ATPIF1 deficiency Significantly Alleviates Citrobacter rodentium-Induced Ulcerative Colitis in Mice

ATPIF1 deficiency Significantly Alleviates Citrobacter rodentium-Induced Ulcerative Colitis in Mice | 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 ATPIF1 deficiency Significantly Alleviates Citrobacter rodentium -Induced Ulcerative Colitis in Mice Haoyu Yang, Ziqi Li, Dong Yan, Min Li, Lingyun Xu, Yuxin Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8938653/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Mitochondrial ATP synthase inhibitory factor 1 (ATPIF1) is a crucial regulator of cellular energy metabolism and has been implicated in inflammatory disorders. However, its role in bacterial infection-driven ulcerative colitis (UC) remains unclear. Purpose This study aimed to investigate the effects of ATPIF1 on host susceptibility and inflammatory responses in a Citrobacter rodentium -induced infectious colitis model. Methods ATPIF1 knock out (KO) and wild type (WT) mice were orally gavaged with C. rodentium to induce infectious colitis. Body weight, disease activity index (DAI), and colon length were recorded. Histopathology, Alcian blue staining, and immunohistochemistry were performed to assess mucosal integrity and barrier function. Inflammatory responses were evaluated through immunohistochemistry, RT-qPCR, and Western blotting, while gut microbiota composition was analyzed via 16S rRNA gene sequencing. Results ATPIF1 deficiency alleviated C. rodentium -induced colitis, as evidenced by reduced weight loss, lower DAI scores, and attenuated colon shortening. KO mice preserved epithelial architecture, exhibited increased numbers of goblet cells and ZO-1 mRNA expression, indicating an intact mucosal barrier. Furthermore, KO mice showed reduced infiltration of inflammatory cells, decreased expression of IL-1β and TNF-α, and suppressed activation of the NLRP3 inflammasome pathway. Microbiota analysis also revealed that ATPIF1 deficiency stabilized bacterial community composition and reduced pathogenic expansion. Conclusion ATPIF1 deficiency significantly alleviates C. rodentium -induced colitis by mitigating inflammation, preserving mucosal barrier function, promoting pathogen clearance, and stabilizing gut microbiota. These findings suggest that ATPIF1 may represent a potential therapeutic target for infection-associated UC. mitochondrial ATPase inhibitory factor 1 (ATPIF1) Ulcerative colitis (UC) Citrobacter rodentium (CR) NOD-like receptor protein 3 (NLRP3) Microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ulcerative colitis (UC) is a major subtype of inflammatory bowel disease (IBD), characterized by chronic inflammation of the colonic mucosa, neutrophilic infiltration, and ulcer formation. These pathological features result from dysregulated immune responses, genetic predisposition, and alterations in gut microbiota composition[ 1 – 4 ]. Despite significant advances in molecular biology that have elucidated multiple several pathways—including cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), immune cell subsets such as T helper 17 (Th17) cells and regulatory T cells (Tregs), and complex host–microbiota interactions[ 5 – 7 ]—the precise mechanisms underlying UC pathogenesis remain incompletely defined[ 8 ]. The murine enteric pathogen Citrobacter rodentium (CR) serves as a well-established model that recapitulates key aspects of human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections. As an attaching and effacing (A/E) pathogen, C . rodentium infection provokes acute host immune responses, including the activation of inflammatory cytokines and recruitment of leukocytes, which facilitate pathogen clearance[ 9 ]. C . rodentium -induced colitis has been extensively characterized as a model of infectious colitis, with pathological and immunological alterations closely mirroring those observed in IBD, thereby providing an ideal platform for investigating immune-mediated bacterial colitis[ 10 ]. Unlike chemically induced colitis models such as dextran sulfate sodium (DSS), C . rodentium infection markedly alters gut microbiota composition and function, indirectly compromising mucosal barrier integrity. Chronic inflammation in IBD patients is hypothesized to result from aberrant immune responses to gut microbiota, particularly in genetically susceptible hosts. Consequently, infectious colitis models offer critical insights into host pathological responses to enteric bacteria[ 10 ]. Emerging evidence further implicates host–microbiota interactions in the regulation of inflammasome activation[ 11 ]. Among the immune mechanisms implicated in UC, the NLRP3 inflammasome plays a central role by sensing microbial and danger-associated signals, activating caspase-1, and promoting the secretion of pro-inflammatory cytokines IL-1β and IL-18, thereby amplifying mucosal inflammation[ 12 , 13 ]. C . rodentium infection activates Toll-like receptor signaling pathways, upregulates NLRP3 and pro-IL-1β expression, and enhances NLRP3 inflammasome activation[ 14 ]. Thus, the C . rodentium -induced colitis model constitutes a valuable tool for dissecting the complex interplay among gut dysbiosis, mucosal immune responses, and epithelial barrier integrity in UC pathogenesis. Mitochondrial ATP synthase inhibitory factor 1 (ATPIF1) is a small (~ 10 kDa) nuclear-encoded protein localized within the mitochondrial matrix. It modulates the activity of the F1Fo-ATP synthase complex, thereby influencing cellular energy metabolism, with established roles in cancer, cardiovascular diseases, and other pathological conditions[ 15 , 16 ]. Notably, ATPIF1 exhibits context-dependent functions: it is overexpressed in various malignancies, where it promotes glycolysis and inhibits apoptosis, facilitating tumor proliferation[ 17 ]; conversely, during ischemic events, ATPIF1 mitigates cellular ATP consumption, reducing reperfusion-induced cell death[ 16 ]. This dual functionality underscores ATPIF1 as a critical regulator of cellular energy homeostasis[ 18 ]. In the context of inflammation, mitochondrial dysfunction-induced oxidative stress correlates positively with NLRP3 inflammasome activation, suggesting that ATPIF1, by modulating ATP synthesis, may influence inflammasome-mediated inflammatory responses[ 19 ]. Given that mitochondrial function modulates host–microbiota interactions and that gut dysbiosis is closely linked to UC pathogenesis, it is imperative to investigate the effects of ATPIF1 deficiency on microbial ecosystems under inflammatory conditions. Previous studies demonstrated that ATPIF1 inactivation ameliorated DSS-induced colitis, although this was limited to a chemical colitis model[ 20 ]. Additionally, inhibition of ATPIF1 activity was reported to enhance murine neutrophil antibacterial function, primarily through increased reactive oxygen species (ROS) and lactic acid production[ 21 ]. These findings suggest a potential association between ATPIF1 deficiency and the development of bacterial infection-driven UC. While ATPIF1 is recognized as a regulator of cellular energy metabolism and inflammation, its specific roles in host defense and mucosal immunity during enteric bacterial infection remain unclear. To address this knowledge gap, the present study employed a C . rodentium -induced murine model to investigate the impact of ATPIF1 deficiency on colonic inflammation, inflammasome activation, and gut microbiota composition. Utilizing histopathological evaluation, quantification of inflammatory markers, and 16S rRNA gene sequencing, this research aims to elucidate the molecular and microbial mechanisms by which ATPIF1 contributes to UC pathogenesis, thereby highlighting its potential as a therapeutic target in bacterial infection-driven UC. Materials and Methods 2.1 Experimental Animals and Housing The generation of ATPIF1 deficiency (ATPIF1 −/− , KO) mice has been previously documented[ 20 ]. Male SPF C57BL/6 wild-type and ATPIF1 −/− mice aged 6 to 8 weeks were maintained under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, relative humidity of 40%–60%, and a temperature of 22 ± 2°C. Mice were provided with unrestricted access to food and sterile water. Following a 7-day acclimation period, experimental procedures were conducted. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Xinxiang Medical University and were performed in accordance with established institutional guidelines. 2.2 Bacterial Culture and Induction of Colitis The Citrobacter rodentium strain (ATCC 51459) was kindly supplied by Professor Zhiping Liu of Gannan Medical University. Frozen stocks of C. rodentium were inoculated into 5 mL of LB broth and incubated overnight at 37°C with agitation at 180 rpm. Subsequently, bacterial cultures were streaked onto MacConkey selective agar plates and incubated overnight. A single colony was then picked and transferred to fresh LB broth for activation[ 22 ]. The mice were randomly assigned to four groups (n = 5 per group): WT (wild type) group, WT + CR ( C. rodentium -induced WT colitis) group, KO (ATPIF1 −/− ) group and KO + CR ( C. rodentium -induced ATPIF1 −/− colitis) group. The experimental duration was 7 days. Mice in the WT + CR and KO + CR groups received a daily oral gavage of 200 µL of C. rodentium suspension (6 × 10⁸ CFU/mice)[ 23 ]. Control groups (WT and KO) were administered an equivalent volume of sterile phosphate-buffered saline (PBS) via oral gavage. At the conclusion of the study (day 7), all mice were euthanized for further analysis. 2.3 Body Weight Monitoring and Disease Activity Index (DAI) Body weight was measured daily, and changes in weight were expressed as a percentage relative to the baseline body weight recorded on day 0. Disease Activity Index (DAI) scores were evaluated according to the criteria established by Murthy et al.[ 24 ], incorporating parameters such as body weight loss, stool consistency, and the presence of fecal occult blood. Rectal bleeding was assessed using a fecal occult blood test kit (Baso Biotechnology Co., Ltd., Guangdong, China). The overall DAI score was calculated as the mean of the three individual component scores. 2.4 Fecal Sample Collection and Colon Length Measurement On the seventh day, fresh fecal samples were collected and promptly stored at − 80°C until further analysis. After euthanasia by cervical dislocation, the colon, including the anal segment, was carefully dissected, placed on a white background, and photographed. The length of the colon was then measured using a ruler. 2.5 16S rRNA Sequencing of Fecal Microbiota Genomic DNA was isolated from fecal specimens using the Soil DNA Isolation Kit (Omega BioTek, Norcross, GA, USA) according to the manufacturer’s instructions. The quality and integrity of the extracted DNA were evaluated by electrophoresis on a 0.8% agarose gel. DNA concentration and purity were measured spectrophotometrically at 260 nm and 280 nm wavelengths using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). DNA samples that met quality criteria were stored at − 20°C for subsequent analyses. To characterize the microbial community, the V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified using the primer pair 338F (5′-ACTCCTACGGGAGGCAGC-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), quantified, and normalized to uniform concentrations. Paired-end sequencing (2 × 300 bp) was performed on the Illumina MiSeq platform by MajorBio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR35154250 to SRR35154272. 2.6 H&E and Alcian Blue Staining of Colon Tissue Tissue samples from the distal colon, obtained proximal to the anal verge, were fixed in 4% paraformaldehyde. Subsequently, the specimens were dehydrated through a graded ethanol series, followed by clearing with xylene. The tissues were then embedded in paraffin and sectioned at a thickness of 5 µm. The resulting sections were stained with hematoxylin and eosin (H&E) as well as Alcian Blue. 2.7 Histological Scoring and Quantification of Goblet Cells Histological scoring of H&E-stained colon sections was conducted using the criteria proposed by Johansson[ 25 ], encompassing five parameters: inflammatory cell infiltration, mucosal thickening, goblet cell depletion, crypt loss, and epithelial damage. Each parameter was assigned a severity score ranging from 0 to 4, and the overall histological score was calculated by summing these individual scores. Additionally, Alcian Blue-stained sections were quantitatively analyzed using ImageJ software to measure the area exhibiting positive goblet cell staining. 2.8 RNA Extraction and RT-qPCR Total RNA was isolated from colon tissues using TRIzol reagent (Takara, Dalian, China). Subsequently, cDNA was synthesized using PrimeScript RT Master Mix and stored at -80℃ for further analysis (Takara, Dalian, China). Quantitative real-time PCR was performed on a StepOne™ Real-Time PCR System (Life Technologies, USA) using SYBR Green PCR Master Mix (Takara) to assess gene expression. Expression levels of key inflammatory cytokines, including TNF-α, IL-1β, and IL-6, as well as the epithelial tight junction marker ZO-1, were quantified using gene-specific primers. GAPDH served as the internal control. Relative gene expression was calculated using the 2 −ΔΔCt method. Primer sequences used in this study are listed in the Table.S1 2.9 Immunohistochemistry Analysis of Colon Tissue Paraffin-embedded tissue sections were first deparaffinized and subjected to antigen retrieval via microwave heating. Endogenous peroxidase activity was inhibited using hydrogen peroxide (H₂O₂), followed by a 30-minute blocking step with ready-to-use goat serum (BosterBio, Wuhan, China). Subsequently, the sections were incubated overnight at 4°C with primary polyclonal antibodies against Cleaved Caspase-1 IgG, ASC IgG, and NLRP3 IgG, each diluted 1:200 (Wanleibio, Shenyang, China). After equilibration to room temperature, the sections were treated with appropriate secondary antibodies and visualized using a diaminobenzidine (DAB) detection kit (ShareBio, Shanghai, China). Nuclear counterstaining was performed with hematoxylin. Following dehydration and clearing through graded ethanol and xylene, the sections were mounted using neutral resin. Imaging was conducted with a fluorescence microscope (Leica, Germany) under standardized exposure conditions. Quantitative analysis of the immunohistochemically positive areas was performed using ImageJ software. 2.10 Statistical Analysis Statistical analyses were performed using GraphPad Prism version 5.0 (Boston, MA, USA). Data are presented as the mean ± standard error of the mean (SEM). Differences between two groups were assessed using an unpaired Student’s t -test, while comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). A p -value less than 0.05 was considered statistically significant. 2.11 Data Availability Raw sequence data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR35154250–SRR35154272. These data correspond to 16S rRNA gene amplicon sequencing of the V3–V4 region, amplified using primers 338F (5′-ACTCCTACGGGAGGCAGC-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). All other data supporting the findings of this study are available within the article and its Supplementary Information files. Result 3.1 ATPIF1 deficiency attenuated disease severity in C . rodentium -induced colitis. To assess colitis severity, we evaluated colorectal length, body weight loss, and disease activity index (DAI) scores across experimental groups. The experimental timeline for C. rodentium -induced colitis is shown in Fig. 1 a. As illustrated in Fig. 1 b and c, colon shortening in the KO + CR group was significantly less pronounced than in the WT + CR group ( P < 0.05). Additionally, the KO + CR group exhibited significantly less weight loss compared to the WT + CR group (Fig. 1 d, P < 0.05). Correspondingly, DAI scores were markedly lower in the KO + CR group than in WT + CR mice (Fig. 1 e, P < 0.01). Collectively, these findings indicate that ATPIF1 deficiency attenuates the severity of C . rodentium -induced colitis. 3.2 ATPIF1 deficiency reduces C. rodentium burden in feces and colon tissue. To assess the impact of ATPIF1 deficiency on bacterial load, we quantified C. rodentium colonization in fecal samples and colonic tissues using MacConkey agar selective medium at the study endpoint. As shown in Fig. 2 a, fecal C. rodentium levels were significantly elevated in the WT + CR group compared to WT controls ( P < 0.001), whereas the KO + CR group exhibited a significant reduction relative to WT + CR mice ( P < 0.05). Similarly, bacterial load within colon tissues was markedly decreased in KO + CR mice compared to WT + CR counterparts (Fig. 2 b, P < 0.001). These findings suggest that ATPIF1 deficiency enhances the host’s ability to clear opportunistic pathogens during C . rodentium -induced colitis. 3.3 ATPIF1 deficiency alleviated tissue damage and preserved mucosal barrier integrity in C . rodentium -induced colitis. Histological analyses using hematoxylin and eosin (H&E) and Alcian Blue staining were performed to asses tissue damage and mucosal barrier integrity. The pathological score in WT + CR mice was significantly elevated (12.5 ± 1.12) compared to WT controls ( P < 0.001), characterized by disrupted colonic epithelium, extensive inflammatory cell infiltration, and pronounced submucosal edema. In contrast, KO + CR mice exhibited a significantly lower pathological score (5.6 ± 0.75) compared with WT + CR mice ( P < 0.01), with preservation of epithelial architecture, reduced edema, and attenuated inflammation (Fig. 3 a, b). In parallel, Alcian Blue staining revealed a significant decrease in goblet cell percentage in WT + CR mice (1.76% ± 0.16) versus WT controls (2.95% ± 0.18, P < 0.001), whereas KO + CR mice showed a significant increase in goblet cell abundance (4.32% ± 0.37) compared to WT + CR ( P < 0.001, Fig. 3 c and d). These data indicate that ATPIF1 deficiency not only mitigates C . rodentium -induced pathological damage but also maintains mucosal barrier integrity by preserving goblet cell populations, thereby suppressing inflammatory responses. 3.4 ATPIF1 deficiency suppresses inflammatory cytokine expression in C . rodentium -induced colitis. To further investigate the impact of ATPIF1 deficiency on intestinal inflammatory responses, we measured the relative mRNA abundance of key inflammatory cytokines in the colonic tissue of C . rodentium -induced UC mice. As shown in Fig. 4 a-c, the expression levels of IL-6 (0.64 ± 0.08), IL-1β (0.46 ± 0.20), and TNF-α (0.0038 ± 0.0013) in the KO + CR group were significantly lower than those in the WT + CR group ( P < 0.001, P < 0.05 and P < 0.001, respectively). Additionally, as illustrated in Fig. 4 d, the relative abundance of the tight junction protein ZO-1 in the KO + CR group (2.35 ± 0.37) was significantly higher than that in the WT + CR group (0.41 ± 0.09, P < 0.001). These findings suggest that ATPIF1 deficiency ameliorates colonic injury and preserves mucosal integrity, likely by maintaining of goblet cell numbers and attenuating inflammatory cytokine production. 3.5 ATPIF1 deficiency enhances the expression of NLRP3 signaling-associated proteins in C . rodentium -induced colitis. When analyzing the mRNA expression levels of inflammatory cytokines, we observed that IL-6, IL-1β and TNF-α levels were significantly downregulated in the KO + CR group. The NLRP3 inflammasome recognizes pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), assembling with ASC and pro-caspase-1 to form a multiprotein complex that activates caspase-1 [ 26 ], leading to the maturation and secretion of IL-1β and IL-18 [ 13 ]. TNF-α can enhance NLRP3 activation and the inflammatory response, with these inflammatory factors constituting a pro-inflammatory positive feedback loop [ 12 ]. To investigate whether ATPIF1 deficiency alleviates intestinal inflammatory responses in the colon of C . rodentium -induced mice via the NLRP3 signaling pathway, we measured the expression of NLRP3 signaling-related proteins ASC, Cleaved Caspase-1, and NLRP3. The results showed that the inflammasome signaling pathway was abnormally active in the WT + CR group, with markedly increased expression of ASC, Cleaved Caspase-1 and NLRP3 (Fig. 5 a-d). In contrast, the expression levels of ASC, Cleaved Caspase-1 and NLRP3 were significantly decreased in the KO + CR group (Fig. 5 a-d, P < 0.0001). These findings suggest that ATPIF1 deficiency mitigates C . rodentium -induced colonic inflammation by inhibiting NLRP3 inflammasome signaling. 3.6 ATPIF1 deficiency alleviates gut microbiota dysbiosis C . rodentium -induced colitis in mice. Gut microbiota dysbiosis is implicated in the pathogenesis of ulcerative colitis. To assess the influence of ATPIF1 deficiency on the gut microbial composition in mice with C. rodentium -induced ulcerative colitis (UC), we performed high-throughput sequencing of the 16S rRNA gene from mouse fecal samples. No significant difference was observed in alpha diversity indices among the WT, WT + CR, KO, and KO + CR groups. However, beta-diversity analysis revealed marked alterations in the gut microbiota composition. Principal coordinates analysis (PCoA) based on Bray-Curtis distances at the ASV level demonstrated that the microbial communities of the KO and WT groups partially overlapped, although distinct differences were observed. In contrast, after C. rodentium infection, the microbial compositions of WT + CR and KO + CR groups were separated from each other obviously, indicating substantial structural divergence in the gut microbiota between these groups (Fig. 6 a). The normalized stochasticity ratio (NST) values indicated that gut microbial assembly in both WT and KO groups under basal conditions was predominantly governed by stochastic processes (Fig. 6 b). The influence of stochasticity was increased in WT + CR group (86.03 ± 9.62%), suggesting disrupted gut environmental selection ( P < 0.001). Conversely, the NST value in the KO + CR group significantly decreased to 53.19 ± 8.65%, indicating a shift in microbial community assembly toward more deterministic processes. This shift may be attributed to the alteration of the host intestinal environment caused by ATPIF1 deficiency, whereby enhanced host-mediated selection pressure led to a more structured and regulated microbial composition. At the genus level, the microbial community composition revealed distinct differences among groups ( Fig. S1 a). After C. rodentium- induced colitis, the abundances of Citrobacter and Roseburia were significantly reduced in the KO + CR group (Fig. 6 c and d, P < 0.05), while Odoribacter was significantly enriched in the WT + CR group Fig. 6 e, P < 0.05). In contrast, Lachnospiraceae_NK4A136_group and Rikenella were markedly decreased in the WT + CR group, with no significant reduction was induced in the KO + CR group (Fig. 6 f and g, P < 0.05). Notably, a genus designated A2 , which was rarely reported, was significantly enriched in the WT + CR group Fig. 6 h). Characteristic genera of each group were further identified using linear discriminant analysis coupled with effect size (LEfSe). As shown in Fig. 6 i, unclassified Clostridia_UCG-014 , RikenellaceaeRC9 group , Roseburia , and Ruminococcus were identified as signature taxa of the WT + CR group, while Muribaculum , Dubosiella , Turicimonas , Tyzzerella, and Rikenella were the characteristic genera enriched in the KO + CR group. The correlations between the top 30 most abundant genera and various environmental factors, including DAI score, colon length, pathological score, and key components of the NLRP3 signaling pathway (ASC, cleaved Caspase-1, and NLRP3), were analyzed to explore the relationship between gut microbiota and host-associated inflammatory parameters Fig. 6 j). Colon length showed positive correlations with Parasutterella , Turicibacter , and Lachnospiraceae_NK4A136_group , but was negatively correlated with Citrobacter , Rikenellaceae_RC9_gut_group , and Odoribacter . In contrast, indicators of colitis severity, including DAI score, pathological score, and NLRP3 pathway-related proteins, were positively associated with Citrobacter , Odoribacter , and unclassified_f__Enterobacteriaceae , and negatively associated with Parasutterella , Dubosiella , and Turicibacter . The network analysis between the top 50 most abundant genus and environmental factors also showed that the severity of colitis was positively related to Citrobacter and Odoribacter , while negatively related to Allobaculum , Turicibacter , Paraprevotella and Rikenella (Fig. 6 k). Predicted functional analysis of microbial metabolic pathways revealed that, at KEGG level 3, Fatty acid biosynthesis, the AMPK signaling pathway and the NOD-like receptor signaling pathway were significantly downregulated in the KO + CR group compared with these in the WT + CR group ( P < 0.05). Interestingly, in the COG functional classification, the ATPase activity pathway was also markedly suppressed in the KO + CR group ( P < 0.05). The reduced ATPase activity observed in KO mice may reflect a mitochondrial adaptation that stabilizes the membrane potential without requiring excessive ATP hydrolysis ( Fig. S1 b and c). Discussion The global incidence of ulcerative colitis (UC) has risen substantially, underscoring the critical need for effective preventive and therapeutic interventions. UC is a complex disorder influenced by genetic predisposition, immune system dysregulation, and environmental factors[ 27 ]. Emerging evidence demonstrates significant differences in gut microbiota composition between UC patients and healthy controls, positioning the microbiota as a pivotal mediator in host-pathogen interactions during disease pathogenesis[ 28 ]. Although our previous research demonstrated that ATPIF1 deficiency effectively ameliorates DSS-induced colitis[ 20 ], its role in infection-driven intestinal inflammation and gut microbiota regulation remains inadequately characterized. In the present investigation, utilizing a C . rodentium infection-induced colitis model, we observed that ATPIF1 deficiency significantly attenuates C . rodentium -induced colitis. This protective effect correlates with suppression of the NLRP3 inflammasome pathway and preservation of gut microbial homeostasis, thereby identifying ATPIF1 as a promising therapeutic target in UC. C. rodentium is a natural murine attaching and effacing (A/E) pathogen capable of inducing transmissible colonic hyperplasia[ 29 ]. Pathogen invasion is closely associated with infectious diarrhea and colitis, with C . rodentium -infected mice exhibiting pronounced colitis symptoms, including weight loss, colon shortening, and fecal occult blood[ 28 ]. Importantly, C . rodentium infection does not require antibiotic pretreatment, establishing it as a primary model for studying A/E pathogen-induced intestinal inflammation[ 30 ]. In C57BL/6 mice, C . rodentium infection progresses through four stages: Establishment, with C . rodentium colonization in cecal patches; Expansion, characterized by distal colon colonization, crypt hyperplasia, and epithelial barrier disruption; Steady-state, with stable C . rodentium shedding (~ 10⁹ CFU/g feces) accompanied by neutrophil and Th17/Th22 cell-mediated IL-17A and IL-22 secretion, the absence of which is lethal; and Clearance, wherein CD4⁺ T cells shift from IL-17A/IL-22 to IFN-γ-dominated responses, and IFN-γ deficiency delays pathogen clearance. C . rodentium is ultimately eliminated through IgG-mediated opsonization, neutrophil phagocytosis, and competition with commensal bacteria. As a primary defense mechanism, activated intestinal epithelial cells secrete antimicrobial peptides, serum amyloid A (SAA), and reactive oxygen species (ROS) to restrict bacterial adherence[ 31 ]. Innate immune recognition is further mediated by Toll-like receptors (TLRs), intracellular NOD-like receptors (NLRs), and signals from damaged epithelial cells, collectively orchestrating infection sensing and response [ 32 ]. Adaptive immunity, particularly involving CD4⁺ T and B lymphocytes, is essential for pathogen clearance[ 33 ]. C . rodentium infection elicits robust innate immune activation, with the NLRP3 inflammasome serving as a central signaling nexus. Consistently, we also observed elevated activation of the NLRP3 inflammasome signaling pathway in the disease group. Lipopolysaccharide (LPS) derived from C . rodentium is detected by epithelial and immune cells, including macrophages and dendritic cells, activating the TLR4/MyD88/NF-κB signaling cascade to induce expression of NLRP3, pro-IL-1β, and pro-IL-18, thereby priming inflammasome assembly[ 34 ]. Additionally, C . rodentium utilizes a Type III Secretion System (T3SS) to translocate effector proteins that disrupt mitochondrial function and compromise tight junction integrity[ 35 ]. Excessive ROS production, originating from mitochondria and NADPH oxidase, further promotes NLRP3 activation. The inflammasome complex, comprising NLRP3, ASC, and pro-Caspase-1, facilitates the secretion of IL-1β and IL-18, recruitment of neutrophils, and propagation of intestinal inflammation. NLRs also mediate pyroptosis, an inflammatory form of programmed cell death in myeloid cells[ 19 ]. Our data demonstrate that ATPIF1 deficiency significantly suppresses NLRP3 signaling in mice, which likely underlies the preservation of mucosal architecture and attenuation of inflammatory infiltration observed (Fig. 5 a-d). Previous studies have indicated that NLRP3 activity is negatively regulated by autophagy but positively modulated by reactive oxygen species (ROS) derived from damaged organelles. Mitochondrial dysfunction leads to ROS accumulation, thereby linking organelle impairment to inflammasome-mediated inflammation[ 21 ]. Mitochondrial ATP synthesis predominantly depends on F1Fo-ATP synthase, whose activity is inhibited by ATPIF1 overexpression, resulting in increased oxidative stress and amplified inflammatory responses[ 20 ]. Consistent with this, ATPIF1 deficiency reduced pro-inflammatory cytokine levels in our study (Fig. 4 a-c) Moreover, prior investigations employing peritonitis models revealed that ATPIF1 loss enhances glycolytic flux and augments neutrophil antimicrobial activity via ROS and lactate production[ 21 ]. Together with the observation that ATPIF1 deficiency mitigates C . rodentium -induced colitis by preserving epithelial barrier integrity, these findings suggest that both the epithelial protection and the enhanced neutrophil function may contribute to disease amelioration. Colitis progression is frequently accompanied by alterations in gut microbiota composition. While α-diversity remained unchanged, β-diversity differed significantly between the WT + CR and KO + CR groups, suggesting that ATPIF1 deficiency reshapes bacterial community composition without affecting richness or evenness. Standardized neutrality ratio (NST) analysis revealed that microbial assembly in KO + CR mice shifted toward a more deterministic pattern, indicating preferential recovery of key functional taxa that may promote SCFA metabolism and enhance host resistance to stress and pathogens. In contrast, microbiota in WT + CR assembly was primarily stochastic. Correlation analyses between bacterial genera and inflammasome components further support bidirectional interactions: microbial metabolites may modulate NLRP3 activation, while inflammation-induced environmental changes can promote the expansion of pathogenic taxa. Under C . rodentium -induced stress, ATPIF1-deficient mice maintained beneficial taxa such as Dubosiella , Rikenella , and Parasutterella compared to WT mice. Dubosiella produces short-chain fatty acids (SCFAs) to balance Treg/Th17 responses and improve mucosal barrier integrity, and through the IDO1-Kynurenine pathway, enhance immune tolerance via dendritic cell-mediated tryptophan metabolism. Rikenella is considered a potential probiotic, while Parasutterella is a core component of murine and human gut microbiota, producing succinate, a critical intermediate in microbial metabolic networks[ 3 , 36 , 37 ]. Preservation of these taxa likely contributes to microbial stability in KO + CR mice. Downregulation of fatty acid metabolism at the KEGG Level 3 pathway indicates reduced inflammation and stabilized microbial composition, whereas suppression of the AMPK pathway reflects diminished cellular stress and inflammatory state, obviating the need for intensive metabolic regulation[ 38 ]. Similarly, NOD-like receptor pathway activity aligns with inflammasome status [ 39 ]. Our KEGG functional predictions corroborate these observations. Additionally, ATP synthase activity was suppressed in KO mice, suggesting a potential mitochondrial energy-conserving adaptation. Collectively, these findings imply that ATPIF1 deficiency modulates host metabolism, indirectly influences microbial function, and that gut microbiota remodeling may mediate its protective effects. Despite observing reduced expression of NLRP3 inflammasome-related proteins, upstream mechanisms—including mitochondrial ROS generation, ASC speck formation, and TLR4 activity—warrant further elucidation. Functional validation of specific microbial taxa, such as Roseburia , will be instrumental in clarifying their contributions to the protective phenotype observed in ATPIF1-deficient mice. In summary, this study provides novel evidence that ATPIF1 deficiency alleviates C . rodentium -induced colitis by suppression of NLRP3 inflammasome activation and modulation of gut microbial composition. This work addresses a critical gap in infection-driven UC models and confirms that ATPIF1 exerts protective effects in both DSS and C . rodentium -induced UC [ 20 ]. These findings reveal a previously unrecognized role of ATPIF1 at the nexus of mitochondrial metabolism, immune regulation, and microbial homeostasis, highlighting its potential as a metabolic therapeutic target for infection-associated UC with significant translational implications. Declarations Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution HY: Writing – original draft, Investigation, Data curation. ZL: Writing – original draft, Investigation, Data curation.LX: Methodology.YX Wang: Methodology.DY: Methodology.ML: Methodology.GZ: Supervision, Funding acquisition, Conceptualization.MW: Supervision, Conceptualization, Data curation, Writing – review & editing. Acknowledgement The author(s) declared that financial support was received for this work and/or its publication. This work was funded by Hunan Natural Science Foundation Project (2026JJ80001) awarded to GZ, the Scientific Research Project of the Education Department of Hunan Province (25A0637) awarded to GZ, and the NSFC-Henan Union grant (No. U1904131) to GZ. Data Availability Raw sequence data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR35154250–SRR35154272. These data correspond to 16S rRNA gene amplicon sequencing of the V3–V4 region, amplified using primers 338F (5′-ACTCCTACGGGAGGCAGC-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). All other data supporting the findings of this study are available within the article and its Supplementary Information files. References Wangchuk, P., K. Yeshi, and A. Loukas, Ulcerative colitis: clinical biomarkers, therapeutic targets, and emerging treatments. Trends Pharmacol Sci, 2024. 45 (10): p. 892-903. Ruel, J., et al., IBD across the age spectrum: is it the same disease? Nat Rev Gastroenterol Hepatol, 2014. 11 (2): p. 88-98. Zhang, X., et al., An Orally-Administered Nanotherapeutics with Carbon Monoxide Supplying for Inflammatory Bowel Disease Therapy by Scavenging Oxidative Stress and Restoring Gut Immune Homeostasis. ACS Nano, 2023. 17 (21): p. 21116-21133. Voelker, R., What Is Ulcerative Colitis? Jama, 2024. 331 (8): p. 716. Levin, A. and O. Shibolet, Toll-like receptors in inflammatory bowel disease-stepping into uncharted territory. World J Gastroenterol, 2008. 14 (33): p. 5149-53. Globig, A.M., et al., Comprehensive intestinal T helper cell profiling reveals specific accumulation of IFN-γ+IL-17+coproducing CD4+ T cells in active inflammatory bowel disease. Inflamm Bowel Dis, 2014. 20 (12): p. 2321-9. Dai, Y., et al., Xianglian Pill attenuates ulcerative colitis through TLR4/MyD88/NF-κB signaling pathway. 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Zhong, G., et al., IF1 inactivation attenuates experimental colitis through downregulation of neutrophil infiltration in colon mucosa. Int Immunopharmacol, 2021. 99 : p. 107980. Zhong, G., et al., Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid. Biochim Biophys Acta Mol Basis Dis, 2023. 1869 (8): p. 166820. Crepin, V.F., et al., Citrobacter rodentium mouse model of bacterial infection. Nat Protoc, 2016. 11 (10): p. 1851-76. Zheng, Y., et al., Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med, 2008. 14 (3): p. 282-9. Brown, E.M., D.J. Kenny, and R.J. Xavier, Gut Microbiota Regulation of T Cells During Inflammation and Autoimmunity. Annu Rev Immunol, 2019. 37 : p. 599-624. Johansson, M.E., et al., Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis. Gut, 2014. 63 (2): p. 281-91. Blevins, H.M., et al., The NLRP3 Inflammasome Pathway: A Review of Mechanisms and Inhibitors for the Treatment of Inflammatory Diseases. Front Aging Neurosci, 2022. 14 : p. 879021. Ungaro, R., et al., Ulcerative colitis. Lancet, 2017. 389 (10080): p. 1756-1770. Feng, Y., et al., Lactobacillus plantarum-derived extracellular vesicles from dietary barley leaf supplementation attenuate Citrobacter rodentium infection and intestinal inflammation. J Nanobiotechnology, 2025. 23 (1): p. 426. Mundy, R., et al., Citrobacter rodentium of mice and man. Cell Microbiol, 2005. 7 (12): p. 1697-706. Wiles, S., et al., Modelling infectious disease - time to think outside the box? Nat Rev Microbiol, 2006. 4 (4): p. 307-12. Sano, T., et al., An IL-23R/IL-22 Circuit Regulates Epithelial Serum Amyloid A to Promote Local Effector Th17 Responses. Cell, 2015. 163 (2): p. 381-93. Goto, Y., Epithelial Cells as a Transmitter of Signals From Commensal Bacteria and Host Immune Cells. Front Immunol, 2019. 10 : p. 2057. Yang, W., et al., GPR120 Inhibits Colitis Through Regulation of CD4(+) T Cell Interleukin 10 Production. Gastroenterology, 2022. 162 (1): p. 150-165. Friedrich, C., et al., MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium. PLoS Pathog, 2017. 13 (5): p. e1006357. Biswas, P., et al., The accessory type III secretion system effectors collectively shape intestinal inflammatory infection outcomes. Gut Microbes, 2025. 17 (1): p. 2526134. Ju, T., et al., Defining the role of Parasutterella, a previously uncharacterized member of the core gut microbiota. Isme j, 2019. 13 (6): p. 1520-1534. Zhang, Y., et al., Dubosiella newyorkensis modulates immune tolerance in colitis via the L-lysine-activated AhR-IDO1-Kyn pathway. Nat Commun, 2024. 15 (1): p. 1333. Malik, N., et al., Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1. Science, 2023. 380 (6642): p. eabj5559. Wen, H., E.A. Miao, and J.P. Ting, Mechanisms of NOD-like receptor-associated inflammasome activation. Immunity, 2013. 39 (3): p. 432-41. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8938653","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597789811,"identity":"813ea6a4-6a27-4e9f-8a33-682de1924200","order_by":0,"name":"Haoyu Yang","email":"","orcid":"","institution":"Changsha University","correspondingAuthor":false,"prefix":"","firstName":"Haoyu","middleName":"","lastName":"Yang","suffix":""},{"id":597789812,"identity":"f0f32dcd-43d6-45ef-94d5-adc718d9519f","order_by":1,"name":"Ziqi Li","email":"","orcid":"","institution":"Changsha University","correspondingAuthor":false,"prefix":"","firstName":"Ziqi","middleName":"","lastName":"Li","suffix":""},{"id":597789813,"identity":"c609e02b-eae6-46fe-a5a9-d4c6f5c7de96","order_by":2,"name":"Dong Yan","email":"","orcid":"","institution":"Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Yan","suffix":""},{"id":597789814,"identity":"3d352552-a474-4401-8bdf-4ad10e67cce0","order_by":3,"name":"Min Li","email":"","orcid":"","institution":"Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Li","suffix":""},{"id":597789815,"identity":"eaff84aa-3f9d-412c-9261-cb2fa4dc1559","order_by":4,"name":"Lingyun Xu","email":"","orcid":"","institution":"Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Xu","suffix":""},{"id":597789816,"identity":"a9187f5f-a265-4de4-bee2-6cdb3ec72e9f","order_by":5,"name":"Yuxin Wang","email":"","orcid":"","institution":"Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Wang","suffix":""},{"id":597789819,"identity":"7c935bbf-a893-4453-a152-a407cc17b8fb","order_by":6,"name":"Genshen Zhong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACCQglB+UyE6/FmHQtiQ1Ea5Gf3Xzs4Zdfden97cefSTBUWCc2sJ89gFcL45xj6cayfYdzZ5zJMZNgOJOe2MCTl4BXC7NEjpm0ZM+B3A0SPGwSjG2HExskeAzwamGDaKlLN5BgfybB+I8ILTxALZIffjAnGEgwmEkwNhChRUIiLU0aqNIQ6BdjiwSgx9p4cvBrkZ+RfEzyx586ef724w9vfKixlu1nP4NfCwgw87ZBWQkg3xFUDwSMP/4Qo2wUjIJRMApGLAAAD0U+uyYbSOcAAAAASUVORK5CYII=","orcid":"","institution":"Changsha University","correspondingAuthor":true,"prefix":"","firstName":"Genshen","middleName":"","lastName":"Zhong","suffix":""},{"id":597789821,"identity":"0764b394-8268-4265-b3a0-e421c59b4ddd","order_by":7,"name":"Minna Wu","email":"","orcid":"","institution":"Changsha University","correspondingAuthor":false,"prefix":"","firstName":"Minna","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-02-22 11:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8938653/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8938653/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103610847,"identity":"bfdddadf-b201-45b6-b613-999ee287488c","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4871603,"visible":true,"origin":"","legend":"\u003cp\u003eATPIF1 deficiency significantly alleviated colitis symptoms in\u003cem\u003e C. rodentium\u003c/em\u003e-induced mice compared to the wild-type colitis model. (a) Experimental procedure during the \u003cem\u003eC. rodentium\u003c/em\u003e-induced colitis experiment. (b) Representative colon images. (c) Colorectal length. (d) Daily body weight. (e) Disease Activity Indices (DAI) scores. WT: wild type; WT+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis; KO: ATPIF1\u003csup\u003e−/−\u003c/sup\u003e; KO+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e−/−\u003c/sup\u003e colitis. Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e = 5). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/6025893e63b219265fd08c2c.jpg"},{"id":103610845,"identity":"d8eef1c2-990a-4060-8304-a0af137b3318","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eC. rodentium\u003c/em\u003e load in the feces (a) and colon (b). WT: wild type; WT+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis; KO: ATPIF1\u003csup\u003e−/−\u003c/sup\u003e; KO+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e−/−\u003c/sup\u003e colitis. Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e = 5). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/bc9b1d0ed49402bfc04a646a.png"},{"id":103610851,"identity":"5239923a-8f5a-479c-bca9-2e00f8870366","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28883946,"visible":true,"origin":"","legend":"\u003cp\u003eATPIF1 deficiency significantly suppressed colonic inflammation and maintained the integrity of the colonic mucosal barrier in \u003cem\u003eC. rodentium\u003c/em\u003e-induced colitis mice. (a) Representative images of Hematoxylin and Eosin (H\u0026amp;E) staining of colonic tissue sections. (b) Pathological scores based on the H\u0026amp;E staining. (c) Representative images of colonic tissue sections stained with Alcian Blue. (d) Statistical analysis of Alcian Blue-stained goblet cells. WT: wild type; WT+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis; KO: ATPIF1\u003csup\u003e−/−\u003c/sup\u003e; KO+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e−/−\u003c/sup\u003e colitis. Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e = 5). ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/a8ec9bd641a47fa80a6325cb.jpg"},{"id":103610846,"identity":"aa8e6e6c-a0b1-4734-8d50-4533cb74fc2d","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77632,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative mRNA expression levels of inflammatory cytokines and tight junction protein in colonic tissues. WT: wild type; WT+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis; KO: ATPIF1\u003csup\u003e−/−\u003c/sup\u003e; KO+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e−/−\u003c/sup\u003e colitis. Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e = 5). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01,***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/1b2dcfec3417e6599fef7df7.png"},{"id":103610852,"identity":"501c333e-18e6-4808-8099-39ec31abe7db","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28366302,"visible":true,"origin":"","legend":"\u003cp\u003eATPIF1 deficiency suppressed the NLRP3 signaling pathway in CR-induced colitis mice. (a) Representative images of NLRP3 signaling pathway-related proteins ASC, Cleaved Caspase-1, and NLRP3 detected by immunohistochemistry. (b)-(d) Percentage of cells positive for ASC, Cleaved Caspase-1, and NLRP3 in (a). WT: wild type; WT+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis; KO: ATPIF1\u003csup\u003e−/−\u003c/sup\u003e; KO+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e−/−\u003c/sup\u003e colitis. Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e = 5). ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/4151200ee3ad62196dcc5b02.jpg"},{"id":103610849,"identity":"64a86e7d-da2f-4514-a5c1-411444c0b71b","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":282145,"visible":true,"origin":"","legend":"\u003cp\u003eModulation of gut microbiota by ATPIF1 deficiency in \u003cem\u003eC. rodentium\u003c/em\u003e-induced UC mice. (a) Principal Coordinates Analysis (PCoA). (b) Normalized Stochasticity Ratio (NST) analysis. (c–h) Relative abundances of some characteristic genera. (i) Linear Discriminant Analysis Effect Size (LEfSe) analysis. (j) Correlation analysis between the top 30 abundant genera and micro-environmental factors. (k) Network analysis of genus-level gut microbiota with inflammatory pathway component and clinical indicators. WT: wild type; WT+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis; KO: ATPIF1\u003csup\u003e−/−\u003c/sup\u003e; KO+CR: \u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e−/−\u003c/sup\u003e colitis. Data are presented as mean ± SEM (\u003cem\u003en\u003c/em\u003e = 5). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/60fb5226f8fdfe564f8458e6.png"},{"id":105856469,"identity":"7df0a46e-636c-4378-9668-85081f418af0","added_by":"auto","created_at":"2026-03-31 22:25:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":63576125,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/c2e8c28a-f832-47c9-a2f4-39404a4341ce.pdf"},{"id":103610848,"identity":"7345d12e-cabe-4367-bc60-ce93f66eb858","added_by":"auto","created_at":"2026-02-27 15:51:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":520267,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8938653/v1/075844a8d63e1f1edac755c7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eATPIF1 deficiency Significantly Alleviates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCitrobacter rodentium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Induced Ulcerative Colitis in Mice\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUlcerative colitis (UC) is a major subtype of inflammatory bowel disease (IBD), characterized by chronic inflammation of the colonic mucosa, neutrophilic infiltration, and ulcer formation. These pathological features result from dysregulated immune responses, genetic predisposition, and alterations in gut microbiota composition[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite significant advances in molecular biology that have elucidated multiple several pathways\u0026mdash;including cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), immune cell subsets such as T helper 17 (Th17) cells and regulatory T cells (Tregs), and complex host\u0026ndash;microbiota interactions[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u0026mdash;the precise mechanisms underlying UC pathogenesis remain incompletely defined[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe murine enteric pathogen \u003cem\u003eCitrobacter rodentium\u003c/em\u003e (CR) serves as a well-established model that recapitulates key aspects of human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections. As an attaching and effacing (A/E) pathogen, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection provokes acute host immune responses, including the activation of inflammatory cytokines and recruitment of leukocytes, which facilitate pathogen clearance[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis has been extensively characterized as a model of infectious colitis, with pathological and immunological alterations closely mirroring those observed in IBD, thereby providing an ideal platform for investigating immune-mediated bacterial colitis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Unlike chemically induced colitis models such as dextran sulfate sodium (DSS), \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection markedly alters gut microbiota composition and function, indirectly compromising mucosal barrier integrity. Chronic inflammation in IBD patients is hypothesized to result from aberrant immune responses to gut microbiota, particularly in genetically susceptible hosts. Consequently, infectious colitis models offer critical insights into host pathological responses to enteric bacteria[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Emerging evidence further implicates host\u0026ndash;microbiota interactions in the regulation of inflammasome activation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among the immune mechanisms implicated in UC, the NLRP3 inflammasome plays a central role by sensing microbial and danger-associated signals, activating caspase-1, and promoting the secretion of pro-inflammatory cytokines IL-1β and IL-18, thereby amplifying mucosal inflammation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection activates Toll-like receptor signaling pathways, upregulates NLRP3 and pro-IL-1β expression, and enhances NLRP3 inflammasome activation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis model constitutes a valuable tool for dissecting the complex interplay among gut dysbiosis, mucosal immune responses, and epithelial barrier integrity in UC pathogenesis.\u003c/p\u003e \u003cp\u003eMitochondrial ATP synthase inhibitory factor 1 (ATPIF1) is a small (~\u0026thinsp;10 kDa) nuclear-encoded protein localized within the mitochondrial matrix. It modulates the activity of the F1Fo-ATP synthase complex, thereby influencing cellular energy metabolism, with established roles in cancer, cardiovascular diseases, and other pathological conditions[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, ATPIF1 exhibits context-dependent functions: it is overexpressed in various malignancies, where it promotes glycolysis and inhibits apoptosis, facilitating tumor proliferation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; conversely, during ischemic events, ATPIF1 mitigates cellular ATP consumption, reducing reperfusion-induced cell death[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This dual functionality underscores ATPIF1 as a critical regulator of cellular energy homeostasis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the context of inflammation, mitochondrial dysfunction-induced oxidative stress correlates positively with NLRP3 inflammasome activation, suggesting that ATPIF1, by modulating ATP synthesis, may influence inflammasome-mediated inflammatory responses[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Given that mitochondrial function modulates host\u0026ndash;microbiota interactions and that gut dysbiosis is closely linked to UC pathogenesis, it is imperative to investigate the effects of ATPIF1 deficiency on microbial ecosystems under inflammatory conditions. Previous studies demonstrated that ATPIF1 inactivation ameliorated DSS-induced colitis, although this was limited to a chemical colitis model[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, inhibition of ATPIF1 activity was reported to enhance murine neutrophil antibacterial function, primarily through increased reactive oxygen species (ROS) and lactic acid production[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These findings suggest a potential association between ATPIF1 deficiency and the development of bacterial infection-driven UC. While ATPIF1 is recognized as a regulator of cellular energy metabolism and inflammation, its specific roles in host defense and mucosal immunity during enteric bacterial infection remain unclear. To address this knowledge gap, the present study employed a \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced murine model to investigate the impact of ATPIF1 deficiency on colonic inflammation, inflammasome activation, and gut microbiota composition. Utilizing histopathological evaluation, quantification of inflammatory markers, and 16S rRNA gene sequencing, this research aims to elucidate the molecular and microbial mechanisms by which ATPIF1 contributes to UC pathogenesis, thereby highlighting its potential as a therapeutic target in bacterial infection-driven UC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental Animals and Housing\u003c/h2\u003e \u003cp\u003eThe generation of ATPIF1 deficiency (ATPIF1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, KO) mice has been previously documented[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Male SPF C57BL/6 wild-type and ATPIF1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice aged 6 to 8 weeks were maintained under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, relative humidity of 40%\u0026ndash;60%, and a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Mice were provided with unrestricted access to food and sterile water. Following a 7-day acclimation period, experimental procedures were conducted. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Xinxiang Medical University and were performed in accordance with established institutional guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Bacterial Culture and Induction of Colitis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCitrobacter rodentium\u003c/em\u003e strain (ATCC 51459) was kindly supplied by Professor Zhiping Liu of Gannan Medical University. Frozen stocks of \u003cem\u003eC. rodentium\u003c/em\u003e were inoculated into 5 mL of LB broth and incubated overnight at 37\u0026deg;C with agitation at 180 rpm. Subsequently, bacterial cultures were streaked onto MacConkey selective agar plates and incubated overnight. A single colony was then picked and transferred to fresh LB broth for activation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The mice were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;5 per group): WT (wild type) group, WT\u0026thinsp;+\u0026thinsp;CR (\u003cem\u003eC. rodentium\u003c/em\u003e-induced WT colitis) group, KO (ATPIF1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) group and KO\u0026thinsp;+\u0026thinsp;CR (\u003cem\u003eC. rodentium\u003c/em\u003e-induced ATPIF1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e colitis) group. The experimental duration was 7 days. Mice in the WT\u0026thinsp;+\u0026thinsp;CR and KO\u0026thinsp;+\u0026thinsp;CR groups received a daily oral gavage of 200 \u0026micro;L of \u003cem\u003eC. rodentium\u003c/em\u003e suspension (6 \u0026times; 10⁸ CFU/mice)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Control groups (WT and KO) were administered an equivalent volume of sterile phosphate-buffered saline (PBS) via oral gavage. At the conclusion of the study (day 7), all mice were euthanized for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Body Weight Monitoring and Disease Activity Index (DAI)\u003c/h2\u003e \u003cp\u003eBody weight was measured daily, and changes in weight were expressed as a percentage relative to the baseline body weight recorded on day 0. Disease Activity Index (DAI) scores were evaluated according to the criteria established by Murthy et al.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], incorporating parameters such as body weight loss, stool consistency, and the presence of fecal occult blood. Rectal bleeding was assessed using a fecal occult blood test kit (Baso Biotechnology Co., Ltd., Guangdong, China). The overall DAI score was calculated as the mean of the three individual component scores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fecal Sample Collection and Colon Length Measurement\u003c/h2\u003e \u003cp\u003eOn the seventh day, fresh fecal samples were collected and promptly stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis. After euthanasia by cervical dislocation, the colon, including the anal segment, was carefully dissected, placed on a white background, and photographed. The length of the colon was then measured using a ruler.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 16S rRNA Sequencing of Fecal Microbiota\u003c/h2\u003e \u003cp\u003eGenomic DNA was isolated from fecal specimens using the Soil DNA Isolation Kit (Omega BioTek, Norcross, GA, USA) according to the manufacturer\u0026rsquo;s instructions. The quality and integrity of the extracted DNA were evaluated by electrophoresis on a 0.8% agarose gel. DNA concentration and purity were measured spectrophotometrically at 260 nm and 280 nm wavelengths using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). DNA samples that met quality criteria were stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent analyses.\u003c/p\u003e \u003cp\u003eTo characterize the microbial community, the V3\u0026ndash;V4 hypervariable region of the bacterial 16S rRNA gene was amplified using the primer pair 338F (5\u0026prime;-ACTCCTACGGGAGGCAGC-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;). PCR products were purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), quantified, and normalized to uniform concentrations. Paired-end sequencing (2 \u0026times; 300 bp) was performed on the Illumina MiSeq platform by MajorBio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR35154250 to SRR35154272.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 H\u0026amp;E and Alcian Blue Staining of Colon Tissue\u003c/h2\u003e \u003cp\u003eTissue samples from the distal colon, obtained proximal to the anal verge, were fixed in 4% paraformaldehyde. Subsequently, the specimens were dehydrated through a graded ethanol series, followed by clearing with xylene. The tissues were then embedded in paraffin and sectioned at a thickness of 5 \u0026micro;m. The resulting sections were stained with hematoxylin and eosin (H\u0026amp;E) as well as Alcian Blue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological Scoring and Quantification of Goblet Cells\u003c/h2\u003e \u003cp\u003eHistological scoring of H\u0026amp;E-stained colon sections was conducted using the criteria proposed by Johansson[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], encompassing five parameters: inflammatory cell infiltration, mucosal thickening, goblet cell depletion, crypt loss, and epithelial damage. Each parameter was assigned a severity score ranging from 0 to 4, and the overall histological score was calculated by summing these individual scores. Additionally, Alcian Blue-stained sections were quantitatively analyzed using ImageJ software to measure the area exhibiting positive goblet cell staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RNA Extraction and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from colon tissues using TRIzol reagent (Takara, Dalian, China). Subsequently, cDNA was synthesized using PrimeScript RT Master Mix and stored at -80℃ for further analysis (Takara, Dalian, China). Quantitative real-time PCR was performed on a StepOne\u0026trade; Real-Time PCR System (Life Technologies, USA) using SYBR Green PCR Master Mix (Takara) to assess gene expression. Expression levels of key inflammatory cytokines, including TNF-α, IL-1β, and IL-6, as well as the epithelial tight junction marker ZO-1, were quantified using gene-specific primers. GAPDH served as the internal control. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. Primer sequences used in this study are listed in the \u003cb\u003eTable.S1\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunohistochemistry Analysis of Colon Tissue\u003c/h2\u003e \u003cp\u003eParaffin-embedded tissue sections were first deparaffinized and subjected to antigen retrieval via microwave heating. Endogenous peroxidase activity was inhibited using hydrogen peroxide (H₂O₂), followed by a 30-minute blocking step with ready-to-use goat serum (BosterBio, Wuhan, China). Subsequently, the sections were incubated overnight at 4\u0026deg;C with primary polyclonal antibodies against Cleaved Caspase-1 IgG, ASC IgG, and NLRP3 IgG, each diluted 1:200 (Wanleibio, Shenyang, China). After equilibration to room temperature, the sections were treated with appropriate secondary antibodies and visualized using a diaminobenzidine (DAB) detection kit (ShareBio, Shanghai, China). Nuclear counterstaining was performed with hematoxylin. Following dehydration and clearing through graded ethanol and xylene, the sections were mounted using neutral resin. Imaging was conducted with a fluorescence microscope (Leica, Germany) under standardized exposure conditions. Quantitative analysis of the immunohistochemically positive areas was performed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism version 5.0 (Boston, MA, USA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences between two groups were assessed using an unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, while comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). A \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Data Availability\u003c/h2\u003e \u003cp\u003eRaw sequence data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR35154250\u0026ndash;SRR35154272. These data correspond to 16S rRNA gene amplicon sequencing of the V3\u0026ndash;V4 region, amplified using primers 338F (5\u0026prime;-ACTCCTACGGGAGGCAGC-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;). All other data supporting the findings of this study are available within the article and its Supplementary Information files.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 ATPIF1 deficiency attenuated disease severity in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis.\u003c/h2\u003e \u003cp\u003eTo assess colitis severity, we evaluated colorectal length, body weight loss, and disease activity index (DAI) scores across experimental groups. The experimental timeline for \u003cem\u003eC. rodentium\u003c/em\u003e-induced colitis is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c, colon shortening in the KO\u0026thinsp;+\u0026thinsp;CR group was significantly less pronounced than in the WT\u0026thinsp;+\u0026thinsp;CR group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the KO\u0026thinsp;+\u0026thinsp;CR group exhibited significantly less weight loss compared to the WT\u0026thinsp;+\u0026thinsp;CR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Correspondingly, DAI scores were markedly lower in the KO\u0026thinsp;+\u0026thinsp;CR group than in WT\u0026thinsp;+\u0026thinsp;CR mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Collectively, these findings indicate that ATPIF1 deficiency attenuates the severity of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 ATPIF1 deficiency reduces \u003cem\u003eC. rodentium\u003c/em\u003e burden in feces and colon tissue.\u003c/h2\u003e \u003cp\u003eTo assess the impact of ATPIF1 deficiency on bacterial load, we quantified \u003cem\u003eC. rodentium\u003c/em\u003e colonization in fecal samples and colonic tissues using MacConkey agar selective medium at the study endpoint. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, fecal \u003cem\u003eC. rodentium\u003c/em\u003e levels were significantly elevated in the WT\u0026thinsp;+\u0026thinsp;CR group compared to WT controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas the KO\u0026thinsp;+\u0026thinsp;CR group exhibited a significant reduction relative to WT\u0026thinsp;+\u0026thinsp;CR mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, bacterial load within colon tissues was markedly decreased in KO\u0026thinsp;+\u0026thinsp;CR mice compared to WT\u0026thinsp;+\u0026thinsp;CR counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings suggest that ATPIF1 deficiency enhances the host\u0026rsquo;s ability to clear opportunistic pathogens during \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 ATPIF1 deficiency alleviated tissue damage and preserved mucosal barrier integrity in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis.\u003c/h2\u003e \u003cp\u003eHistological analyses using hematoxylin and eosin (H\u0026amp;E) and Alcian Blue staining were performed to asses tissue damage and mucosal barrier integrity. The pathological score in WT\u0026thinsp;+\u0026thinsp;CR mice was significantly elevated (12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12) compared to WT controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), characterized by disrupted colonic epithelium, extensive inflammatory cell infiltration, and pronounced submucosal edema. In contrast, KO\u0026thinsp;+\u0026thinsp;CR mice exhibited a significantly lower pathological score (5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75) compared with WT\u0026thinsp;+\u0026thinsp;CR mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with preservation of epithelial architecture, reduced edema, and attenuated inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In parallel, Alcian Blue staining revealed a significant decrease in goblet cell percentage in WT\u0026thinsp;+\u0026thinsp;CR mice (1.76% \u0026plusmn; 0.16) versus WT controls (2.95% \u0026plusmn; 0.18, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas KO\u0026thinsp;+\u0026thinsp;CR mice showed a significant increase in goblet cell abundance (4.32% \u0026plusmn; 0.37) compared to WT\u0026thinsp;+\u0026thinsp;CR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d). These data indicate that ATPIF1 deficiency not only mitigates \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced pathological damage but also maintains mucosal barrier integrity by preserving goblet cell populations, thereby suppressing inflammatory responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 ATPIF1 deficiency suppresses inflammatory cytokine expression in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis.\u003c/h2\u003e \u003cp\u003eTo further investigate the impact of ATPIF1 deficiency on intestinal inflammatory responses, we measured the relative mRNA abundance of key inflammatory cytokines in the colonic tissue of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced UC mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c, the expression levels of IL-6 (0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08), IL-1β (0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20), and TNF-α (0.0038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0013) in the KO\u0026thinsp;+\u0026thinsp;CR group were significantly lower than those in the WT\u0026thinsp;+\u0026thinsp;CR group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Additionally, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, the relative abundance of the tight junction protein ZO-1 in the KO\u0026thinsp;+\u0026thinsp;CR group (2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37) was significantly higher than that in the WT\u0026thinsp;+\u0026thinsp;CR group (0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings suggest that ATPIF1 deficiency ameliorates colonic injury and preserves mucosal integrity, likely by maintaining of goblet cell numbers and attenuating inflammatory cytokine production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 ATPIF1 deficiency enhances the expression of NLRP3 signaling-associated proteins in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis.\u003c/h2\u003e \u003cp\u003eWhen analyzing the mRNA expression levels of inflammatory cytokines, we observed that IL-6, IL-1β and TNF-α levels were significantly downregulated in the KO\u0026thinsp;+\u0026thinsp;CR group. The NLRP3 inflammasome recognizes pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), assembling with ASC and pro-caspase-1 to form a multiprotein complex that activates caspase-1 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], leading to the maturation and secretion of IL-1β and IL-18 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. TNF-α can enhance NLRP3 activation and the inflammatory response, with these inflammatory factors constituting a pro-inflammatory positive feedback loop [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To investigate whether ATPIF1 deficiency alleviates intestinal inflammatory responses in the colon of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced mice via the NLRP3 signaling pathway, we measured the expression of NLRP3 signaling-related proteins ASC, Cleaved Caspase-1, and NLRP3. The results showed that the inflammasome signaling pathway was abnormally active in the WT\u0026thinsp;+\u0026thinsp;CR group, with markedly increased expression of ASC, Cleaved Caspase-1 and NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). In contrast, the expression levels of ASC, Cleaved Caspase-1 and NLRP3 were significantly decreased in the KO\u0026thinsp;+\u0026thinsp;CR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These findings suggest that ATPIF1 deficiency mitigates \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colonic inflammation by inhibiting NLRP3 inflammasome signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 ATPIF1 deficiency alleviates gut microbiota dysbiosis \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis in mice.\u003c/h2\u003e \u003cp\u003eGut microbiota dysbiosis is implicated in the pathogenesis of ulcerative colitis. To assess the influence of ATPIF1 deficiency on the gut microbial composition in mice with \u003cem\u003eC. rodentium\u003c/em\u003e-induced ulcerative colitis (UC), we performed high-throughput sequencing of the 16S rRNA gene from mouse fecal samples. No significant difference was observed in alpha diversity indices among the WT, WT\u0026thinsp;+\u0026thinsp;CR, KO, and KO\u0026thinsp;+\u0026thinsp;CR groups. However, beta-diversity analysis revealed marked alterations in the gut microbiota composition. Principal coordinates analysis (PCoA) based on Bray-Curtis distances at the ASV level demonstrated that the microbial communities of the KO and WT groups partially overlapped, although distinct differences were observed. In contrast, after \u003cem\u003eC. rodentium\u003c/em\u003e infection, the microbial compositions of WT\u0026thinsp;+\u0026thinsp;CR and KO\u0026thinsp;+\u0026thinsp;CR groups were separated from each other obviously, indicating substantial structural divergence in the gut microbiota between these groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The normalized stochasticity ratio (NST) values indicated that gut microbial assembly in both WT and KO groups under basal conditions was predominantly governed by stochastic processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The influence of stochasticity was increased in WT\u0026thinsp;+\u0026thinsp;CR group (86.03\u0026thinsp;\u0026plusmn;\u0026thinsp;9.62%), suggesting disrupted gut environmental selection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, the NST value in the KO\u0026thinsp;+\u0026thinsp;CR group significantly decreased to 53.19\u0026thinsp;\u0026plusmn;\u0026thinsp;8.65%, indicating a shift in microbial community assembly toward more deterministic processes. This shift may be attributed to the alteration of the host intestinal environment caused by ATPIF1 deficiency, whereby enhanced host-mediated selection pressure led to a more structured and regulated microbial composition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the genus level, the microbial community composition revealed distinct differences among groups (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003ea). After \u003cem\u003eC. rodentium-\u003c/em\u003einduced colitis, the abundances of \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003eRoseburia\u003c/em\u003e were significantly reduced in the KO\u0026thinsp;+\u0026thinsp;CR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and d, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while \u003cem\u003eOdoribacter\u003c/em\u003e was significantly enriched in the WT\u0026thinsp;+\u0026thinsp;CR group Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e and \u003cem\u003eRikenella\u003c/em\u003e were markedly decreased in the WT\u0026thinsp;+\u0026thinsp;CR group, with no significant reduction was induced in the KO\u0026thinsp;+\u0026thinsp;CR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and g, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, a genus designated \u003cem\u003eA2\u003c/em\u003e, which was rarely reported, was significantly enriched in the WT\u0026thinsp;+\u0026thinsp;CR group Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eCharacteristic genera of each group were further identified using linear discriminant analysis coupled with effect size (LEfSe). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, unclassified \u003cem\u003eClostridia_UCG-014\u003c/em\u003e, \u003cem\u003eRikenellaceaeRC9 group\u003c/em\u003e, \u003cem\u003eRoseburia\u003c/em\u003e, and \u003cem\u003eRuminococcus\u003c/em\u003e were identified as signature taxa of the WT\u0026thinsp;+\u0026thinsp;CR group, while \u003cem\u003eMuribaculum\u003c/em\u003e, \u003cem\u003eDubosiella\u003c/em\u003e, \u003cem\u003eTuricimonas\u003c/em\u003e, Tyzzerella, and \u003cem\u003eRikenella\u003c/em\u003e were the characteristic genera enriched in the KO\u0026thinsp;+\u0026thinsp;CR group.\u003c/p\u003e \u003cp\u003eThe correlations between the top 30 most abundant genera and various environmental factors, including DAI score, colon length, pathological score, and key components of the NLRP3 signaling pathway (ASC, cleaved Caspase-1, and NLRP3), were analyzed to explore the relationship between gut microbiota and host-associated inflammatory parameters Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej). Colon length showed positive correlations with \u003cem\u003eParasutterella\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, and \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, but was negatively correlated with \u003cem\u003eCitrobacter\u003c/em\u003e, \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e, and \u003cem\u003eOdoribacter\u003c/em\u003e. In contrast, indicators of colitis severity, including DAI score, pathological score, and NLRP3 pathway-related proteins, were positively associated with \u003cem\u003eCitrobacter\u003c/em\u003e, \u003cem\u003eOdoribacter\u003c/em\u003e, and \u003cem\u003eunclassified_f__Enterobacteriaceae\u003c/em\u003e, and negatively associated with \u003cem\u003eParasutterella\u003c/em\u003e, \u003cem\u003eDubosiella\u003c/em\u003e, and \u003cem\u003eTuricibacter\u003c/em\u003e. The network analysis between the top 50 most abundant genus and environmental factors also showed that the severity of colitis was positively related to \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003eOdoribacter\u003c/em\u003e, while negatively related to \u003cem\u003eAllobaculum\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eParaprevotella\u003c/em\u003e and \u003cem\u003eRikenella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek).\u003c/p\u003e \u003cp\u003ePredicted functional analysis of microbial metabolic pathways revealed that, at KEGG level 3, Fatty acid biosynthesis, the AMPK signaling pathway and the NOD-like receptor signaling pathway were significantly downregulated in the KO\u0026thinsp;+\u0026thinsp;CR group compared with these in the WT\u0026thinsp;+\u0026thinsp;CR group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, in the COG functional classification, the ATPase activity pathway was also markedly suppressed in the KO\u0026thinsp;+\u0026thinsp;CR group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The reduced ATPase activity observed in KO mice may reflect a mitochondrial adaptation that stabilizes the membrane potential without requiring excessive ATP hydrolysis (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003eb and c).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global incidence of ulcerative colitis (UC) has risen substantially, underscoring the critical need for effective preventive and therapeutic interventions. UC is a complex disorder influenced by genetic predisposition, immune system dysregulation, and environmental factors[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Emerging evidence demonstrates significant differences in gut microbiota composition between UC patients and healthy controls, positioning the microbiota as a pivotal mediator in host-pathogen interactions during disease pathogenesis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although our previous research demonstrated that ATPIF1 deficiency effectively ameliorates DSS-induced colitis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], its role in infection-driven intestinal inflammation and gut microbiota regulation remains inadequately characterized. In the present investigation, utilizing a \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection-induced colitis model, we observed that ATPIF1 deficiency significantly attenuates \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis. This protective effect correlates with suppression of the NLRP3 inflammasome pathway and preservation of gut microbial homeostasis, thereby identifying ATPIF1 as a promising therapeutic target in UC.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. rodentium\u003c/em\u003e is a natural murine attaching and effacing (A/E) pathogen capable of inducing transmissible colonic hyperplasia[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Pathogen invasion is closely associated with infectious diarrhea and colitis, with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-infected mice exhibiting pronounced colitis symptoms, including weight loss, colon shortening, and fecal occult blood[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Importantly, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection does not require antibiotic pretreatment, establishing it as a primary model for studying A/E pathogen-induced intestinal inflammation[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In C57BL/6 mice, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection progresses through four stages: Establishment, with \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e colonization in cecal patches; Expansion, characterized by distal colon colonization, crypt hyperplasia, and epithelial barrier disruption; Steady-state, with stable \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e shedding (~\u0026thinsp;10⁹ CFU/g feces) accompanied by neutrophil and Th17/Th22 cell-mediated IL-17A and IL-22 secretion, the absence of which is lethal; and Clearance, wherein CD4⁺ T cells shift from IL-17A/IL-22 to IFN-γ-dominated responses, and IFN-γ deficiency delays pathogen clearance. \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e is ultimately eliminated through IgG-mediated opsonization, neutrophil phagocytosis, and competition with commensal bacteria. As a primary defense mechanism, activated intestinal epithelial cells secrete antimicrobial peptides, serum amyloid A (SAA), and reactive oxygen species (ROS) to restrict bacterial adherence[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Innate immune recognition is further mediated by Toll-like receptors (TLRs), intracellular NOD-like receptors (NLRs), and signals from damaged epithelial cells, collectively orchestrating infection sensing and response [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Adaptive immunity, particularly involving CD4⁺ T and B lymphocytes, is essential for pathogen clearance[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e infection elicits robust innate immune activation, with the NLRP3 inflammasome serving as a central signaling nexus. Consistently, we also observed elevated activation of the NLRP3 inflammasome signaling pathway in the disease group.\u003c/p\u003e \u003cp\u003eLipopolysaccharide (LPS) derived from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e is detected by epithelial and immune cells, including macrophages and dendritic cells, activating the TLR4/MyD88/NF-κB signaling cascade to induce expression of NLRP3, pro-IL-1β, and pro-IL-18, thereby priming inflammasome assembly[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e utilizes a Type III Secretion System (T3SS) to translocate effector proteins that disrupt mitochondrial function and compromise tight junction integrity[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Excessive ROS production, originating from mitochondria and NADPH oxidase, further promotes NLRP3 activation. The inflammasome complex, comprising NLRP3, ASC, and pro-Caspase-1, facilitates the secretion of IL-1β and IL-18, recruitment of neutrophils, and propagation of intestinal inflammation. NLRs also mediate pyroptosis, an inflammatory form of programmed cell death in myeloid cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our data demonstrate that ATPIF1 deficiency significantly suppresses NLRP3 signaling in mice, which likely underlies the preservation of mucosal architecture and attenuation of inflammatory infiltration observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d).\u003c/p\u003e \u003cp\u003ePrevious studies have indicated that NLRP3 activity is negatively regulated by autophagy but positively modulated by reactive oxygen species (ROS) derived from damaged organelles. Mitochondrial dysfunction leads to ROS accumulation, thereby linking organelle impairment to inflammasome-mediated inflammation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mitochondrial ATP synthesis predominantly depends on F1Fo-ATP synthase, whose activity is inhibited by ATPIF1 overexpression, resulting in increased oxidative stress and amplified inflammatory responses[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consistent with this, ATPIF1 deficiency reduced pro-inflammatory cytokine levels in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c) Moreover, prior investigations employing peritonitis models revealed that ATPIF1 loss enhances glycolytic flux and augments neutrophil antimicrobial activity via ROS and lactate production[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Together with the observation that ATPIF1 deficiency mitigates \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis by preserving epithelial barrier integrity, these findings suggest that both the epithelial protection and the enhanced neutrophil function may contribute to disease amelioration.\u003c/p\u003e \u003cp\u003eColitis progression is frequently accompanied by alterations in gut microbiota composition. While α-diversity remained unchanged, β-diversity differed significantly between the WT\u0026thinsp;+\u0026thinsp;CR and KO\u0026thinsp;+\u0026thinsp;CR groups, suggesting that ATPIF1 deficiency reshapes bacterial community composition without affecting richness or evenness. Standardized neutrality ratio (NST) analysis revealed that microbial assembly in KO\u0026thinsp;+\u0026thinsp;CR mice shifted toward a more deterministic pattern, indicating preferential recovery of key functional taxa that may promote SCFA metabolism and enhance host resistance to stress and pathogens. In contrast, microbiota in WT\u0026thinsp;+\u0026thinsp;CR assembly was primarily stochastic. Correlation analyses between bacterial genera and inflammasome components further support bidirectional interactions: microbial metabolites may modulate NLRP3 activation, while inflammation-induced environmental changes can promote the expansion of pathogenic taxa. Under \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced stress, ATPIF1-deficient mice maintained beneficial taxa such as \u003cem\u003eDubosiella\u003c/em\u003e, \u003cem\u003eRikenella\u003c/em\u003e, and \u003cem\u003eParasutterella\u003c/em\u003e compared to WT mice. \u003cem\u003eDubosiella\u003c/em\u003e produces short-chain fatty acids (SCFAs) to balance Treg/Th17 responses and improve mucosal barrier integrity, and through the IDO1-Kynurenine pathway, enhance immune tolerance via dendritic cell-mediated tryptophan metabolism. \u003cem\u003eRikenella\u003c/em\u003e is considered a potential probiotic, while \u003cem\u003eParasutterella\u003c/em\u003e is a core component of murine and human gut microbiota, producing succinate, a critical intermediate in microbial metabolic networks[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Preservation of these taxa likely contributes to microbial stability in KO\u0026thinsp;+\u0026thinsp;CR mice. Downregulation of fatty acid metabolism at the KEGG Level 3 pathway indicates reduced inflammation and stabilized microbial composition, whereas suppression of the AMPK pathway reflects diminished cellular stress and inflammatory state, obviating the need for intensive metabolic regulation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Similarly, NOD-like receptor pathway activity aligns with inflammasome status [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our KEGG functional predictions corroborate these observations. Additionally, ATP synthase activity was suppressed in KO mice, suggesting a potential mitochondrial energy-conserving adaptation. Collectively, these findings imply that ATPIF1 deficiency modulates host metabolism, indirectly influences microbial function, and that gut microbiota remodeling may mediate its protective effects.\u003c/p\u003e \u003cp\u003eDespite observing reduced expression of NLRP3 inflammasome-related proteins, upstream mechanisms\u0026mdash;including mitochondrial ROS generation, ASC speck formation, and TLR4 activity\u0026mdash;warrant further elucidation. Functional validation of specific microbial taxa, such as \u003cem\u003eRoseburia\u003c/em\u003e, will be instrumental in clarifying their contributions to the protective phenotype observed in ATPIF1-deficient mice.\u003c/p\u003e \u003cp\u003eIn summary, this study provides novel evidence that ATPIF1 deficiency alleviates \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced colitis by suppression of NLRP3 inflammasome activation and modulation of gut microbial composition. This work addresses a critical gap in infection-driven UC models and confirms that ATPIF1 exerts protective effects in both DSS and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003erodentium\u003c/em\u003e-induced UC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These findings reveal a previously unrecognized role of ATPIF1 at the nexus of mitochondrial metabolism, immune regulation, and microbial homeostasis, highlighting its potential as a metabolic therapeutic target for infection-associated UC with significant translational implications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eHY: Writing \u0026ndash; original draft, Investigation, Data curation. ZL: Writing \u0026ndash; original draft, Investigation, Data curation.LX: Methodology.YX Wang: Methodology.DY: Methodology.ML: Methodology.GZ: Supervision, Funding acquisition, Conceptualization.MW: Supervision, Conceptualization, Data curation, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe author(s) declared that financial support was received for this work and/or its publication. This work was funded by Hunan Natural Science Foundation Project (2026JJ80001) awarded to GZ, the Scientific Research Project of the Education Department of Hunan Province (25A0637) awarded to GZ, and the NSFC-Henan Union grant (No. U1904131) to GZ.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eRaw sequence data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR35154250\u0026ndash;SRR35154272. These data correspond to 16S rRNA gene amplicon sequencing of the V3\u0026ndash;V4 region, amplified using primers 338F (5\u0026prime;-ACTCCTACGGGAGGCAGC-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;). All other data supporting the findings of this study are available within the article and its Supplementary Information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWangchuk, P., K. Yeshi, and A. 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Miao, and J.P. Ting, \u003cem\u003eMechanisms of NOD-like receptor-associated inflammasome activation.\u003c/em\u003e Immunity, 2013. \u003cstrong\u003e39\u003c/strong\u003e(3): p. 432-41.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mitochondrial ATPase inhibitory factor 1 (ATPIF1), Ulcerative colitis (UC), Citrobacter rodentium (CR), NOD-like receptor protein 3 (NLRP3), Microbiota","lastPublishedDoi":"10.21203/rs.3.rs-8938653/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8938653/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e Mitochondrial ATP synthase inhibitory factor 1 (ATPIF1) is a crucial regulator of cellular energy metabolism and has been implicated in inflammatory disorders. However, its role in bacterial infection-driven ulcerative colitis (UC) remains unclear.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePurpose\u003c/b\u003e This study aimed to investigate the effects of ATPIF1 on host susceptibility and inflammatory responses in a \u003cem\u003eCitrobacter rodentium\u003c/em\u003e-induced infectious colitis model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e ATPIF1 knock out (KO) and wild type (WT) mice were orally gavaged with \u003cem\u003eC. rodentium\u003c/em\u003e to induce infectious colitis. Body weight, disease activity index (DAI), and colon length were recorded. Histopathology, Alcian blue staining, and immunohistochemistry were performed to assess mucosal integrity and barrier function. Inflammatory responses were evaluated through immunohistochemistry, RT-qPCR, and Western blotting, while gut microbiota composition was analyzed via 16S rRNA gene sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e ATPIF1 deficiency alleviated \u003cem\u003eC. rodentium\u003c/em\u003e-induced colitis, as evidenced by reduced weight loss, lower DAI scores, and attenuated colon shortening. KO mice preserved epithelial architecture, exhibited increased numbers of goblet cells and ZO-1 mRNA expression, indicating an intact mucosal barrier. Furthermore, KO mice showed reduced infiltration of inflammatory cells, decreased expression of IL-1β and TNF-α, and suppressed activation of the NLRP3 inflammasome pathway. Microbiota analysis also revealed that ATPIF1 deficiency stabilized bacterial community composition and reduced pathogenic expansion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e ATPIF1 deficiency significantly alleviates \u003cem\u003eC. rodentium\u003c/em\u003e-induced colitis by mitigating inflammation, preserving mucosal barrier function, promoting pathogen clearance, and stabilizing gut microbiota. These findings suggest that ATPIF1 may represent a potential therapeutic target for infection-associated UC.\u003c/p\u003e","manuscriptTitle":"ATPIF1 deficiency Significantly Alleviates Citrobacter rodentium-Induced Ulcerative Colitis in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 15:51:44","doi":"10.21203/rs.3.rs-8938653/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7fef8417-eff5-469d-8b9f-f560e2532110","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T22:24:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 15:51:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8938653","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8938653","identity":"rs-8938653","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}