cloned into the pMG36e plasmid vector through homologous recombination to construct recombinant strains. This construct was first transformed into chemically competent BL21 cells for plasmid amplification and then electroporated into chemically competent L. plantarum C8 cells. L. plantarum C8 was inoculated at 1% into 100 mL of MRS liquid medium, while the empty vector strain and the recombinant strain were inoculated at 1% into 100 mL of MRS liquid medium containing 20 µg/mL erythromycin. The cultures were incubated statically in a 37°C constant temperature and humidity incubator for 10–12 hours and activated to the third generation. Then, at the same 1% inoculation rate, the strains were inoculated under co-culture conditions into a new 100 mL MRS broth and continued to be cultured at 37°C for 12 hours. The cultured bacterial suspension was centrifuged to remove the supernatant, washed three times with sterile PBS, and then diluted with sterile physiological saline to an OD600 nm = 1.00 ± 0.05, ready for immediate use. Construction of Animal Experimental Model SPF-grade male KM mice (approximately 6 weeks old), weighing 25 ± 2 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. A total of 56 mice (6 weeks old, n = 8 per group) were acclimatized for 7 days and then randomly assigned to a control group (n = 8) and a model group (n = 48). The model group received 300 mg/mL lincomycin hydrochloride via oral gavage twice daily for 3 consecutive days to induce intestinal dysbiosis, and was subsequently divided into six treatment subgroups: natural recovery group, L. plantarum C8 intervention group, blank strain intervention group, recombinant strain intervention group, L. plantarum C8 + blank strain co-culture group, and L. plantarum C8 + recombinant strain co-culture group ( Table 1 ) . Throughout the 25-day experiment, all mice had ad libitum access to standard diet and water, with the blank and natural recovery groups receiving saline (0.2 mL/10 g body weight) while intervention groups received equivalent volumes of their respective bacterial suspensions. Daily body weight measurements and fecal sample collections were performed to monitor physiological changes. Following an overnight fast on day 24, all mice were humanely euthanized by cervical dislocation on day 25, at which point serum samples were collected and colon, liver, and spleen tissues were excised and weighed. All experimental procedures were conducted in strict accordance with the guidelines of Ningbo University Animal Experiment Center and were approved by the University's Animal Ethics Committee (Approval No.: NBU20240314). Table 1 Mice group in the experiment No. Group Diet Gavage Volume per Day 1 Blank Control Group (8 males) Regular feed + water 0.2 mL/10 g physiological saline 2 Natural Recovery Group (8 males) Regular feed + water 0.2 mL/10 g physiological saline 3 L. plantarum C8 Intervention Group (8 males) Regular feed + water 0.2 mL/10 g bacterial suspension 4 Empty Vector Strain Intervention Group (8 males) Regular feed + water 0.2 mL/10 g bacterial suspension 5 Recombinant Strain Intervention Group (8 males) Regular feed + water 0.2 mL/10 g bacterial suspension 6 Co-culture Intervention Group ( L. plantarum C8 + Empty Vector Strain) (8 males) Regular feed + water 0.2 mL/10 g bacterial suspension 7 Co-culture Intervention Group ( L. plantarum C8 + Recombinant Strain) (8 males) Regular feed + water 0.2 mL/10 g bacterial suspension Physiological Parameter Monitoring and Organ Collection in Mice During the experimental period, daily body weight measurements were conducted on all mice. At the conclusion of the recovery phase, fecal samples (4–5 pellets per mouse) were aseptically collected into sterile 1.5 mL microcentrifuge tubes and immediately stored at -80 ℃ for subsequent analysis. Blood samples were obtained through retro-orbital puncture under anesthesia, allowed to clot at room temperature for 30 minutes, then centrifuged at 12,000 × g for 10 minutes at 4 ℃ to separate serum. Following euthanasia, a complete necropsy was performed which included measurement of colon length prior to fixation in 4% paraformaldehyde, precise weighing of liver and spleen tissues, and snap-freezing of all collected organs in liquid nitrogen for preservation at -80 ℃. All procedures were conducted in accordance with standard laboratory protocols to ensure sample integrity. Histomorphological Observation Following euthanasia, the target intestinal segments were promptly excised and thoroughly rinsed with PBS before fixation in 4% paraformaldehyde for 24–48 hours. The samples subsequently underwent ethanol gradient dehydration and xylene clearing, followed by paraffin infiltration, embedding, and sectioning (4–7 µm). Sections were expanded in 45 ℃ warm water and baked at 37 ℃ for 2 hours. Deparaffinization was performed using xylene I and II (10 minutes each), followed by gradient ethanol dehydration (100%, 95%, 85%, and 75%; 5 minutes each). Hematoxylin staining was conducted for 8–10 minutes, differentiated with 1% acid alcohol for 3–5 seconds, and blued in running water for 15 minutes. Eosin staining lasted 1–2 minutes, followed by rapid dehydration with 95% and 100% ethanol, xylene clearing for 5 minutes, and final mounting with neutral balsam. Cytokine Measurement The levels of IL-6, IL-10 and TNF-α in mouse serum were detected by ELISA (Thermo Fisher Scientific). The 96-well plate was firstly coated with capture antibody overnight at 4 ℃, washed and then closed with 1% BSA for 1 hour. Sequentially, standards or samples to be tested were added and incubated for 2 hours. After washing, detection antibodies were added and incubated for 2 hours. Then the plates were reacted with HRP-labeled secondary antibody for 20 minutes avoiding light. Finally, TMB substrate was added to develop the color for 20 minutes, and the absorbance value at 450 nm was measured after terminating the reaction to calculate the cytokine concentration. Immunohistochemical Tissue sections were dewaxed in xylene (2 × 20 minutes), dehydrated through an ethanol series (100% − 75%), and subjected to antigen retrieval in citrate buffer (pH 6.0) using microwave heating. After peroxidase blocking (3% H₂O₂, 15 min), sections were incubated with primary antibody (4 ℃ overnight) and HRP-conjugated secondary antibody (20 minutes RT), with PBS washes between steps. Following DAB development (1–5 minutes) and hematoxylin counterstaining, sections were dehydrated (70% − 100% ethanol), cleared in xylene, and mounted with rhamsan gum. Gut Microbiota Analysis Total genomic DNA from the mouse fecal microbial community was extracted using the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China) according to the manufacturer’s instructions. The integrity of the extracted DNA was verified by 1% agarose gel electrophoresis, and its concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The V3-V4 hypervariable regions of the 16S rRNA gene were amplified by PCR using barcoded primers and the extracted DNA as a template. Amplification was performed under the following conditions: initial denaturation at 95°C for 3 minutes; 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; followed by a final extension at 72°C for 10 minutes. Statistical Analysis All experimental data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS Statistics 19.1 (IBM Corp., Armonk, NY, USA). For multiple comparisons among treatment groups, one-way ANOVA followed by Duncan's post hoc test was performed, with statistical significance set at p < 0.05. Results and discussion Weighing Index of Mice No mortality was observed throughout the animal experiments. During the 0–7 day adaptation phase, mice in all groups exhibited a stable increase in body weight. In contrast, during the modeling period, treatment with lincomycin hydrochloride led to a gradual decrease in body weight in the experimental group, whereas the blank control group continued to gain weight steadily (Fig. 1A). During the recovery phase, mice in the natural recovery group showed significantly lower body weight gain than those receiving bacterial interventions, indicating that L. plantarum C8 effectively promoted weight recovery. This difference highlights the negative impact of antibiotics on body weight, likely resulting from LCM-induced disruption of the gut microbiota. Among the intervention groups, treatment with wild-type L. plantarum C8 resulted in improved weight recovery, while the co-culture group of the recombinant strain and L. plantarum C8 exhibited the most pronounced effect. This enhanced recovery is likely attributable to rapid intestinal colonization mediated by LPxTG protein–dependent adhesion of the recombinant strain. Consistent with this, previous studies have shown that surface display of functional proteins via engineered signal peptides and LPxTG motifs significantly enhances bacterial adhesion and persistence in the gut [ 43 ]. In contrast, the saline control group displayed delayed recovery, underscoring the limited capacity of spontaneous microbiota reconstitution after antibiotic withdrawal. Collectively, these results suggest that antibiotic-associated weight loss is closely linked to impaired microbiota function and that engineered probiotics expressing LPxTG motifs can effectively mitigate this adverse effect. Length of Colon in Mice Related studies have demonstrated that intestinal dysbiosis is a key factor driving inflammatory responses in the colon, with colon shortening being a significant symptom of colonic inflammation. Measurements of colon length revealed a marked reduction in the natural recovery group (5.43 ± 0.86 cm), a phenomenon likely linked to antibiotic-induced gut microbiota dysbiosis (Fig. 1B and 1C) . Notably, all probiotic-treated groups—especially those supplemented with L. plantarum C8, the recombinant strain, or their co-culture preparation—exhibited restored colon lengths (7.06–8.37 cm) that approximated or exceeded the blank control values (7.26 ± 0.63 cm). The restoration of colonic architecture not only ensures adequate nutrient absorption to support weight recovery but also reduces hepatic metabolic burden by maintaining intestinal barrier integrity and minimizing endotoxin translocation. Furthermore, the re-established colonic microbial homeostasis appears to modulate systemic immune responses, which explains the subsequent normalization of the spleen index observed in probiotic-treated mice. Notably, the co-culture group of L. plantarum C8 and recombinant strains demonstrated the most pronounced effects, suggesting that specific strain combinations may synergistically repair antibiotic-induced multisystem damage more effectively, providing valuable insights for clinical interventions. These findings are consistent with previous studies showing that oral administration of L. plantarum 12 can ameliorate colitis in DSS-induced mouse models [ 31 ]. Liver and Spleen Index in Mice The liver index of mice in the natural recovery group, which were gavaged with saline, was significantly elevated (> 5%), exceeding the normal range (Fig. 1D). This indicates an increased hepatic metabolic burden and inflammatory response during recovery. In contrast, all probiotic intervention groups exhibited reduced liver indices, suggesting effective alleviation of liver metabolic stress and inflammation. Notably, treatment with the wild-type strain, recombinant strain, or their co-culture reduced the liver index to levels comparable to those of the blank control group ( p < 0.05), reflecting improved hepatic metabolic function. However, strains carrying empty plasmids and their co-culture groups showed relatively weaker recovery effects in both liver and spleen indices. This may be attributed to the additional metabolic burden imposed by plasmid maintenance, which can impair bacterial growth and probiotic efficacy [ 26 ]. Previous studies have demonstrated that L. plantarum strains can enhance Nrf2-mediated antioxidant responses and alleviate liver injury [ 22 ]. Accordingly, the hepatoprotective effects of L. plantarum C8 and its co-cultured strains observed in this study may similarly involve activation of the Nrf2 signaling pathway. Furthermore, the natural recovery group exhibited significantly elevated spleen indices (Fig. 1E) , indicative of antibiotic-induced immune dysregulation. In contrast, the recombinant strain and co-culture groups demonstrated optimal spleen recovery, highlighting the superior immunomodulatory capacity conferred by their LPxTG-anchored proteins and signal peptides. These findings are consistent with previous work by Assad Moon et al., who developed recombinant lactobacilli expressing the PRV gD antigen via an LPxTG (LP3065) anchoring system. Their study showed enhanced CD4 + T cell populations in murine splenocytes, thereby promoting helper T cell activation and a more robust immune response [ 18 ]. These results suggest that the introduction of LPxTG-anchored proteins and signal peptides in the recombinant strain not only enhances its intestinal colonization ability but may also regulate liver metabolic function, alleviate antibiotic-induced metabolic disorders, and restore immune homeostasis by modulating the balance of T cell subsets. Although the wild-type L. plantarum itself demonstrates certain probiotic functions, such as alleviating antibiotic-induced weight loss and abnormalities in intestinal, liver, and spleen indices, the co-culture group of the engineered strain overexpressing LPxTG demonstrated superior effects. The recombinant single strain produced effects similar to those of the wild-type, whereas the co-culture group of the engineered strain overexpressing LPxTG showed enhanced results. This suggests that the efficacy of a single strain may be limited by its individual capabilities, while the co-culture group, through increased microbial interactions, exerts a stronger overall health benefit. Figure 1 Physiological indicators of mice.Changes in average daily body weight ( A ); representative photograph of colons from mice ( B ); quantitative analysis of colon length (cm) ( C ); relativate liver weight ( D ) and relative spleen weight ( E ). Different lowercase letters above bars denote significant differences at p < 0.05. Colonic Histological Morphology To better understand the effects of antibiotics on the colon, we conducted histological analyses. Hematoxylin and eosin (H&E) staining revealed intact colonic architecture, clear intestinal walls, and evenly distributed goblet cells in the blank control group, with no evident inflammatory infiltration ( Fig. 2 ) . In contrast, the natural recovery group exhibited disrupted intestinal villi, abnormal goblet cell morphology, and substantial inflammatory cell infiltration, indicating significant antibiotic-induced intestinal damage. All intervention groups showed varying degrees of histological improvement, with the co-culture group demonstrating the most remarkable restoration—its tissue structure closely resembled normal, with almost no pathological alterations. This demonstrated that the engineered bacteria could alleviate histopathological damage in the intestinal, liver, and spleen tissues, with the co-cultivation group showing the most pronounced effect. Inflammation is a fundamental defensive response. The severe inflammatory infiltration observed in the natural recovery group likely reflects sustained pro-inflammatory activity, whereas the therapeutic effects seen in the intervention groups—particularly the co-culture group—may result from probiotic-mediated modulation of inflammatory pathways [ 11 ]. Notably, the superior efficacy of the co-culture group underscores a synergistic interaction between strains, which may optimize microbiota reconstitution and strengthen host defense mechanisms, thereby providing enhanced protection against antibiotic-induced tissue injury. Effect of Strain C8 on Serum Inflammatory Cytokines Cytokine analysis revealed that antibiotic treatment induced a strong pro-inflammatory response in the colon of mice undergoing natural recovery. Compared with the blank control group, IL-6 and TNF-α levels increased significantly by 98.34% and 83.99%, respectively ( p < 0.05), while IL-10 levels decreased by 73.43% ( p < 0.05) (Fig. 3 A–C). All groups treated with L. plantarum C8 exhibited reduced inflammation. IL-6 and TNF-α levels were lowered to 164.17/156.08 pg/mg in the monotherapy group, 175.08/169.64 pg/mg in the empty vector group, and 153.50/146.57 pg/mg in the recombinant strain group. Notably, combined treatment with L. plantarum C8 and the recombinant strain produced the most pronounced effect, further reducing IL-6 and TNF-α to 132.00 and 131.83 pg/mg, respectively, while increasing IL-10 to 226.47 pg/mg—the highest level among all intervention groups. These findings suggest that L. plantarum C8 alleviates antibiotic-induced intestinal inflammation by modulating immune responses, consistent with histopathological observations. The notably enhanced efficacy of the combined strain intervention highlights the potential therapeutic advantage of multi-strain probiotic formulations in mitigating antibiotic-induced intestinal inflammation [ 35 ]. Research has demonstrated that administering L. plantarum to cyclophosphamide-induced immunosuppressed mice produces beneficial regulatory effects on immunity, gut microbiota composition, and jejunal inflammatory cytokines [ 41 , 28 ]. Furthermore, the LPxTG protein may contribute to inflammation alleviation through specific mechanisms. Compared to other intervention groups, L. plantarum C8 carrying the recombinant plasmid and the co-culture group of L. plantarum C8 with the wild-type strain exhibit superior regulation of inflammatory factors. Previous studies have shown that an LPxTG motif protein from Lactiplantibacillus reuteri SH23 alleviated DSS-induced colitis by suppressing MAPK/NF-κB signaling, reducing TNF-α and IL-6 levels while increasing IL-10, and reshaping gut microbiota composition—particularly by increasing Lactiplantibacillus and Akkermansia [ 44 ]. These findings support the hypothesis that the LPxTG protein in our current study may operate via analogous pathways to modulate inflammatory cytokine profiles and subsequently improve gut microbiota composition, potentially explaining the observed therapeutic effects of our recombinant strain. The consistent outcomes across these independent studies reinforce the concept that LPxTG-containing surface proteins represent a promising therapeutic strategy for inflammation-related gut disorders through their dual action on both host immune responses and microbial ecosystems. Immunohistochemical Analysis Probiotic interventions demonstrated critical protective effects on intestinal barrier integrity. Immunohistochemical analysis revealed that antibiotic treatment severely compromised the gut barrier in the natural recovery group ( Fig. 4 A and C) , significantly reducing the expression of ZO-1 and Occludin to only 14.77% and 15.42% of the blank control levels, respectively. This disruption of tight junction proteins, which are essential for maintaining paracellular permeability, is a well-established mechanism by which certain pathogens impair the gut barrier [ 7 ]. All probiotic intervention groups significantly upregulated the expression of these key proteins ( p < 0.05), with the co-culture group of L. plantarum C8 and the recombinant strain showing the most pronounced restorative effects—achieving 31.54% and 31.33% recovery of ZO-1 and Occludin expression, respectively, representing approximately a 2.1-fold improvement over the natural recovery group ( p < 0.05). Notably, the co-culture group demonstrated significantly superior restorative effects compared to single-strain interventions (22.99% − 28.11%, p < 0.05) ( Fig. 4 B and D) . The protein expression levels of Occludin and ZO-1 followed the same trend, indicating that the recombinant strain can better induce quorum sensing effects between L. plantarum C8, thereby promoting communication within the microbiota and more effectively repairing the intestinal barrier. This conclusion is supported by HE staining results, which revealed the mildest pathological damage in colonic tissues and the most intact crypt and villus structures in the co-culture group. This synergistic effect may originate from enhanced interaction efficiency between strains via quorum sensing, coupled with the coordinated regulation of tight junction protein expression in epithelial cells by microbial metabolites. These molecular findings corroborate our previous cytokine results, providing mechanistic insights into the superior anti-inflammatory efficacy of the co-culture formulation. Supporting evidence comes from Jiang et al., who demonstrated that L. plantarum HM-22 significantly enhanced Occludin and Claudin-1 expression in α-LA-induced allergic mice, thereby reducing intestinal permeability and improving barrier function. Furthermore, NF-κB and MAPK signaling pathways are known to mediate microbial stimulus-induced proinflammatory cytokine production, while LPxTG-anchored or sortase-dependent proteins (SDPs), which are covalently attached to peptidoglycan, play pivotal roles in Lactiplantibacillus -host interactions [ 6 ]. Similarly, Wang et al. demonstrated that L. plantarum DPUL-S164-TM enhanced intestinal barrier function and alleviated inflammation by promoting tight junction protein expression, activating AhR/Nrf2 signaling, and inhibiting the NF-κB pathway [ 34 ]. Our findings collectively demonstrate that engineered bacteria-probiotic synergism can specifically upregulate the expression of ZO-1 and Occludin, thereby reinforcing intestinal barrier function [ 16 ]. The concurrent regulation of tight junction integrity and inflammatory signaling highlights the multifaceted therapeutic potential of rationally designed probiotic consortia. Enhancing the intestinal barrier limits the translocation of luminal bacteria and their metabolites into systemic circulation, consequently attenuating systemic low-grade inflammation. Consistent with these effects, histological analysis of colonic tissues revealed markedly reduced inflammatory cell infiltration and improved tissue repair in the co-culture group. Furthermore, cytokine analysis showed that TNF-α expression in colonic tissues was lowest in the co-culture group. Analysis of Differences in Gut Microbiota Composition between Groups Immunohistochemical analysis revealed significant alterations in mucosal immune markers; therefore, we further investigated whether these changes correlated with shifts in microbial ecology. Analysis of gut microbiota α-diversity showed that the blank control group exhibited the highest Chao1 and Shannon indices ( p < 0.05), indicating optimal species richness and community equilibrium in their intestinal microbiota (Fig. 5 A and B) . Following antibiotic intervention, the natural recovery group demonstrated significant reductions in both diversity indices ( p < 0.05), confirming that lincomycin hydrochloride induces severe disruption of gut microecological balance [ 25 ]. Notably, all probiotic intervention groups effectively ameliorated dysbiosis ( p < 0.05); however, the effects of the single strain L. plantarum C8 carrying the empty plasmid and its co-culture group were comparatively weaker than those of the wild-type strain. The presence of the empty plasmid can negatively impact microbial diversity development. Due to the metabolic burden imposed by the plasmid, the empty plasmid strain may struggle to establish effective colonization in the gut [ 38 ]. Additionally, the absence of specific immune-regulating factors in the empty plasmid strain may hinder effective interaction with the host immune system, resulting in a less pronounced anti-inflammatory effect [ 17 ]. Among the groups, the co-culture of L. plantarum C8 and the recombinant strain exhibited the most significant recovery effects—its diversity index showed no statistically significant difference compared to the blank control group ( p < 0.05). The enhanced diversity indices in the co-culture group suggest that the combined action of the recombinant strain and L. plantarum C8 creates a more favorable microenvironment that supports increased microbial diversity and community stability. In the LP.SP-pMG36e- L. C8 group, the observed increase in Firmicutes relative abundance may result from L. plantarum C8 overexpression, which could selectively inhibit certain harmful Firmicutes species through nutrient competition or antimicrobial substance production while simultaneously promoting the proliferation of beneficial Firmicutes. This finding aligns with previous research by Miaopeng Ma et al., who demonstrated that pEGF overexpression in Clostridium butyricum enhanced intestinal protection by improving gut development, antimicrobial activity, and anti-inflammatory effects, thereby better maintaining the balance between pathogen challenge and inflammatory response. Such targeted microbial regulation not only improved community richness but also enhanced the stability of the microecosystem, which likely represents the key mechanism underlying this group's superior performance in restoring colonic morphology, inflammatory markers, and metabolic parameters [ 14 ]. These results collectively suggest that precisely engineered probiotic combinations can effectively reconstruct gut microbiota architecture through multiple synergistic mechanisms, offering significant therapeutic potential for addressing antibiotic-induced dysbiosis and its associated complications. Principal coordinates analysis (PCoA) results ( Fig. 5 C ) revealed that lincomycin hydrochloride treatment significantly altered the β-diversity of the murine gut microbiota (P = 0.001, R = 0.193), indicating substantial structural shifts in microbial communities following antibiotic intervention [ 23 ]. Notably, the co-culture intervention group ( L. plantarum C8 with the recombinant strain) exhibited microbiota profiles most closely resembling those of the blank control group, suggesting a superior capacity for restoring gut microecological equilibrium. This restorative effect corresponds with the group’s optimal α-diversity indices, implying that strain synergism may facilitate microecological reconstruction through the concurrent restoration of species richness and remodeling of community architecture [ 30 ]. Such dual regulatory mechanisms likely underlie the group’ s superior performance in physiological parameters (e.g., colon length, liver index). These modifications may result from either the recombinant strain’ s secretion of specialized metabolites that influence the growth of other microbes or altered chemical signaling pathways driven by modified metabolic activities that reshape interspecies ecological relationships. Gut microbiota-derived metabolites function as signaling molecules that regulate host gene expression and immune responses. Under co-culture conditions, recombinant strains may alter these metabolic signals, thereby modulating host–microbiota communication. Supporting this concept, Hwang et al. reported that L. plantarum –based mucosal vaccination reshaped gut microbiota composition and metabolic pathways, leading to enhanced immune responses and highlighting the role of microbial metabolism in host immune regulation [ 5 ]. This suggests that both direct contact between bacterial cells and intestinal cells, as well as indirect stimulation through secreted molecules, are involved in this process. The comprehensive restructuring of microbial networks through such multifaceted interactions explains the co-culture group’ s exceptional efficacy in restoring gut homeostasis after antibiotic disruption [ 8 ]. Overall, the co-culture group exhibited the highest α-diversity index, and its microbial community structure (β-diversity) most closely resembled that of the healthy group. Network analysis revealed more complex and stable microbial interactions in the co-culture group. Relative Abundance of Intestinal Flora in Mice 16S rRNA sequencing revealed that lincomycin hydrochloride significantly altered the composition of the murine gut microbiota, while probiotic interventions variably restored microbial communities. At the phylum level, six dominant phyla were identified in both groups of mice: Bacteroidota, Firmicutes, Proteobacteria, Verrucomicrobiota, Actinobacteria, and Campilobacterota. Among these, Bacteroidota was the predominant phylum, with Verrucomicrobiota as the third most abundant. The natural recovery group exhibited a typical dysbiotic pattern, characterized by a significant 30.51% reduction in Bacteroidota abundance and a 17.63% increase in Verrucomicrobiota, disrupting the critical phylum-level balance necessary for intestinal homeostasis (Fig. 5 D). Notably, compared to the wild-type strain and the single recombinant strain, the co-culture group of L. plantarum C8 with the recombinant strain demonstrated superior regulatory effects, showing a significant 50.96% increase in Bacteroidota abundance alongside a marked enrichment of Firmicutes. Ultimately, this group restored a microbial community profile most similar to that of the blank control group. The synergistic mechanism between recombinant strains and wild-type L. plantarum C8 likely involves three coordinated actions of LPxTG proteins and signal peptides: enhanced bacterial adhesion prolongs intestinal colonization of both strains; overexpression of signal peptides persistently activates histidine kinase (HK), amplifying quorum sensing signals and stimulating increased antimicrobial production by C8[ 27 ]; and this dual action not only directly suppresses pathogens but also modulates short-chain fatty acid (SCFA) production, promoting the proliferation of probiotics such as Bifidobacterium [ 1 ]. Furthermore, LPxTG proteins facilitate bacterial aggregation through homologous or heterologous interactions, which serves as the foundation for biofilm microcolony formation [ 29 ]. Biofilms provide bacteria with a protective niche against external stressors, enabling more stable and persistent colonization within the gut [ 9 , 33 ]. This mechanism also plausibly explains the enhanced efficacy observed under co-culture conditions: the engineered strain acts as a structural “scaffold,” allowing wild-type bacteria to adhere and integrate, thereby collectively forming a more robust and functionally synergistic consortium. At the genus level, Fig. 5 E displays the top 20 most abundant genera in the mice. The natural recovery group exhibited significant microbial alterations, characterized by a marked reduction in beneficial bacteria—specifically, Bacteroides , Bifidobacterium , and Lactobacillus decreased by 32.28%, 41.95%, and 72.62%, respectively—alongside a substantial proliferation of potential pathogens. Notably, the abundance of Escherichia-Shigella increased significantly by 85.20%, and Klebsiella by 91.37%. Previous studies have indicated that an increase in Klebsiella abundance can exacerbate intestinal inflammation, impair the gut barrier, and negatively affect the commensal microbiota in the mouse gut [ 37 ]. Administration of the L. plantarum C8 strain led to a partial recovery of beneficial microbial taxa and contributed to the modulation of gut microbiota structure. Notably, the recombinant engineered strain exerted superior modulatory effects, as evidenced by a significant elevation in the abundance of Bacteroides and Lactobacillus . This synergistic effect may be attributed to the co-expression system of the LPxTG protein and signal peptide, which confers unique biological properties to the recombinant strains. The LPxTG protein enhances the adaptive capacity of recombinant strains to the intestinal environment, facilitating better colonization and growth in the gut, thereby enabling more effective participation in quorum sensing and microbial regulation. The LPxTG anchoring mechanism enhances intestinal colonization of recombinant lactic acid bacteria through interactions between surface-anchored proteins and host epithelial receptors [ 15 ]. COG analysis ( Fig. 5 F ) reveals key functional adaptations supporting this process: cell wall/membrane biogenesis (Category M, 0.6–0.8 abundance) indicates active peptidoglycan remodeling, while posttranslational modification (Category O, 0.4–0.6) reflects sortase-mediated protein processing. These coordinated changes demonstrate the molecular basis for enhanced bacterial colonization. Studies have shown that the LPxT-GYLEQ protein significantly enhances the adhesion of Lactiplantibacillus reuteri ( L. reuteri ) to intestinal epithelial cells, improving the colonization capacity of lactic acid bacteria strains [ 2 , 12 ]. Experimental results revealed that the relative abundance of Firmicutes significantly increased by 31.97% and 50.96% in the recombinant strain intervention group and the L. plantarum C8 + recombinant strain co-intervention group, respectively ( p < 0.05). The abundance of Lactiplantibacillus exhibited a more pronounced elevation, reaching 241.94% and 331.34% in these groups ( p < 0.05). Collectively, these findings provide compelling evidence that LPxTG proteins promote more effective intestinal colonization in a co-cultivation model of wild-type probiotics and engineered strains by modulating microbial ecological niches. Beyond promoting colonization, the LPxTG motif enhances probiotic functionality through multiple mechanisms. In addition to improving the intestinal survival and adhesion properties of L. reuteri , it amplifies the role of autoinducer-2 (AI-2) signaling molecules in quorum sensing [ 39 ]. However, monotherapy with LPxTG-overexpressing engineered strains may exert excessive competitive pressure, limiting the recovery of beneficial commensal microbes and the overall restoration of gut microbiota following antibiotic exposure. In contrast, a co-cultivation strategy combining the engineered strain with its wild-type parental counterpart may establish an ecological buffering system. These findings align with those of Sophie et al., who demonstrated that overexpression of sRNA Ern0160 leads to reduced growth rates in vitro and a competitive disadvantage during intestinal colonization in vivo. This suggests that sRNA overexpression imposes a significant metabolic burden or fitness cost, thereby limiting efficacy in monoculture [ 24 ]. Consequently, the co-presence of the wild-type strain likely mitigates these deleterious effects at the population level. Through niche partitioning, functional complementarity, and metabolic cooperation, this two-strain consortium effectively occupies and defends key ecological niches against pathogen invasion while preserving resources and spatial availability for indigenous microbiota recolonization [ 32 ]. These synergistic effects enable the recombinant strain not only to better adapt to the gut microbial environment but also to engage in complex microbial interactions and regulate metabolic processes within co-culture systems involving the wild-type strain, ultimately resulting in superior probiotic efficacy. Conclusion In summary, this study confirms that LPxTG proteins and signal peptides significantly enhance the colonization capacity of probiotics in the gut and improve microbiota modulation within co-culture systems. The genetically engineered L.plantarum C8 strain, displaying surface LPxTG domains, markedly increases the abundance of beneficial bacteria and effectively promotes the restoration of gut microbiota disrupted by antibiotics while inhibiting pathogen proliferation when co-cultured with the wild-type strain. This co-culture system alleviates antibiotic-induced dysbiosis through multiple mechanisms, including enhanced gut adaptability, improved villi development, and microbiota regulation. These findings provide a robust scientific foundation for developing functional probiotics as innovative alternatives to antibiotics in fermented foods, highlighting their significant potential for practical application. Declarations Notes The authors declare no competing financial interest. Author Contribution J. Liang and J. Liu performed the research and Writing-original draft, A. Zhang and S. Zheng required the data and Formal analysis, T. Jiang performed the Visualization work, and L. Yang and X. Qi performed the Methodology, X. Zeng and Y. Guo required the Validation, D. Pan and T. Zhang performed the Supervision, and Z. Wu required the Funding acquisition. Acknowledgements This study was supported by This work was supported by the National Natural Science Foundation of China (32472356, 32402125), the Key Project of Ningbo Science and Technology (2023Z127), General Research Project of Zhejiang Provincial Education Department (Y202352124), Natural Science Foundation of Ningbo (2023J103), Open Fund for the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (2021DG700024-KF202423). References Ding, Q. C., F. W. Cao, S. L. Lai, H. Zhuge, K. X. Chang, T. G. Valencak, J. X. Liu, S. T. Li, and D. X. Ren. 2022. ZY08 relieves chronic alcohol-induced hepatic steatosis and liver injury in mice via restoring intestinal flora homeostasis. Food Res. Int. 157:111259. http://doi.org/10.1016/j.foodres.2022.111259 . Fredriksen, L., G. 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20:19:52","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36188,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/5a5552225289377ce422a5ed.png"},{"id":100858686,"identity":"f8ad5df9-62bf-40dc-bcbd-764eba55cc09","added_by":"auto","created_at":"2026-01-22 07:24:36","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":208167,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/9be4514f6ec8069747ceb956.png"},{"id":100858925,"identity":"cfa90a18-8353-488a-96a5-0e411268d3e5","added_by":"auto","created_at":"2026-01-22 07:24:57","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27686,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/4558ea0cdfc8341c9cbe6279.png"},{"id":100829228,"identity":"78f35111-250a-4f40-a81f-916274ade7c5","added_by":"auto","created_at":"2026-01-21 20:19:52","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92742,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/d3da2539a5c0c3c1fb6c0020.png"},{"id":100859178,"identity":"2e12330d-792a-41ef-986a-a669da06d10a","added_by":"auto","created_at":"2026-01-22 07:25:31","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72655,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/986250c12ed0f30eaba8420a.png"},{"id":100829232,"identity":"9e0a8071-f054-4c1a-9454-014ec03a0e79","added_by":"auto","created_at":"2026-01-21 20:19:52","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146917,"visible":true,"origin":"","legend":"","description":"","filename":"f6df05c757b74e108c4a49df987dc4321structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/9a8a2cf65af2adb479d4e015.xml"},{"id":100829231,"identity":"164e57e8-b940-4439-a1a0-85ef75c39681","added_by":"auto","created_at":"2026-01-21 20:19:52","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161363,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/3decfac9415aa3b4531ed6aa.html"},{"id":100859112,"identity":"cc6d89fe-e532-4cf7-9ef2-13402bcedd40","added_by":"auto","created_at":"2026-01-22 07:25:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":220669,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological indicators of mice.Changes in average daily body weight (\u003cstrong\u003eA\u003c/strong\u003e); representative photograph of colons from mice (\u003cstrong\u003eB\u003c/strong\u003e); quantitative analysis of colon length (cm) (\u003cstrong\u003eC\u003c/strong\u003e); relativate liver weight (\u003cstrong\u003eD\u003c/strong\u003e) and relative spleen weight (\u003cstrong\u003eE\u003c/strong\u003e) . Different lowercase letters above bars denote significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/64955a201e11b6a6942ef7d3.png"},{"id":100859088,"identity":"9ce289d8-c45e-47ae-8f9e-76292aa643e9","added_by":"auto","created_at":"2026-01-22 07:25:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":439822,"visible":true,"origin":"","legend":"\u003cp\u003eHistomorphology of the colon (hematoxylin and eosin staining). Representative photomicrographs showing the colonic tissue structure across different experimental groups. The scale bar represents 300 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/2b6135d8118969801a76a082.png"},{"id":100858440,"identity":"00c591f0-bb6b-4412-aa4b-3a4d8483cebc","added_by":"auto","created_at":"2026-01-22 07:24:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183596,"visible":true,"origin":"","legend":"\u003cp\u003eSerum cytokine levels in mice. Serum levels of IL-6 in mice (\u003cstrong\u003eA\u003c/strong\u003e); Serum levels of IL-10 in mice (\u003cstrong\u003eB\u003c/strong\u003e); Serum levels of TNF-α in mice (\u003cstrong\u003eC\u003c/strong\u003e). Different lowercase letters above bars denote significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/0ce9c8e1820193dc022cf835.png"},{"id":100858607,"identity":"f5230b30-0e9c-48ed-bcc6-8196174285cf","added_by":"auto","created_at":"2026-01-22 07:24:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":334120,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of colonic tissues from experimental mice. Representative images show the localization and expression levels of ZO-1 (\u003cstrong\u003eA\u003c/strong\u003e) and Occludin (\u003cstrong\u003eC\u003c/strong\u003e) across treatment groups. Scale bar, 100 μm (applicable to \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e). Quantitative analysis of ZO-1 (\u003cstrong\u003eB\u003c/strong\u003e) and Occludin (\u003cstrong\u003eD\u003c/strong\u003e) protein expression is presented. Different lowercase letters above bars denote significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/68be90df9565fd347bf2f8ac.png"},{"id":100859066,"identity":"5a3b322d-0d64-43ad-9580-b516e8913c93","added_by":"auto","created_at":"2026-01-22 07:25:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":522159,"visible":true,"origin":"","legend":"\u003cp\u003eChao index (\u003cstrong\u003eA\u003c/strong\u003e)；Shannon index (B) of intestinal flora；PCoA analysis of intestinal flora (\u003cstrong\u003eC\u003c/strong\u003e), P=0.001, R=0.193. Effect of different treatment groups on gut microbiota composition. Histogram of relative abundance at phylum level (\u003cstrong\u003eD\u003c/strong\u003e) and at genus level (\u003cstrong\u003eE\u003c/strong\u003e), COG functional prediction of gut microbiota (\u003cstrong\u003eF\u003c/strong\u003e). Each bar represents the average relative abundance of each bacterial taxon within a treatment group. Different lowercase letters above bars denote significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/514c0f50a9b122726d978212.png"},{"id":101202431,"identity":"092747d3-b615-432d-ac57-388ca4f07fc6","added_by":"auto","created_at":"2026-01-27 09:33:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2618026,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/52899b23-42ee-422e-9b2e-b664fc8d9392.pdf"},{"id":100829220,"identity":"a73ccaec-d215-4a3c-9383-fdec83d37d28","added_by":"auto","created_at":"2026-01-21 20:19:52","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":310540,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8618174/v1/28e1119fb8b17af40f3716d5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"LPxTG Proteins Mediate Microbial Interactions to Modulate Lactiplantibacillus plantarum C8 in Alleviating Antibiotic-Associated Diarrhea","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotic-associated diarrhea (AAD) is a common adverse effect during antimicrobial therapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Its occurrence is not only related to the abnormal overgrowth of pathogenic microorganisms but is also closely associated with disruption of gut microbiota composition and imbalance of mucosal immune homeostasis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Recent studies demonstrate that antibiotic-induced gut microbiota alterations correlate with immune dysfunction and various diseases, including diabetes, obesity, and cardiovascular disorders. Notably, antibiotic-mediated disruption of the gut microbiota may persist for months or even years after treatment, thereby increasing susceptibility to diseases[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, it is necessary to explore better approaches to prevent or mitigate the adverse effects of antibiotics on the gut microbiota.\u003c/p\u003e \u003cp\u003eIn recent years, LAB have attracted increasing research interest due to their long history of safe use and demonstrated therapeutic benefits for human health[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eL. plantarum\u003c/em\u003e possesses multiple probiotic characteristics, including the ability to ferment a wide spectrum of plant-derived carbohydrates, achieve high-density growth, tolerate bile salts and low pH conditions, and exert antagonistic effects against intestinal pathogens [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. As live microorganisms, probiotics can modulate gut microbiota composition and correct aberrant mucosal immune responses associated with chronic intestinal inflammation. Furthermore, they enhance intestinal barrier function through multiple mechanisms: regulating cytokine production, stimulating regulatory T cell (Treg) release, and promoting intestinal epithelial cell survival [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the efficacy of probiotics in AAD intervention varies markedly among individuals, largely due to insufficient intestinal adhesion and unstable functional performance of probiotic strains in vivo.\u003c/p\u003e \u003cp\u003eProteins containing the LPxTG motif are a class of adhesion-related surface proteins predominantly anchored to \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These proteins enhance interactions with intestinal epithelial cells, thereby promoting stable gut colonization, and their expression is regulated by quorum sensing, contributing to colonization capacity and functional persistence in the intestinal environment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. LPxTG-containing surface proteins have been reported to improve resistance to gastrointestinal stresses, enhancing bacterial survival and colonization stability [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, studies in Listeria monocytogenes indicate that LPxTG-anchored proteins are involved in environmental stress adaptation and host cell adhesion, supporting the functional conservation of this protein family in microbe\u0026ndash;host interactions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the context of antibiotic-induced dysbiosis, ecological niches within the gut microbiota become highly unstable, and single-strain interventions often fail to achieve sustained colonization and functional recovery. Emerging evidence suggests that probiotics derived from co-cultivation strategies may exhibit improved adaptability and activity in the gastrointestinal environment compared with those obtained from monoculture [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We therefore hypothesize that cooperative colonization between wild-type and engineered strains may confer survival and functional advantages in alleviating antibiotic-associated diarrhea and promoting gut microbiota restoration. LPxTG-anchored surface proteins may act as structural or adhesive factors that facilitate such inter-bacterial cooperative interactions, thereby enhancing collective persistence and functional stability within the intestinal ecosystem.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Strains\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eL. plantarum\u003c/em\u003e C8 (CGMCC NO.30504) strain used in this study was independently isolated by our laboratory and is currently preserved at the China General Microbiological Culture Collection Center. In this study, signal peptides were identified from the proteome of \u003cem\u003eL. plantarum\u003c/em\u003e C8 using the NCBI and UniProt databases. Subsequently, the LPxTG motif protein gene and the signal peptide gene were cloned into the pMG36e plasmid vector through homologous recombination to construct recombinant strains. This construct was first transformed into chemically competent BL21 cells for plasmid amplification and then electroporated into chemically competent \u003cem\u003eL. plantarum\u003c/em\u003e C8 cells. \u003cem\u003eL. plantarum\u003c/em\u003e C8 was inoculated at 1% into 100 mL of MRS liquid medium, while the empty vector strain and the recombinant strain were inoculated at 1% into 100 mL of MRS liquid medium containing 20 \u0026micro;g/mL erythromycin. The cultures were incubated statically in a 37\u0026deg;C constant temperature and humidity incubator for 10\u0026ndash;12 hours and activated to the third generation. Then, at the same 1% inoculation rate, the strains were inoculated under co-culture conditions into a new 100 mL MRS broth and continued to be cultured at 37\u0026deg;C for 12 hours. The cultured bacterial suspension was centrifuged to remove the supernatant, washed three times with sterile PBS, and then diluted with sterile physiological saline to an OD600 nm\u0026thinsp;=\u0026thinsp;1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, ready for immediate use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction of Animal Experimental Model\u003c/h3\u003e\n\u003cp\u003eSPF-grade male KM mice (approximately 6 weeks old), weighing 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. A total of 56 mice (6 weeks old, n\u0026thinsp;=\u0026thinsp;8 per group) were acclimatized for 7 days and then randomly assigned to a control group (n\u0026thinsp;=\u0026thinsp;8) and a model group (n\u0026thinsp;=\u0026thinsp;48). The model group received 300 mg/mL lincomycin hydrochloride via oral gavage twice daily for 3 consecutive days to induce intestinal dysbiosis, and was subsequently divided into six treatment subgroups: natural recovery group, \u003cem\u003eL. plantarum\u003c/em\u003e C8 intervention group, blank strain intervention group, recombinant strain intervention group, \u003cem\u003eL. plantarum\u003c/em\u003e C8\u0026thinsp;+\u0026thinsp;blank strain co-culture group, and \u003cem\u003eL. plantarum\u003c/em\u003e C8\u0026thinsp;+\u0026thinsp;recombinant strain co-culture group \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Throughout the 25-day experiment, all mice had ad libitum access to standard diet and water, with the blank and natural recovery groups receiving saline (0.2 mL/10 g body weight) while intervention groups received equivalent volumes of their respective bacterial suspensions. Daily body weight measurements and fecal sample collections were performed to monitor physiological changes. Following an overnight fast on day 24, all mice were humanely euthanized by cervical dislocation on day 25, at which point serum samples were collected and colon, liver, and spleen tissues were excised and weighed. All experimental procedures were conducted in strict accordance with the guidelines of Ningbo University Animal Experiment Center and were approved by the University's Animal Ethics Committee (Approval No.: NBU20240314).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMice group in the experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGavage Volume per Day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlank Control Group (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g physiological saline\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNatural Recovery Group (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g physiological saline\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e C8 Intervention Group (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g bacterial suspension\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEmpty Vector Strain Intervention Group (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g bacterial suspension\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecombinant Strain Intervention Group (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g bacterial suspension\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCo-culture Intervention Group\u003c/p\u003e \u003cp\u003e(\u003cem\u003eL. plantarum\u003c/em\u003e C8\u0026thinsp;+\u0026thinsp;Empty Vector Strain) (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g bacterial suspension\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCo-culture Intervention Group\u003c/p\u003e \u003cp\u003e(\u003cem\u003eL. plantarum\u003c/em\u003e\u0026nbsp;C8\u0026thinsp;+\u0026thinsp;Recombinant Strain) (8 males)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegular feed\u0026thinsp;+\u0026thinsp;water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2 mL/10 g bacterial suspension\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePhysiological Parameter Monitoring and Organ Collection in Mice\u003c/h3\u003e\n\u003cp\u003eDuring the experimental period, daily body weight measurements were conducted on all mice. At the conclusion of the recovery phase, fecal samples (4\u0026ndash;5 pellets per mouse) were aseptically collected into sterile 1.5 mL microcentrifuge tubes and immediately stored at -80 ℃ for subsequent analysis. Blood samples were obtained through retro-orbital puncture under anesthesia, allowed to clot at room temperature for 30 minutes, then centrifuged at 12,000 \u0026times; g for 10 minutes at 4 ℃ to separate serum. Following euthanasia, a complete necropsy was performed which included measurement of colon length prior to fixation in 4% paraformaldehyde, precise weighing of liver and spleen tissues, and snap-freezing of all collected organs in liquid nitrogen for preservation at -80 ℃. All procedures were conducted in accordance with standard laboratory protocols to ensure sample integrity.\u003c/p\u003e\n\u003ch3\u003eHistomorphological Observation\u003c/h3\u003e\n\u003cp\u003eFollowing euthanasia, the target intestinal segments were promptly excised and thoroughly rinsed with PBS before fixation in 4% paraformaldehyde for 24\u0026ndash;48 hours. The samples subsequently underwent ethanol gradient dehydration and xylene clearing, followed by paraffin infiltration, embedding, and sectioning (4\u0026ndash;7 \u0026micro;m). Sections were expanded in 45 ℃ warm water and baked at 37 ℃ for 2 hours. Deparaffinization was performed using xylene I and II (10 minutes each), followed by gradient ethanol dehydration (100%, 95%, 85%, and 75%; 5 minutes each). Hematoxylin staining was conducted for 8\u0026ndash;10 minutes, differentiated with 1% acid alcohol for 3\u0026ndash;5 seconds, and blued in running water for 15 minutes. Eosin staining lasted 1\u0026ndash;2 minutes, followed by rapid dehydration with 95% and 100% ethanol, xylene clearing for 5 minutes, and final mounting with neutral balsam.\u003c/p\u003e\n\u003ch3\u003eCytokine Measurement\u003c/h3\u003e\n\u003cp\u003eThe levels of IL-6, IL-10 and TNF-α in mouse serum were detected by ELISA (Thermo Fisher Scientific). The 96-well plate was firstly coated with capture antibody overnight at 4 ℃, washed and then closed with 1% BSA for 1 hour. Sequentially, standards or samples to be tested were added and incubated for 2 hours. After washing, detection antibodies were added and incubated for 2 hours. Then the plates were reacted with HRP-labeled secondary antibody for 20 minutes avoiding light. Finally, TMB substrate was added to develop the color for 20 minutes, and the absorbance value at 450 nm was measured after terminating the reaction to calculate the cytokine concentration.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical\u003c/h2\u003e \u003cp\u003eTissue sections were dewaxed in xylene (2 \u0026times; 20 minutes), dehydrated through an ethanol series (100% \u0026minus;\u0026thinsp;75%), and subjected to antigen retrieval in citrate buffer (pH 6.0) using microwave heating. After peroxidase blocking (3% H₂O₂, 15 min), sections were incubated with primary antibody (4 ℃ overnight) and HRP-conjugated secondary antibody (20 minutes RT), with PBS washes between steps. Following DAB development (1\u0026ndash;5 minutes) and hematoxylin counterstaining, sections were dehydrated (70% \u0026minus;\u0026thinsp;100% ethanol), cleared in xylene, and mounted with rhamsan gum.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGut Microbiota Analysis\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA from the mouse fecal microbial community was extracted using the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China) according to the manufacturer\u0026rsquo;s instructions. The integrity of the extracted DNA was verified by 1% agarose gel electrophoresis, and its concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The V3-V4 hypervariable regions of the 16S rRNA gene were amplified by PCR using barcoded primers and the extracted DNA as a template. Amplification was performed under the following conditions: initial denaturation at 95\u0026deg;C for 3 minutes; 30 cycles of 95\u0026deg;C for 30 seconds, 55\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds; followed by a final extension at 72\u0026deg;C for 10 minutes.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS Statistics 19.1 (IBM Corp., Armonk, NY, USA). For multiple comparisons among treatment groups, one-way ANOVA followed by Duncan's post hoc test was performed, with statistical significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWeighing Index of Mice\u003c/h2\u003e \u003cp\u003eNo mortality was observed throughout the animal experiments. During the 0\u0026ndash;7 day adaptation phase, mice in all groups exhibited a stable increase in body weight. In contrast, during the modeling period, treatment with lincomycin hydrochloride led to a gradual decrease in body weight in the experimental group, whereas the blank control group continued to gain weight steadily (Fig.\u0026nbsp;1A). During the recovery phase, mice in the natural recovery group showed significantly lower body weight gain than those receiving bacterial interventions, indicating that \u003cem\u003eL. plantarum\u003c/em\u003e C8 effectively promoted weight recovery. This difference highlights the negative impact of antibiotics on body weight, likely resulting from LCM-induced disruption of the gut microbiota. Among the intervention groups, treatment with wild-type \u003cem\u003eL. plantarum\u003c/em\u003e C8 resulted in improved weight recovery, while the co-culture group of the recombinant strain and \u003cem\u003eL. plantarum\u003c/em\u003e C8 exhibited the most pronounced effect. This enhanced recovery is likely attributable to rapid intestinal colonization mediated by LPxTG protein\u0026ndash;dependent adhesion of the recombinant strain. Consistent with this, previous studies have shown that surface display of functional proteins via engineered signal peptides and LPxTG motifs significantly enhances bacterial adhesion and persistence in the gut [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In contrast, the saline control group displayed delayed recovery, underscoring the limited capacity of spontaneous microbiota reconstitution after antibiotic withdrawal. Collectively, these results suggest that antibiotic-associated weight loss is closely linked to impaired microbiota function and that engineered probiotics expressing LPxTG motifs can effectively mitigate this adverse effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLength of Colon in Mice\u003c/h2\u003e \u003cp\u003eRelated studies have demonstrated that intestinal dysbiosis is a key factor driving inflammatory responses in the colon, with colon shortening being a significant symptom of colonic inflammation. Measurements of colon length revealed a marked reduction in the natural recovery group (5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 cm), a phenomenon likely linked to antibiotic-induced gut microbiota dysbiosis \u003cb\u003e(Fig.\u0026nbsp;1B and 1C)\u003c/b\u003e. Notably, all probiotic-treated groups\u0026mdash;especially those supplemented with \u003cem\u003eL. plantarum\u003c/em\u003e C8, the recombinant strain, or their co-culture preparation\u0026mdash;exhibited restored colon lengths (7.06\u0026ndash;8.37 cm) that approximated or exceeded the blank control values (7.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 cm). The restoration of colonic architecture not only ensures adequate nutrient absorption to support weight recovery but also reduces hepatic metabolic burden by maintaining intestinal barrier integrity and minimizing endotoxin translocation. Furthermore, the re-established colonic microbial homeostasis appears to modulate systemic immune responses, which explains the subsequent normalization of the spleen index observed in probiotic-treated mice. Notably, the co-culture group of \u003cem\u003eL. plantarum\u003c/em\u003e C8 and recombinant strains demonstrated the most pronounced effects, suggesting that specific strain combinations may synergistically repair antibiotic-induced multisystem damage more effectively, providing valuable insights for clinical interventions. These findings are consistent with previous studies showing that oral administration of \u003cem\u003eL. plantarum\u003c/em\u003e 12 can ameliorate colitis in DSS-induced mouse models [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLiver and Spleen Index in Mice\u003c/h2\u003e \u003cp\u003eThe liver index of mice in the natural recovery group, which were gavaged with saline, was significantly elevated (\u0026gt;\u0026thinsp;5%), exceeding the normal range (Fig.\u0026nbsp;1D). This indicates an increased hepatic metabolic burden and inflammatory response during recovery. In contrast, all probiotic intervention groups exhibited reduced liver indices, suggesting effective alleviation of liver metabolic stress and inflammation. Notably, treatment with the wild-type strain, recombinant strain, or their co-culture reduced the liver index to levels comparable to those of the blank control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting improved hepatic metabolic function. However, strains carrying empty plasmids and their co-culture groups showed relatively weaker recovery effects in both liver and spleen indices. This may be attributed to the additional metabolic burden imposed by plasmid maintenance, which can impair bacterial growth and probiotic efficacy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have demonstrated that \u003cem\u003eL. plantarum\u003c/em\u003e strains can enhance Nrf2-mediated antioxidant responses and alleviate liver injury [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Accordingly, the hepatoprotective effects of \u003cem\u003eL. plantarum\u003c/em\u003e C8 and its co-cultured strains observed in this study may similarly involve activation of the Nrf2 signaling pathway.\u003c/p\u003e \u003cp\u003eFurthermore, the natural recovery group exhibited significantly elevated spleen indices \u003cb\u003e(Fig.\u0026nbsp;1E)\u003c/b\u003e, indicative of antibiotic-induced immune dysregulation. In contrast, the recombinant strain and co-culture groups demonstrated optimal spleen recovery, highlighting the superior immunomodulatory capacity conferred by their LPxTG-anchored proteins and signal peptides. These findings are consistent with previous work by Assad Moon et al., who developed recombinant lactobacilli expressing the PRV gD antigen via an LPxTG (LP3065) anchoring system. Their study showed enhanced CD4\u0026thinsp;+\u0026thinsp;T cell populations in murine splenocytes, thereby promoting helper T cell activation and a more robust immune response [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These results suggest that the introduction of LPxTG-anchored proteins and signal peptides in the recombinant strain not only enhances its intestinal colonization ability but may also regulate liver metabolic function, alleviate antibiotic-induced metabolic disorders, and restore immune homeostasis by modulating the balance of T cell subsets.\u003c/p\u003e \u003cp\u003eAlthough the wild-type \u003cem\u003eL. plantarum\u003c/em\u003e itself demonstrates certain probiotic functions, such as alleviating antibiotic-induced weight loss and abnormalities in intestinal, liver, and spleen indices, the co-culture group of the engineered strain overexpressing LPxTG demonstrated superior effects. The recombinant single strain produced effects similar to those of the wild-type, whereas the co-culture group of the engineered strain overexpressing LPxTG showed enhanced results. This suggests that the efficacy of a single strain may be limited by its individual capabilities, while the co-culture group, through increased microbial interactions, exerts a stronger overall health benefit.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1\u003c/b\u003e Physiological indicators of mice.Changes in average daily body weight (\u003cb\u003eA\u003c/b\u003e); representative photograph of colons from mice (\u003cb\u003eB\u003c/b\u003e); quantitative analysis of colon length (cm) (\u003cb\u003eC\u003c/b\u003e); relativate liver weight (\u003cb\u003eD\u003c/b\u003e) and relative spleen weight (\u003cb\u003eE\u003c/b\u003e). Different lowercase letters above bars denote significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColonic Histological Morphology\u003c/h2\u003e \u003cp\u003eTo better understand the effects of antibiotics on the colon, we conducted histological analyses. Hematoxylin and eosin (H\u0026amp;E) staining revealed intact colonic architecture, clear intestinal walls, and evenly distributed goblet cells in the blank control group, with no evident inflammatory infiltration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In contrast, the natural recovery group exhibited disrupted intestinal villi, abnormal goblet cell morphology, and substantial inflammatory cell infiltration, indicating significant antibiotic-induced intestinal damage. All intervention groups showed varying degrees of histological improvement, with the co-culture group demonstrating the most remarkable restoration\u0026mdash;its tissue structure closely resembled normal, with almost no pathological alterations.\u003c/p\u003e \u003cp\u003eThis demonstrated that the engineered bacteria could alleviate histopathological damage in the intestinal, liver, and spleen tissues, with the co-cultivation group showing the most pronounced effect. Inflammation is a fundamental defensive response. The severe inflammatory infiltration observed in the natural recovery group likely reflects sustained pro-inflammatory activity, whereas the therapeutic effects seen in the intervention groups\u0026mdash;particularly the co-culture group\u0026mdash;may result from probiotic-mediated modulation of inflammatory pathways [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, the superior efficacy of the co-culture group underscores a synergistic interaction between strains, which may optimize microbiota reconstitution and strengthen host defense mechanisms, thereby providing enhanced protection against antibiotic-induced tissue injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Strain C8 on Serum Inflammatory Cytokines\u003c/h2\u003e \u003cp\u003eCytokine analysis revealed that antibiotic treatment induced a strong pro-inflammatory response in the colon of mice undergoing natural recovery. Compared with the blank control group, IL-6 and TNF-α levels increased significantly by 98.34% and 83.99%, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while IL-10 levels decreased by 73.43% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). All groups treated with \u003cem\u003eL. plantarum\u003c/em\u003e C8 exhibited reduced inflammation. IL-6 and TNF-α levels were lowered to 164.17/156.08 pg/mg in the monotherapy group, 175.08/169.64 pg/mg in the empty vector group, and 153.50/146.57 pg/mg in the recombinant strain group. Notably, combined treatment with \u003cem\u003eL. plantarum\u003c/em\u003e C8 and the recombinant strain produced the most pronounced effect, further reducing IL-6 and TNF-α to 132.00 and 131.83 pg/mg, respectively, while increasing IL-10 to 226.47 pg/mg\u0026mdash;the highest level among all intervention groups. These findings suggest that \u003cem\u003eL. plantarum\u003c/em\u003e C8 alleviates antibiotic-induced intestinal inflammation by modulating immune responses, consistent with histopathological observations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe notably enhanced efficacy of the combined strain intervention highlights the potential therapeutic advantage of multi-strain probiotic formulations in mitigating antibiotic-induced intestinal inflammation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Research has demonstrated that administering \u003cem\u003eL. plantarum\u003c/em\u003e to cyclophosphamide-induced immunosuppressed mice produces beneficial regulatory effects on immunity, gut microbiota composition, and jejunal inflammatory cytokines [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, the LPxTG protein may contribute to inflammation alleviation through specific mechanisms. Compared to other intervention groups, \u003cem\u003eL. plantarum\u003c/em\u003e C8 carrying the recombinant plasmid and the co-culture group of \u003cem\u003eL. plantarum\u003c/em\u003e C8 with the wild-type strain exhibit superior regulation of inflammatory factors. Previous studies have shown that an LPxTG motif protein from \u003cem\u003eLactiplantibacillus reuteri\u003c/em\u003e SH23 alleviated DSS-induced colitis by suppressing MAPK/NF-κB signaling, reducing TNF-α and IL-6 levels while increasing IL-10, and reshaping gut microbiota composition\u0026mdash;particularly by increasing \u003cem\u003eLactiplantibacillus\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These findings support the hypothesis that the LPxTG protein in our current study may operate via analogous pathways to modulate inflammatory cytokine profiles and subsequently improve gut microbiota composition, potentially explaining the observed therapeutic effects of our recombinant strain. The consistent outcomes across these independent studies reinforce the concept that LPxTG-containing surface proteins represent a promising therapeutic strategy for inflammation-related gut disorders through their dual action on both host immune responses and microbial ecosystems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eProbiotic interventions demonstrated critical protective effects on intestinal barrier integrity. Immunohistochemical analysis revealed that antibiotic treatment severely compromised the gut barrier in the natural recovery group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand C)\u003c/b\u003e, significantly reducing the expression of ZO-1 and Occludin to only 14.77% and 15.42% of the blank control levels, respectively. This disruption of tight junction proteins, which are essential for maintaining paracellular permeability, is a well-established mechanism by which certain pathogens impair the gut barrier [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. All probiotic intervention groups significantly upregulated the expression of these key proteins (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the co-culture group of \u003cem\u003eL. plantarum\u003c/em\u003e C8 and the recombinant strain showing the most pronounced restorative effects\u0026mdash;achieving 31.54% and 31.33% recovery of ZO-1 and Occludin expression, respectively, representing approximately a 2.1-fold improvement over the natural recovery group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the co-culture group demonstrated significantly superior restorative effects compared to single-strain interventions (22.99% \u0026minus;\u0026thinsp;28.11%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u003cb\u003eand D)\u003c/b\u003e. The protein expression levels of Occludin and ZO-1 followed the same trend, indicating that the recombinant strain can better induce quorum sensing effects between \u003cem\u003eL. plantarum\u003c/em\u003e C8, thereby promoting communication within the microbiota and more effectively repairing the intestinal barrier. This conclusion is supported by HE staining results, which revealed the mildest pathological damage in colonic tissues and the most intact crypt and villus structures in the co-culture group. This synergistic effect may originate from enhanced interaction efficiency between strains via quorum sensing, coupled with the coordinated regulation of tight junction protein expression in epithelial cells by microbial metabolites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese molecular findings corroborate our previous cytokine results, providing mechanistic insights into the superior anti-inflammatory efficacy of the co-culture formulation. Supporting evidence comes from Jiang et al., who demonstrated that \u003cem\u003eL. plantarum\u003c/em\u003e HM-22 significantly enhanced Occludin and Claudin-1 expression in α-LA-induced allergic mice, thereby reducing intestinal permeability and improving barrier function. Furthermore, NF-κB and MAPK signaling pathways are known to mediate microbial stimulus-induced proinflammatory cytokine production, while LPxTG-anchored or sortase-dependent proteins (SDPs), which are covalently attached to peptidoglycan, play pivotal roles in \u003cem\u003eLactiplantibacillus\u003c/em\u003e-host interactions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Similarly, Wang et al. demonstrated that \u003cem\u003eL. plantarum\u003c/em\u003e DPUL-S164-TM enhanced intestinal barrier function and alleviated inflammation by promoting tight junction protein expression, activating AhR/Nrf2 signaling, and inhibiting the NF-κB pathway [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings collectively demonstrate that engineered bacteria-probiotic synergism can specifically upregulate the expression of ZO-1 and Occludin, thereby reinforcing intestinal barrier function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The concurrent regulation of tight junction integrity and inflammatory signaling highlights the multifaceted therapeutic potential of rationally designed probiotic consortia. Enhancing the intestinal barrier limits the translocation of luminal bacteria and their metabolites into systemic circulation, consequently attenuating systemic low-grade inflammation. Consistent with these effects, histological analysis of colonic tissues revealed markedly reduced inflammatory cell infiltration and improved tissue repair in the co-culture group. Furthermore, cytokine analysis showed that TNF-α expression in colonic tissues was lowest in the co-culture group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Differences in Gut Microbiota Composition between Groups\u003c/h2\u003e \u003cp\u003eImmunohistochemical analysis revealed significant alterations in mucosal immune markers; therefore, we further investigated whether these changes correlated with shifts in microbial ecology. Analysis of gut microbiota α-diversity showed that the blank control group exhibited the highest Chao1 and Shannon indices (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating optimal species richness and community equilibrium in their intestinal microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand B)\u003c/b\u003e. Following antibiotic intervention, the natural recovery group demonstrated significant reductions in both diversity indices (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming that lincomycin hydrochloride induces severe disruption of gut microecological balance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Notably, all probiotic intervention groups effectively ameliorated dysbiosis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); however, the effects of the single strain \u003cem\u003eL. plantarum\u003c/em\u003e C8 carrying the empty plasmid and its co-culture group were comparatively weaker than those of the wild-type strain. The presence of the empty plasmid can negatively impact microbial diversity development. Due to the metabolic burden imposed by the plasmid, the empty plasmid strain may struggle to establish effective colonization in the gut [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, the absence of specific immune-regulating factors in the empty plasmid strain may hinder effective interaction with the host immune system, resulting in a less pronounced anti-inflammatory effect [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the groups, the co-culture of \u003cem\u003eL. plantarum\u003c/em\u003e C8 and the recombinant strain exhibited the most significant recovery effects\u0026mdash;its diversity index showed no statistically significant difference compared to the blank control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The enhanced diversity indices in the co-culture group suggest that the combined action of the recombinant strain and \u003cem\u003eL. plantarum\u003c/em\u003e C8 creates a more favorable microenvironment that supports increased microbial diversity and community stability. In the LP.SP-pMG36e-\u003cem\u003eL.\u003c/em\u003eC8 group, the observed increase in Firmicutes relative abundance may result from \u003cem\u003eL. plantarum\u003c/em\u003e C8 overexpression, which could selectively inhibit certain harmful Firmicutes species through nutrient competition or antimicrobial substance production while simultaneously promoting the proliferation of beneficial Firmicutes. This finding aligns with previous research by Miaopeng Ma et al., who demonstrated that pEGF overexpression in Clostridium butyricum enhanced intestinal protection by improving gut development, antimicrobial activity, and anti-inflammatory effects, thereby better maintaining the balance between pathogen challenge and inflammatory response. Such targeted microbial regulation not only improved community richness but also enhanced the stability of the microecosystem, which likely represents the key mechanism underlying this group's superior performance in restoring colonic morphology, inflammatory markers, and metabolic parameters [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These results collectively suggest that precisely engineered probiotic combinations can effectively reconstruct gut microbiota architecture through multiple synergistic mechanisms, offering significant therapeutic potential for addressing antibiotic-induced dysbiosis and its associated complications.\u003c/p\u003e \u003cp\u003ePrincipal coordinates analysis (PCoA) results \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e revealed that lincomycin hydrochloride treatment significantly altered the β-diversity of the murine gut microbiota (P\u0026thinsp;=\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.193), indicating substantial structural shifts in microbial communities following antibiotic intervention [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Notably, the co-culture intervention group (\u003cem\u003eL. plantarum\u003c/em\u003e C8 with the recombinant strain) exhibited microbiota profiles most closely resembling those of the blank control group, suggesting a superior capacity for restoring gut microecological equilibrium. This restorative effect corresponds with the group\u0026rsquo;s optimal α-diversity indices, implying that strain synergism may facilitate microecological reconstruction through the concurrent restoration of species richness and remodeling of community architecture [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Such dual regulatory mechanisms likely underlie the group\u0026rsquo; s superior performance in physiological parameters (e.g., colon length, liver index). These modifications may result from either the recombinant strain\u0026rsquo; s secretion of specialized metabolites that influence the growth of other microbes or altered chemical signaling pathways driven by modified metabolic activities that reshape interspecies ecological relationships.\u003c/p\u003e \u003cp\u003eGut microbiota-derived metabolites function as signaling molecules that regulate host gene expression and immune responses. Under co-culture conditions, recombinant strains may alter these metabolic signals, thereby modulating host\u0026ndash;microbiota communication. Supporting this concept, Hwang et al. reported that \u003cem\u003eL. plantarum\u003c/em\u003e\u0026ndash;based mucosal vaccination reshaped gut microbiota composition and metabolic pathways, leading to enhanced immune responses and highlighting the role of microbial metabolism in host immune regulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This suggests that both direct contact between bacterial cells and intestinal cells, as well as indirect stimulation through secreted molecules, are involved in this process. The comprehensive restructuring of microbial networks through such multifaceted interactions explains the co-culture group\u0026rsquo; s exceptional efficacy in restoring gut homeostasis after antibiotic disruption [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Overall, the co-culture group exhibited the highest α-diversity index, and its microbial community structure (β-diversity) most closely resembled that of the healthy group. Network analysis revealed more complex and stable microbial interactions in the co-culture group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRelative Abundance of Intestinal Flora in Mice\u003c/h2\u003e \u003cp\u003e16S rRNA sequencing revealed that lincomycin hydrochloride significantly altered the composition of the murine gut microbiota, while probiotic interventions variably restored microbial communities. At the phylum level, six dominant phyla were identified in both groups of mice: Bacteroidota, Firmicutes, Proteobacteria, Verrucomicrobiota, Actinobacteria, and Campilobacterota. Among these, Bacteroidota was the predominant phylum, with Verrucomicrobiota as the third most abundant. The natural recovery group exhibited a typical dysbiotic pattern, characterized by a significant 30.51% reduction in Bacteroidota abundance and a 17.63% increase in Verrucomicrobiota, disrupting the critical phylum-level balance necessary for intestinal homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, compared to the wild-type strain and the single recombinant strain, the co-culture group of \u003cem\u003eL. plantarum\u003c/em\u003e C8 with the recombinant strain demonstrated superior regulatory effects, showing a significant 50.96% increase in Bacteroidota abundance alongside a marked enrichment of Firmicutes. Ultimately, this group restored a microbial community profile most similar to that of the blank control group.\u003c/p\u003e \u003cp\u003eThe synergistic mechanism between recombinant strains and wild-type \u003cem\u003eL. plantarum\u003c/em\u003e C8 likely involves three coordinated actions of LPxTG proteins and signal peptides: enhanced bacterial adhesion prolongs intestinal colonization of both strains; overexpression of signal peptides persistently activates histidine kinase (HK), amplifying quorum sensing signals and stimulating increased antimicrobial production by C8[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; and this dual action not only directly suppresses pathogens but also modulates short-chain fatty acid (SCFA) production, promoting the proliferation of probiotics such as \u003cem\u003eBifidobacterium\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Furthermore, LPxTG proteins facilitate bacterial aggregation through homologous or heterologous interactions, which serves as the foundation for biofilm microcolony formation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Biofilms provide bacteria with a protective niche against external stressors, enabling more stable and persistent colonization within the gut [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This mechanism also plausibly explains the enhanced efficacy observed under co-culture conditions: the engineered strain acts as a structural \u0026ldquo;scaffold,\u0026rdquo; allowing wild-type bacteria to adhere and integrate, thereby collectively forming a more robust and functionally synergistic consortium.\u003c/p\u003e \u003cp\u003eAt the genus level, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE displays the top 20 most abundant genera in the mice. The natural recovery group exhibited significant microbial alterations, characterized by a marked reduction in beneficial bacteria\u0026mdash;specifically, \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e decreased by 32.28%, 41.95%, and 72.62%, respectively\u0026mdash;alongside a substantial proliferation of potential pathogens. Notably, the abundance of \u003cem\u003eEscherichia-Shigella\u003c/em\u003e increased significantly by 85.20%, and \u003cem\u003eKlebsiella\u003c/em\u003e by 91.37%. Previous studies have indicated that an increase in Klebsiella abundance can exacerbate intestinal inflammation, impair the gut barrier, and negatively affect the commensal microbiota in the mouse gut [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Administration of the \u003cem\u003eL. plantarum\u003c/em\u003e C8 strain led to a partial recovery of beneficial microbial taxa and contributed to the modulation of gut microbiota structure. Notably, the recombinant engineered strain exerted superior modulatory effects, as evidenced by a significant elevation in the abundance of \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e. This synergistic effect may be attributed to the co-expression system of the LPxTG protein and signal peptide, which confers unique biological properties to the recombinant strains. The LPxTG protein enhances the adaptive capacity of recombinant strains to the intestinal environment, facilitating better colonization and growth in the gut, thereby enabling more effective participation in quorum sensing and microbial regulation.\u003c/p\u003e \u003cp\u003eThe LPxTG anchoring mechanism enhances intestinal colonization of recombinant lactic acid bacteria through interactions between surface-anchored proteins and host epithelial receptors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. COG analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e reveals key functional adaptations supporting this process: cell wall/membrane biogenesis (Category M, 0.6\u0026ndash;0.8 abundance) indicates active peptidoglycan remodeling, while posttranslational modification (Category O, 0.4\u0026ndash;0.6) reflects sortase-mediated protein processing. These coordinated changes demonstrate the molecular basis for enhanced bacterial colonization. Studies have shown that the LPxT-GYLEQ protein significantly enhances the adhesion of \u003cem\u003eLactiplantibacillus reuteri\u003c/em\u003e (\u003cem\u003eL. reuteri\u003c/em\u003e) to intestinal epithelial cells, improving the colonization capacity of lactic acid bacteria strains [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Experimental results revealed that the relative abundance of Firmicutes significantly increased by 31.97% and 50.96% in the recombinant strain intervention group and the \u003cem\u003eL. plantarum\u003c/em\u003e C8\u0026thinsp;+\u0026thinsp;recombinant strain co-intervention group, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The abundance of \u003cem\u003eLactiplantibacillus\u003c/em\u003e exhibited a more pronounced elevation, reaching 241.94% and 331.34% in these groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Collectively, these findings provide compelling evidence that LPxTG proteins promote more effective intestinal colonization in a co-cultivation model of wild-type probiotics and engineered strains by modulating microbial ecological niches.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBeyond promoting colonization, the LPxTG motif enhances probiotic functionality through multiple mechanisms. In addition to improving the intestinal survival and adhesion properties of \u003cem\u003eL. reuteri\u003c/em\u003e, it amplifies the role of autoinducer-2 (AI-2) signaling molecules in quorum sensing [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, monotherapy with LPxTG-overexpressing engineered strains may exert excessive competitive pressure, limiting the recovery of beneficial commensal microbes and the overall restoration of gut microbiota following antibiotic exposure. In contrast, a co-cultivation strategy combining the engineered strain with its wild-type parental counterpart may establish an ecological buffering system. These findings align with those of Sophie et al., who demonstrated that overexpression of sRNA Ern0160 leads to reduced growth rates in vitro and a competitive disadvantage during intestinal colonization in vivo. This suggests that sRNA overexpression imposes a significant metabolic burden or fitness cost, thereby limiting efficacy in monoculture [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, the co-presence of the wild-type strain likely mitigates these deleterious effects at the population level. Through niche partitioning, functional complementarity, and metabolic cooperation, this two-strain consortium effectively occupies and defends key ecological niches against pathogen invasion while preserving resources and spatial availability for indigenous microbiota recolonization [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These synergistic effects enable the recombinant strain not only to better adapt to the gut microbial environment but also to engage in complex microbial interactions and regulate metabolic processes within co-culture systems involving the wild-type strain, ultimately resulting in superior probiotic efficacy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study confirms that LPxTG proteins and signal peptides significantly enhance the colonization capacity of probiotics in the gut and improve microbiota modulation within co-culture systems. The genetically engineered \u003cem\u003eL.plantarum\u003c/em\u003e C8 strain, displaying surface LPxTG domains, markedly increases the abundance of beneficial bacteria and effectively promotes the restoration of gut microbiota disrupted by antibiotics while inhibiting pathogen proliferation when co-cultured with the wild-type strain. This co-culture system alleviates antibiotic-induced dysbiosis through multiple mechanisms, including enhanced gut adaptability, improved villi development, and microbiota regulation. These findings provide a robust scientific foundation for developing functional probiotics as innovative alternatives to antibiotics in fermented foods, highlighting their significant potential for practical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eNotes\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ. Liang and J. Liu performed the research and Writing-original draft, A. Zhang and S. Zheng required the data and Formal analysis, T. Jiang performed the Visualization work, and L. Yang and X. Qi performed the Methodology, X. Zeng and Y. Guo required the Validation, D. Pan and T. Zhang performed the Supervision, and Z. Wu required the Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by This work was supported by the National Natural Science Foundation of China (32472356, 32402125), the Key Project of Ningbo Science and Technology (2023Z127), General Research Project of Zhejiang Provincial Education Department (Y202352124), Natural Science Foundation of Ningbo (2023J103), Open Fund for the State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (2021DG700024-KF202423).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDing, Q. C., F. W. Cao, S. L. Lai, H. Zhuge, K. X. Chang, T. G. Valencak, J. X. Liu, S. T. Li, and D. X. Ren. 2022. ZY08 relieves chronic alcohol-induced hepatic steatosis and liver injury in mice via restoring intestinal flora homeostasis. Food Res. 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The FASEB Journal 37(5):e22895.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1096/fj.2022-0252-RR\u003c/span\u003e\u003cspan address=\"10.1096/fj.2022-0252-RR\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"agricultural-products-processing-and-storage","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Agricultural Products Processing and Storage](https://link.springer.com/journal/44462)","snPcode":"44462","submissionUrl":"https://submission.springernature.com/new-submission/44462/3","title":"Agricultural Products Processing and Storage","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"antibiotic-associated diarrhea (AAD), Lactiplantibacillus plantarum C8, LPxTG protein, gut microbiome, co-culture system","lastPublishedDoi":"10.21203/rs.3.rs-8618174/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8618174/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo address the challenges of intestinal dysbiosis and antibiotic-associated diarrhea (AAD) caused by antibiotic treatment, this study investigated the potential of an LPxTG surface protein overexpression strategy for probiotic modulation. Specifically, we established a co-culture model comprising a recombinant \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e C8 strain (overexpressing LPxTG proteins) and the wild-type strain, and evaluated its efficacy in alleviating clindamycin-induced AAD in mice. Our findings indicate that the overexpression of LPxTG proteins enhanced the adhesive colonization capabilities and anti-inflammatory properties of the strain. Crucially, this co-culture model demonstrated significantly superior efficacy in regulating the intestinal microecology compared to single-strain models. Experimental data revealed that the co-culture system not only effectively restored the abundance and diversity of the compromised gut microbiota (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but also exerted protective effects via a dual mechanism: it significantly improved the immune microenvironment (reducing IL-6 and TNF-α while elevating IL-10) and repaired the intestinal physical barrier by upregulating Occludin and Claudin-1. These results confirm that constructing a co-culture microecological system utilizing LPxTG-overexpressing recombinant strains represents a highly efficient and superior probiotic therapeutic strategy for managing antibiotic-induced intestinal dysbiosis.\u003c/p\u003e","manuscriptTitle":"LPxTG Proteins Mediate Microbial Interactions to Modulate Lactiplantibacillus plantarum C8 in Alleviating Antibiotic-Associated Diarrhea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 20:19:47","doi":"10.21203/rs.3.rs-8618174/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-16T03:08:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-11T06:15:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2836719197933679394523817403424802488","date":"2026-03-02T11:30:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-24T03:30:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86595571470653420542328309512062504210","date":"2026-01-21T05:27:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254722576301572043874240178449129932366","date":"2026-01-19T03:15:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T02:33:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T01:27:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-16T11:04:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Agricultural Products Processing and Storage","date":"2026-01-16T09:44:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"agricultural-products-processing-and-storage","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Agricultural Products Processing and Storage](https://link.springer.com/journal/44462)","snPcode":"44462","submissionUrl":"https://submission.springernature.com/new-submission/44462/3","title":"Agricultural Products Processing and Storage","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca85e147-9c2c-4204-b290-b0e9220ede1e","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-15T05:55:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 20:19:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8618174","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8618174","identity":"rs-8618174","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}