Epithelial Hypusination Regulates Helicobacter pylori-induced Gastric Inflammation

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Gobert, Kara M. McNamara, Caroline V. Hawkins, Mohammad Asim, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7906468/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Feb, 2026 Read the published version in Amino Acids → Version 1 posted 11 You are reading this latest preprint version Abstract Hypusine is a unique amino acid synthesized on the eukaryotic initiation factor 5A (EIF5A) from the polyamine spermidine by deoxyhypusine synthase (DHPS). Hypusination of EIF5A plays a key role in translation. Here, we examined the contribution of the epithelial hypusination pathway to gastric inflammation induced by Helicobacter pylori . Immunohistochemical analyses revealed increased expression of DHPS and hypusinated EIF5A (EIF5A Hyp ) in the gastric mucosa of patients with H. pylori gastritis compared to uninfected individuals, notably within gastric epithelial cells (GECs) and immune infiltrates. Then, we created a mouse model with epithelial-specific deletion of Dhps ( Dhps Δepi ) and confirmed the reduction of DHPS and EIF5A Hyp in GECs. H. pylori -infected Dhps Δepi mice exhibited an attenuation of gastric histologic inflammation scores compared with infected Dhps fl/+ controls, without alteration in bacterial colonization levels. Quantitative proteomics of isolated GECs showed that Dhps deletion altered the expression of proteins involved in organismal injury, cancer, and gastrointestinal diseases in naïve mice. Upon H. pylori infection, inflammatory and immune response proteins, including signaling factors and immunoglobulin mediators, were less induced in Dhps Δepi GECs, and pathways linked to tissue injury and inflammation were selectively downregulated. Together, these findings demonstrate that epithelial hypusination supports H. pylori -driven gastric inflammation without affecting bacterial persistence. Targeting DHPS-dependent EIF5A hypusination may thus represent a novel therapeutic strategy to limit H. pylori -associated mucosal injury and disease progression. Infection Gastritis Polyamines Hypusine Proteome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The polyamine spermidine serves an essential role as the substrate for the synthesis of hypusine, a unique amino acid found only in the highly conserved eukaryotic protein eukaryotic translation initiation factor 5A (EIF5A) (Park et al. 1981 ). The hypusine modification is catalyzed by the sequential action of two enzymes, deoxyhypusine synthase (DHPS), the rate-limiting enzyme, which transfers the 4-aminobutyl moiety of the polyamine spermidine to the Lys50 residue of EIF5A (Joe et al. 1995 ; Park et al. 2006 ), and deoxyhypusine hydroxylase (DOHH), which hydroxylases the intermediate deoxyhypusine residue (Abbruzzese et al. 1986 ). This post-translational modification is essential for the function of EIF5A, as hypusinated EIF5A (EIF5A Hyp ) can bind mRNAs that contain a 5′-AAAUGU-3′ consensus sequence (Maier et al. 2010 ; Xu et al. 2004 ). EIF5A Hyp can also alleviate ribosome stalling at polyproline repeats during translation elongation and at other non-polyproline motifs, such as peptides enriched in basic amino acids (Pelechano and Alepuz 2017 ; Schuller et al. 2017 ). Hypusination has been implicated in regulating inflammatory responses, specifically in myeloid and T cells (Gobert et al. 2020 ; Puleston et al. 2021 ). We have reported that hypusination in myeloid cells supports the antimicrobial response of macrophages to pathogenic bacteria including Helicobacter pylori (Gobert et al. 2020 ), a bacteria that colonizes the human stomach and causes diseases ranging from non-atrophic gastritis to the precancerous lesions of multifocal atrophic gastritis, intestinal metaplasia (IM), dysplasia, and gastric adenocarcinoma (Correa 1988 ; Piazuelo et al. 2021 ). Notably, mice with a myeloid-specific deletion of Dhps exhibited increased bacterial burden and inflammation, implicating hypusination in host defense against pathogenic bacteria (Gobert et al. 2020 ). However, the effect of hypusination in gastric epithelial cells (GECs) on H. pylori pathogenesis remains unknown. In this report, we showed that patients with H. pylori gastritis exhibit increased level of DHPS and hypusinated EIF5A. We then created mice with specific deletion of Dhps in intestinal epithelial cells including in the stomach to assess the role of hypusination in GECs in H. pylori pathogenesis. We found that these infected mice develop less gastritis, demonstrating that the activity of DHPS in GECs supports stomach inflammation. Further, the proteome of the GECs in Dhps -deficient mice is reshaped toward a less inflammatory and carcinogenic profile. Materials and methods Ethics statement Endoscopic gastric biopsies were obtained from patients at the Nashville Veterans Affairs Medical Center of the VA Tennessee Valley Healthcare System. Patients were undergoing esophagogastroduodenoscopy for clinically indicated reasons and provided informed consent for obtaining research biopsies under VA IRB protocol 1571167. The mice were used under protocols V2000018 and V2300022 approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee and the Research and Development Committee of the Veterans Affairs Tennessee Valley Healthcare System. Procedures followed institutional policies, AAALAC guidelines, the AVMA Guidelines on Euthanasia, NIH regulations regarding the Guide for the Care and Use of Laboratory Animals, and the United States Animal Welfare Act of 1996. Bacteria H. pylori PMSS1, a cagA + strain with intact type IV secretion system function, was grown on Trypticase soy agar plates containing 10% sheep’s blood. Bacteria were harvested from the plates and grown overnight in Brucella broth containing 10% fetal bovine serum (FBS). This culture was resuspended in fresh Brucella broth-FBS and then collected at the exponential phase to infect the mice. Mice and infections We used Foxa3 -cre mice that we crossed with Dhps fl/fl mice to obtain C57BL/6 Dhps fl/+ ;Foxa3 +/+ ( Dhps fl/+ ) and Dhps fl/+ ; Foxa3 cre/+ ( Dhps Δepi ) mice. Note that Dhps fl/fl ; Foxa3 cre/+ mice were embryonically lethal or only survived a few weeks out of utero. Mice were housed in a pathogen-free facility, with ventilated cage racks and were on a 12 h light-dark cycle. Male and female mice between 8 and 12 weeks were used for all studies. Animals were infected by oral gavage with 10 9 colony forming units (CFU) of H. pylori PMSS1 in 200 µL Brucella broth, two times, on days 0 and 2. The control mice were gavaged with only broth on both days. Eight weeks after the first infection, mice were euthanized and stomachs were harvested. Colonization was determined in all infected mice by counting the CFUs cultured after plating serial dilutions of homogenized gastric tissues (Latour et al. 2022 ; McNamara et al. 2025 ; Sierra et al. 2020 ). Histopathology Human biopsies from the gastric antrum and corpus, and longitudinal strips of murine stomach tissue including the corpus and antrum, were fixed in 10% neutral buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin (H&E). Biopsies were scored as reported (Latour et al. 2022 ; McNamara et al. 2025 ; Sierra et al. 2020 ). Histology was scored by our gastrointestinal pathologist (M.B.P.) who was blinded to the experimental groups. H. pylori infection of patient tissues was confirmed by culture of gastric biopsies as described above for the mouse tissues. Epithelial cell isolation Stomachs were removed from Dhps fl/+ and Dhps Δepi animals and incubated in a solution of cold 0.5 mM DTT and 3 mM EDTA for 30 min on ice. After incubation, the tissues were placed in 3 mM EDTA and vigorously shaken to release gastric glands. The cell suspension was poured over a 70 µm Strainer (Falcon) and the resulting isolated GECs were pelleted through centrifugation at 1500 rpm for 10 min at 4 °C. Proteomics analysis Isolated epithelial cells were lysed in 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 2 mM EDTA, and 1% SDS; protein concentration was measured by the BCA Protein Assay (Pierce) and samples from the same group were pooled. Protein extracts were reduced with 10 mM TCEP (tris(2-carboxyethyl)phosphine), alkylated with 20 mM iodoacetamide, and protein samples were prepared by S-Trap™ (ProtiFi) digestion with trypsin (1:10) similar to methods described in Howard et al (Howard et al. 2024 ). TMT-based quantitative proteomics was performed as described (Latour et al. 2022 ). Labeled peptides (5 µg per sample) were combined, fractionated using high pH reversed phase fractionation, and elution steps were performed with 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 60% acetonitrile with 0.1% triethylamine. Fractions were dried and reconstituted in 0.2% formic acid for LC-MS/MS analysis. Peptides were gradient-eluted at a flow rate of 350 nl/min, using varied reverse phase gradients over 90 min. For fraction 1, peptides were analyzed with the following gradient: 5–18% B in 75 min, 18–50% B in 6 min, 50–70% B in 3 min, 70 − 2% B in 1 min, 2% B for 5 min. For fractions 2–4, the first 2 steps of the gradient were adjusted to 5–25% B in 75 min and 25–50% B in 6 min, with the subsequent three steps identical to fraction 1. For fraction 5, the gradient included 2–8% B in 0.5 min, 8–30% B in 74.5 min, 30–50% B in 6 min, 50–70% B in 2 min, followed by the same final two steps. For fraction 6, the gradient included 2–8% B in 2 min, 8–30% B in 73 min, 30–50% B in 7 min, 50–70%B in 1 min, followed by the same final two steps. For fraction 7–8, the gradient included 5–45% B in 75 min, 45–90% B in 8 min, 90% B for 1 min, 90 − 2% B in 1 min, and 2% B for 5 min. Peptides were analyzed using a data-dependent acquisition method on an Orbitrap Exploris 240 mass spectrometer (Thermo Scientific), equipped with a nanoelectrospray ionization source. The instrument method consisted of MS1, followed by up to 20 MS/MS scans, with an automatic gain control target of 2x10 5 . Higher-energy collisional dissociation was set to 35 nce and dynamic exclusion (15 sec) was enabled. Data were searched in Proteome Discoverer 2.2 (Thermo Scientific) using SequestHT for database searching against a mouse database created from the UniProtKB database. Search parameters and quantitative analysis was performed as reported (Latour et al. 2022 ). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2025 ) partner repository with the dataset identifier PXD069622 Ingenuity pathway analysis (IPA) software (QIAGEN) was used for the functional interpretation of differential expression results obtained from the proteomic analyses. The pathways related to Diseases and Functions were generated. Western blot analysis Western blot analysis Proteins were extracted from isolated GECs as reported (Latour et al. 2022 ) and concentrations were determined using the BCA Protein Assay (Pierce). Western blots were performed using 10 µg protein per lane using a rabbit polyclonal anti-DHPS antibody (Ab; Abcam, Cat#ab202133; 1:5000), a rabbit polyclonal anti-EIF5A Hyp Ab (Millipore, Cat#ABS1064-I; 1:8000), or a mouse monoclonal anti-b-actin Ab (MilliporeSigma, Cat#A5316; 1:10000). The Peroxidase AffiniPure® Goat Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, Cat#111-035-003; 1:5000) or the goat anti-mouse IgG, HRP-labeled (Jackson ImmunoResearch, Cat#115-035-003; 1:5000) were the used as secondary Abs. Immunostaining Immunofluorescence was performed on human gastric biopsies and murine gastric tissues. Sections were deparaffinized and incubated at room temperature with 3% hydrogen peroxide in phosphate-buffered saline to block endogenous peroxidase. Tissues were then blocked for 1 h in Protein Block, Serum-Free (Dako, Cat#X0909). Slides were sequentially incubated with a rabbit polyclonal anti-DHPS Ab (Proteintech, Cat#11184-1-AP; 1:1000) or a rabbit anti-EIF5A Hyp Ab (MilliporeSigma Cat#ABS1064; 1:2000) overnight at 4°C and with a donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (ThermoFisher Scientific, Cat#A32790; 1:700) 45 min at room temperature. Slides were mounted with VECTASHIELD HardSet™ Antifade Mounting Medium with DAPI (Vector Laboratories, Cat#H-1500-10) and visualized using a Nikon E800 microscope and a SPOT Imaging CMOS camera. Statistics Prism 10.6.0 (GraphPad Inc.) was used for figure design and statistical analysis. All the data are expressed as mean ± SEM. Data that were not normally distributed according to the D’Agostino & Pearson normality test were log transformed. Student’s t test was used to determine significant differences between two groups, whereas a one-way ANOVA followed by a Tukey’s test or Šídák’s test was used for multiple groups. Results Increased levels of DHPS and EIF5A Hyp in H. pylori -infected patients Using immunostaining, we evidenced that that the level of DHPS and hypusinated EIF5A were increased overall in the gastric mucosa of endoscopic biopsies from patients with H. pylori gastritis compared to individuals without infection (Fig. 1 ). Of importance, the staining was abundant in GECs in H. pylori -infected patients but also present in the immune infiltrates, as we reported (Gobert et al. 2020 ). Deletion of epithelial hypusination reduces the inflammatory response to H. pylori To investigate the role of hypusination in epithelial cells during H. pylori infection we utilized a genetic approach by generating C57BL/6 animals with a gastric epithelial-specific knockout of Dhps . First, we verified by immunoblots and densitometry that Dhps Δepi mice exhibited reduced expression of DHPS and EIF5A Hyp levels in the gastric epithelium compared to Dhps fl/+ control mice (Fig. 2 A and 2 B). Then, we infected Dhps fl/+ and Dhps Δepi mice with H. pylori PMSS1 for 8 weeks. We confirmed by immunofluorescence that DHPS and EIF5A Hyp were less expressed in GECs from naive Dhps Δepi mice (Fig. 3 A). Upon infection, the levels of DHPS and EIF5A Hyp were increased in GECs and immune infiltrates of Dhps fl/+ mice compared to uninfected animals (Fig. 3 A); there was markedly less staining in the GECs of Dhps Δepi mice, whereas immune cells were still positive for DHPS (Fig. 3 A). All 30 Dhps Δepi mice were colonized and only 1 of the 21 Dhps fl/+ mice was not colonized and was thus removed from the analysis. We observed no difference in gastric bacterial burden between both genotypes (Fig. 3 B). An inflammatory infiltrate and mild foveolar hyperplasia were mainly observed at the antrocorporal transitional mucosa of H. pylori -infected Dhps fl/+ mice (Fig. 3 C). These parameters were less observed in the stomach of infected Dhps Δepi mice (Fig. 3 C). Using a comprehensive score, we found increased inflammation in both genotypes compared to uninfected animals, but also significantly less inflammation in the gastric tissue of infected Dhps Δepi mice compared to the infected Dhps +/fl mice (Fig. 3 D). Proteome of H. pylori -infected animals with Dhps deficiency To determine the role of the hypusination pathway during H. pylori -mediated inflammation, we performed TMT proteomics on isolated GECs from uninfected and infected Dhps fl/+ and Dhps Δepi mice. First, we analyzed the proteome in GECs from naïve mice. There were 79 proteins significantly induced by the specific Dhps deletion in epithelial cells (Supplementary Table S1 ). These proteins were signaling molecules, such as cyclin-dependent kinase 1 (CDK1) or Src substrate cortactin (SRC8), the superoxide dismutase SODM, and numerous heterogeneous nuclear ribonucleoproteins (ROA1/A2/A3/AA), which are RNA-binding proteins playing critical roles in multiple cellular processes such as DNA repair and regulation of gene expression (Fig. 4 A and Supplementary Table S1 ). We also found 183 proteins downregulated in GECs from Dhps Δepi mice. Among them we found numerous ribosomal proteins (e.g., RM24, RS4X, RT33), as expected, different cytochromes P450 (e.g. CP3AB, CP2E1, CP1A2), and one glutathione-S-transferase (MGST1) (Fig. 4 A and Supplementary Table S1 ). The changes in the level of these proteins in GECs were overall associated with a lessening of the pathways associated with organismal injury, cancer, and gastrointestinal diseases in Dhps Δepi mice (Fig. 4 B and Supplementary Table S2 ); pathways associated with cell movement were mainly induced in Dhps Δepi mice (Fig. 4 B and Supplementary Table S2 ). We identified 110 and 174 proteins significantly induced by H. pylori infection in the GECs from the stomach tissues from Dhps fl/+ and Dhps Δepi mice, respectively. Among them, 37 were in common to both genotypes (Fig. 5 A). These included mediators of adaptive immunity (IGHA, HB2A/2I, HG2A, and TGTP2), and regulators of the innate immune response, such as DOXA2, I23O1, and STAT1 (Fig. 5 B and Supplementary Table S1 ). However overall, the level of expression of these proteins in H. pylori -infected Dhps Δepi mice was lower compared to infected Dhps fl/+ animals (Fig. 5 B). Moreover, there were 170 proteins significantly downregulated in GECs from Dhps fl/+ mice with infection (Fig. 5 A), whereas only 61 proteins were less expressed in infected Dhps Δepi GECs (Fig. 5 A); only 18 proteins were similar in both genotypes (Fig. 5 A). The proteins downregulated by H. pylori infection in Dhps fl/+ animals included numerous cytochromes, e.g., CP2F, CP2E1, CP3AB, CP2DA, and CP240, which are known to be downregulated during infection and inflammation, TFF1, the stabilizer of the mucous gel overlying the gastrointestinal mucosa that provides a physical barrier against bacteria, and the marker of M2/Mreg macrophages, ARGI1 (Fig. 5 C and Supplementary Table S1 ); most of these effectors were less altered in infected Dhps Δepi mice (Fig. 5 C and Supplementary Table S1 ). When we analyzed the functional clusters corresponding to the proteins differentially expressed between infected Dhps Δepi versus infected Dhps fl/+ mice (see Supplementary Table S1 ), we evidenced that numerous pathways related to infections were significantly upregulated, whereas the biological processes related to inflammation were mainly downregulated (Fig. 5 D and Supplementary Table S3 ). Discussion DHPS is the rate-limiting enzyme for the synthesis of hypusine on EIF5A, thus controlling its activation and the translation of specific proteins. In this report, we found that DHPS was induced in GECs of mice infected by the gastric pathogen H. pylori ; consequently, the level of EIF5A Hyp was also increased in the gastric epithelium. Interestingly, when we specifically knocked-down DHPS in intestinal epithelial cells, we found less H. pylori -induced gastritis in the stomach, suggesting that hypusination in GECs supports inflammation. Our proteomic investigation in isolated GECs from the mice confirmed that the reduction of hypusination was associated with reduced expression of proteins involved in pathophysiological processes in both naïve and infected mice. Lastly, the increased level of hypusination in GECs from patients with H. pylori gastritis underlines the clinical relevance of our findings and highlights DHPS as a potential target to reduce the development of the diseases associated with H. pylori infection. The homozygous deletion of the Dhps gene results in embryonic lethality (Nishimura et al. 2012 ) and biallelic variants in the DHPS gene in humans have been linked to a neurodevelopmental disorder (Ganapathi et al. 2019 ), evidencing that DHPS activity is globally essential for embryogenesis and homeostasis, and therefore constitutively expressed. Notably, this gene can be induced, as we reported increased DHPS levels in macrophages infected with pathogenic bacteria, including H. pylori (Gobert et al. 2020 ), in human colonic epithelial cells (CECs) infected in vitro with enteropathogenic Escherichia coli , and in the colon of mice infected with Citrobacter rodentium , a bacterial pathogen of the colon that induces colitis in mice (Gobert et al. 2024 ). Moreover, the expression of DHPS is also increased in adipose tissue macrophages of obese mice and in bone marrow-derived macrophages from C57BL/6J mice stimulated toward an M1 phenotype with LPS + IFN-g (Anderson-Baucum et al. 2021 ). Similarly, we found increased expression of DHPS in GECs from H. pylori -infected humans and mice; this was associated with an enhanced level of EIF5A Hyp , as expected. We previously reported that the specific deletion of DHPS in myeloid cells using a Lyz2-Cre driver yields an increased colonization of the colon by the rodent pathogen C. rodentium and of the stomach by H. pylori (Gobert et al. 2020 ). This observation led us to propose that hypusination supports macrophage activity, which was consistent with the loss of expression of innate proteins with antimicrobial functions in infected Dhps Dmye mice (Gobert et al. 2020 ). Moreover, we have reported that mice with knock-down of Dhps in intestinal epithelial cells also exhibit increased C. rodentium burden in the colon (Gobert et al. 2024 ), although the hypusine-dependent proteome of macrophages differed from that of CECs in infected mice (Gobert et al. 2020 ; Gobert et al. 2024 ). Herein, we found that Dhps deletion in the stomach has no impact on gastric colonization by H. pylori , demonstrating that hypusination in GECs does not play a major role in the antimicrobial effect of the gastric mucosa and the cell-specificity of DHPS activity. Moreover, we found reduced inflammation and histological damage in H. pylori -infected Dhps ∆epi mice. In contrast, mice with specific Dhps deletion in intestinal epithelial cells exhibited spontaneous colitis and inflammation of the small intestine, increased susceptibility to dextran sulfate sodium-induced and C. rodentium -mediated colitis, and exacerbated tumorigenesis in response to the carcinogen azoxymethane (Gobert et al. 2024 ; Gobert et al. 2023 ) compared to Dhps fl/fl animals. In this context, we propose that the role of hypusination in epithelial cells throughout the gastrointestinal tract is clearly organ specific, being protective in the colon and deleterious in the stomach. The reason behind this discrepancy is likely related to the nature of the transcriptomes of GECs and CECs that are drastically different due to their distinct physiological functions and microenvironments; therefore, the proteins regulated by hypusination in these organs are different, as we have observed in our previous report (Gobert et al. 2023 ) and in the present study. DHPS activity is also controlled by the availability of its substrate spermidine (Gobert et al. 2023 ). The concentration of this polyamine is regulated by the enzyme spermine oxidase (SMOX) in the gastrointestinal tract (Gobert et al. 2022 ). Interestingly, we reported that deletion of Smox in C57BL6 mice and in cancer-prone transgenic FVB/N mice overexpressing the human gastrin gene reduces the development of gastritis and gastric carcinoma, respectively (McNamara et al. 2025 ; Sierra et al. 2020 ), demonstrating that SMOX activity mediates H. pylori pathogenesis. Although we attributed the deleterious effects of SMOX on the synthesis of the monocarbonyl electrophile acrolein in the stomach (McNamara et al. 2025 ), it is also possible that DHPS activity can be enhanced by the generation of spermidine by SMOX. Thus, collectively these data indicate that the spermidine/hypusine pathway is a critical mediator of H. pylori pathogenesis. The development of precancerous lesions in H. pylori -infected patients often occurs in the context of chronic gastritis (Correa 1988 ; Piazuelo et al. 2021 ). Moreover, eradication of H. pylori does not necessarily reduce cancer risk once precancerous lesions are present (Ma et al. 2012 ; Mera et al. 2005 ). Therefore, the inhibition of hypusination in the stomach might represent a therapeutic approach to dampen gastritis, but also a preventive strategy to reduce the risk of gastric cancer development. Further, it has been reported that hypusination supports the growth and proliferation of various established cancer cell lines (Bandino et al. 2014 ; Fang et al. 2018 ; Zhao et al. 2025 ), including from the gastrointestinal tract (Coni et al. 2020 ). In this context, the study of the role of the spermidine/hypusine pathway on gastric cancer cells is warranted and is underway in our laboratory. Declarations Competing interest: The authors disclose no conflicts. Grant Support This study was funded by NIH grants R01DK128200, P01CA116087, and P01CA028842 (KTW); Veterans Affairs Merit Review grants I01CX002171 and I01CX002473 (KTW) and I01BX004366 (LAC); Department of Defense Peer Reviewed Cancer Research Program Impact Award W81XWH-21-1-0617 (KTW); a gift from CURE for IBD (KTW); the Thomas F Frist Sr. Endowment (KTW); and the Vanderbilt Center for Mucosal Inflammation and Cancer (KTW). KMM was supported by T32CA009592 and F31CA278330. The Tissue Morphology Core (MBP) of the Vanderbilt Digestive Disease Research Center is supported by P30DK058404, and the proteomics studies were supported by the Proteomics and Metabolomics Core of P01CA116087 and the Mass Spectrometry Cores of P30DK058404 and the Vanderbilt Ingram Cancer Center supported by NIH grant P30CA068485. Author Contribution Conceptualization, A.P.G., K.M.M., and K.T.W.; Formal Analysis, K.L.R.; Investigation, K.M.M., C.V.H., M.A., D.P.B., A.G.D., P.P., R.N.T., K.S.C., L.A.C., M.B.P., and K.T.W.; Writing – Original Draft, A.P.G. and K.M.M.; Writing – Review & Editing, A.P.G. and K.T.W.; Visualization, A.P.G. and K.M.M.; Supervision, A.P.G. and K.T.W.; Funding Acquisition, A.P.G. and K.T.W. Data Availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2025) partner repository with the dataset identifier PXD069622. 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Supplementary Files DhpsDgecTableS3.xls DhpsDgecTableS2.xls DhpsDgecTableS1.xlsx Cite Share Download PDF Status: Published Journal Publication published 02 Feb, 2026 Read the published version in Amino Acids → Version 1 posted Editorial decision: Revision requested 10 Dec, 2025 Reviews received at journal 10 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviews received at journal 19 Nov, 2025 Reviewers agreed at journal 19 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 23 Oct, 2025 Editor assigned by journal 21 Oct, 2025 Submission checks completed at journal 21 Oct, 2025 First submitted to journal 20 Oct, 2025 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-7906468","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":539921542,"identity":"380af4f0-467d-46fb-a7ef-bd07012a5f3b","order_by":0,"name":"Alain P. 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1","display":"","copyAsset":false,"role":"figure","size":4752190,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e in patients infected with \u003cem\u003eH. pylori\u003c/em\u003e. Gastric biopsies were obtained from patients with gastritis and \u003cem\u003eH. pylori\u003c/em\u003e infection, determined by histology and culture of ground tissues, and from normal individuals. Tissues were immunostained for DHPS or EIF5A\u003csup\u003eHyp\u003c/sup\u003e (green); nucleus were stained with DAPI and appear in blue. These data are representative immunofluorescence images of n = 3-4 patients per group.\u003c/p\u003e","description":"","filename":"DhpsDgecFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/541740fb8b6808130731acda.png"},{"id":95227283,"identity":"3c168a46-af00-4cf7-8cd9-114a7cd56031","added_by":"auto","created_at":"2025-11-05 16:32:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":555459,"visible":true,"origin":"","legend":"\u003cp\u003eLoss of DHPS and hypusination in \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e mice. GECs were isolated from the stomach of \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e mice. Proteins were extracted and the levels of DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e were determined by Western blots (\u003cstrong\u003eA\u003c/strong\u003e) and densitometry (\u003cstrong\u003eB\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"DhpsDgecFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/ce11f29973510406befb294c.png"},{"id":95205792,"identity":"f8da6866-1a4c-406c-b798-5f32fa24d317","added_by":"auto","created_at":"2025-11-05 13:13:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6731771,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eDhps\u003c/em\u003e deletion in GECs on \u003cem\u003eH. pylori\u003c/em\u003e pathogenesis. \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e = 21) and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e = 30) mice were infected or not with \u003cem\u003eH. pylori\u003c/em\u003e PMSS1 for 8 weeks. (\u003cstrong\u003eA\u003c/strong\u003e) The presence of DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e in the gastric tissues was assessed by immunofluroresence (green); representative images of \u003cem\u003en\u003c/em\u003e = 5 mice per group are shown. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eH. pylori\u003c/em\u003e colonization of gastric tissues was determined by culture of serial dilutions of homogenized tissues. (\u003cstrong\u003eC-D\u003c/strong\u003e) H\u0026amp;E images (\u003cstrong\u003eC\u003c/strong\u003e; scale bars: 50 μm) were used to establish the histologic gastritis scores (\u003cstrong\u003eD\u003c/strong\u003e); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 by one-way ANOVA and Šídák’s test.\u003c/p\u003e","description":"","filename":"DhpsDgecFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/26b72c946ad3f263ea9925f2.png"},{"id":95228779,"identity":"3cb579db-3505-4b55-87b1-91fa0c7001e9","added_by":"auto","created_at":"2025-11-05 16:34:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1941863,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic changes in GECs of \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e mice. (\u003cstrong\u003eA\u003c/strong\u003e) Proteins from isolated GECs of uninfected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e mice were analyzed by TMT and the 40 proteins that were the most significantly upregulated or downregulated in \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e GECs are shown (\u003cem\u003en\u003c/em\u003e = 2 per group). The complete list of proteins identified is provided in Supplementary Table S1. (\u003cstrong\u003eB\u003c/strong\u003e) The differential proteomic dataset comparing GECs of \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e to \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice was used to determine the “Disease \u0026amp; Functions” pathways using IPA. See Supplementary Table S1 for the complete list of pathways.\u003c/p\u003e","description":"","filename":"DhpsDgecFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/8e2d71ddc5433cfd3589b6d9.png"},{"id":95205789,"identity":"6e0c1a4a-f65e-4277-b3a4-9f9ef3626016","added_by":"auto","created_at":"2025-11-05 13:13:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1060760,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation by hypusination of the proteome of GECs during \u003cem\u003eH. pylori\u003c/em\u003e infection. \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e mice were infected or not with \u003cem\u003eH. pylori\u003c/em\u003e PMSS1 for 8 weeks. Proteins from isolated GECs were analyzed by TMT (\u003cem\u003en\u003c/em\u003e = 2 per group). (\u003cstrong\u003eA\u003c/strong\u003e) The number of proteins significantly upregulated or downregulated in infected mice compared to uninfected animals in both genotypes is depicted as a Venn diagram. (\u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e) The heatmaps depict the level of expression of the 40 proteins that were the most significantly upregulated (\u003cstrong\u003eB\u003c/strong\u003e) or downregulated (\u003cstrong\u003eC\u003c/strong\u003e) in infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e compared to cells from uninfected mice (column \u003cem\u003ea\u003c/em\u003e); the fold change in infected vs. uninfected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e GECs is shown in column \u003cem\u003eb\u003c/em\u003e. (\u003cstrong\u003eD\u003c/strong\u003e) Functional analysis of the proteins differentially expressed in GECs from infected\u003cem\u003e Dhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDepi\u003c/em\u003e\u003c/sup\u003e compared to infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice using IPA; the complete list of pathways related to “Disease \u0026amp; Functions” is provided in Supplementary Table S3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"DhpsDgecFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/54db3be4fa7ab761756bfa8b.png"},{"id":102234264,"identity":"519adfca-0161-497f-9130-6cffbb839284","added_by":"auto","created_at":"2026-02-09 16:08:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14530324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/74cf83a6-a3cf-4132-bdfa-b9fbb806c7df.pdf"},{"id":95205782,"identity":"ede98fa8-074f-4331-937e-0bc045915cdc","added_by":"auto","created_at":"2025-11-05 13:13:12","extension":"xls","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":335360,"visible":true,"origin":"","legend":"","description":"","filename":"DhpsDgecTableS3.xls","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/a6031567d3fba58905d01791.xls"},{"id":95228478,"identity":"3894c7b7-1db7-4590-9e37-18eaeb229cbd","added_by":"auto","created_at":"2025-11-05 16:33:48","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":271872,"visible":true,"origin":"","legend":"","description":"","filename":"DhpsDgecTableS2.xls","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/ff7af531ebab398437bbf635.xls"},{"id":95228536,"identity":"826cbe8c-d4a5-4b64-a827-3a27de700933","added_by":"auto","created_at":"2025-11-05 16:33:53","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8264523,"visible":true,"origin":"","legend":"","description":"","filename":"DhpsDgecTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7906468/v1/f6cad9e2a2fb306b6776f649.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epithelial Hypusination Regulates Helicobacter pylori-induced Gastric Inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe polyamine spermidine serves an essential role as the substrate for the synthesis of hypusine, a unique amino acid found only in the highly conserved eukaryotic protein eukaryotic translation initiation factor 5A (EIF5A) (Park et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The hypusine modification is catalyzed by the sequential action of two enzymes, deoxyhypusine synthase (DHPS), the rate-limiting enzyme, which transfers the 4-aminobutyl moiety of the polyamine spermidine to the Lys50 residue of EIF5A (Joe et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and deoxyhypusine hydroxylase (DOHH), which hydroxylases the intermediate deoxyhypusine residue (Abbruzzese et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). This post-translational modification is essential for the function of EIF5A, as hypusinated EIF5A (EIF5A\u003csup\u003eHyp\u003c/sup\u003e) can bind mRNAs that contain a 5\u0026prime;-AAAUGU-3\u0026prime;\u0026ensp;consensus sequence (Maier et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). EIF5A\u003csup\u003eHyp\u003c/sup\u003e can also alleviate ribosome stalling at polyproline repeats during translation elongation and at other non-polyproline motifs, such as peptides enriched in basic amino acids (Pelechano and Alepuz \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Schuller et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHypusination has been implicated in regulating inflammatory responses, specifically in myeloid and T cells (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Puleston et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We have reported that hypusination in myeloid cells supports the antimicrobial response of macrophages to pathogenic bacteria including \u003cem\u003eHelicobacter pylori\u003c/em\u003e (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a bacteria that colonizes the human stomach and causes diseases ranging from non-atrophic gastritis to the precancerous lesions of multifocal atrophic gastritis, intestinal metaplasia (IM), dysplasia, and gastric adenocarcinoma (Correa \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Piazuelo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, mice with a myeloid-specific deletion of \u003cem\u003eDhps\u003c/em\u003e exhibited increased bacterial burden and inflammation, implicating hypusination in host defense against pathogenic bacteria (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the effect of hypusination in gastric epithelial cells (GECs) on \u003cem\u003eH. pylori\u003c/em\u003e pathogenesis remains unknown.\u003c/p\u003e\u003cp\u003eIn this report, we showed that patients with \u003cem\u003eH. pylori\u003c/em\u003e gastritis exhibit increased level of DHPS and hypusinated EIF5A. We then created mice with specific deletion of \u003cem\u003eDhps\u003c/em\u003e in intestinal epithelial cells including in the stomach to assess the role of hypusination in GECs in \u003cem\u003eH. pylori\u003c/em\u003e pathogenesis. We found that these infected mice develop less gastritis, demonstrating that the activity of DHPS in GECs supports stomach inflammation. Further, the proteome of the GECs in \u003cem\u003eDhps\u003c/em\u003e-deficient mice is reshaped toward a less inflammatory and carcinogenic profile.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003eEndoscopic gastric biopsies were obtained from patients at the Nashville Veterans Affairs Medical Center of the VA Tennessee Valley Healthcare System. Patients were undergoing esophagogastroduodenoscopy for clinically indicated reasons and provided informed consent for obtaining research biopsies under VA IRB protocol 1571167.\u003c/p\u003e\u003cp\u003e The mice were used under protocols V2000018 and V2300022 approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee and the Research and Development Committee of the Veterans Affairs Tennessee Valley Healthcare System. Procedures followed institutional policies, AAALAC guidelines, the AVMA Guidelines on Euthanasia, NIH regulations regarding the Guide for the Care and Use of Laboratory Animals, and the United States Animal Welfare Act of 1996.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBacteria\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eH. pylori\u003c/em\u003e PMSS1, a \u003cem\u003ecagA\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e strain with intact type IV secretion system function, was grown on Trypticase soy agar plates containing 10% sheep\u0026rsquo;s blood. Bacteria were harvested from the plates and grown overnight in Brucella broth containing 10% fetal bovine serum (FBS). This culture was resuspended in fresh Brucella broth-FBS and then collected at the exponential phase to infect the mice.\u003c/p\u003e\n\u003ch3\u003eMice and infections\u003c/h3\u003e\n\u003cp\u003eWe used \u003cem\u003eFoxa3\u003c/em\u003e-cre mice that we crossed with \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice to obtain C57BL/6 \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e;Foxa3\u003csup\u003e+/+\u003c/sup\u003e (\u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e) and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eFoxa3\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e) mice. Note that \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eFoxa3\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e mice were embryonically lethal or only survived a few weeks out of utero. Mice were housed in a pathogen-free facility, with ventilated cage racks and were on a 12 h light-dark cycle. Male and female mice between 8 and 12 weeks were used for all studies. Animals were infected by oral gavage with 10\u003csup\u003e9\u003c/sup\u003e colony forming units (CFU) of \u003cem\u003eH. pylori\u003c/em\u003e PMSS1 in 200 \u0026micro;L Brucella broth, two times, on days 0 and 2. The control mice were gavaged with only broth on both days. Eight weeks after the first infection, mice were euthanized and stomachs were harvested. Colonization was determined in all infected mice by counting the CFUs cultured after plating serial dilutions of homogenized gastric tissues (Latour et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; McNamara et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sierra et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eHistopathology\u003c/h3\u003e\n\u003cp\u003eHuman biopsies from the gastric antrum and corpus, and longitudinal strips of murine stomach tissue including the corpus and antrum, were fixed in 10% neutral buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin (H\u0026amp;E). Biopsies were scored as reported (Latour et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; McNamara et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sierra et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Histology was scored by our gastrointestinal pathologist (M.B.P.) who was blinded to the experimental groups. H. pylori infection of patient tissues was confirmed by culture of gastric biopsies as described above for the mouse tissues.\u003c/p\u003e\n\u003ch3\u003eEpithelial cell isolation\u003c/h3\u003e\n\u003cp\u003eStomachs were removed from \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e animals and incubated in a solution of cold 0.5 mM DTT and 3 mM EDTA for 30 min on ice. After incubation, the tissues were placed in 3 mM EDTA and vigorously shaken to release gastric glands. The cell suspension was poured over a 70 \u0026micro;m Strainer (Falcon) and the resulting isolated GECs were pelleted through centrifugation at 1500 rpm for 10 min at 4 \u0026deg;C.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eProteomics analysis\u003c/h2\u003e\u003cp\u003eIsolated epithelial cells were lysed in 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 2 mM EDTA, and 1% SDS; protein concentration was measured by the BCA Protein Assay (Pierce) and samples from the same group were pooled. Protein extracts were reduced with 10 mM TCEP (tris(2-carboxyethyl)phosphine), alkylated with 20 mM iodoacetamide, and protein samples were prepared by S-Trap\u0026trade; (ProtiFi) digestion with trypsin (1:10) similar to methods described in Howard \u003cem\u003eet al\u003c/em\u003e (Howard et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). TMT-based quantitative proteomics was performed as described (Latour et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Labeled peptides (5 \u0026micro;g per sample) were combined, fractionated using high pH reversed phase fractionation, and elution steps were performed with 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 60% acetonitrile with 0.1% triethylamine. Fractions were dried and reconstituted in 0.2% formic acid for LC-MS/MS analysis. Peptides were gradient-eluted at a flow rate of 350 nl/min, using varied reverse phase gradients over 90 min. For fraction 1, peptides were analyzed with the following gradient: 5\u0026ndash;18% B in 75 min, 18\u0026ndash;50% B in 6 min, 50\u0026ndash;70% B in 3 min, 70\u0026thinsp;\u0026minus;\u0026thinsp;2% B in 1 min, 2% B for 5 min. For fractions 2\u0026ndash;4, the first 2 steps of the gradient were adjusted to 5\u0026ndash;25% B in 75 min and 25\u0026ndash;50% B in 6 min, with the subsequent three steps identical to fraction 1. For fraction 5, the gradient included 2\u0026ndash;8% B in 0.5 min, 8\u0026ndash;30% B in 74.5 min, 30\u0026ndash;50% B in 6 min, 50\u0026ndash;70% B in 2 min, followed by the same final two steps. For fraction 6, the gradient included 2\u0026ndash;8% B in 2 min, 8\u0026ndash;30% B in 73 min, 30\u0026ndash;50% B in 7 min, 50\u0026ndash;70%B in 1 min, followed by the same final two steps. For fraction 7\u0026ndash;8, the gradient included 5\u0026ndash;45% B in 75 min, 45\u0026ndash;90% B in 8 min, 90% B for 1 min, 90\u0026thinsp;\u0026minus;\u0026thinsp;2% B in 1 min, and 2% B for 5 min. Peptides were analyzed using a data-dependent acquisition method on an Orbitrap Exploris 240 mass spectrometer (Thermo Scientific), equipped with a nanoelectrospray ionization source. The instrument method consisted of MS1, followed by up to 20 MS/MS scans, with an automatic gain control target of 2x10\u003csup\u003e5\u003c/sup\u003e. Higher-energy collisional dissociation was set to 35 nce and dynamic exclusion (15 sec) was enabled. Data were searched in Proteome Discoverer 2.2 (Thermo Scientific) using SequestHT for database searching against a mouse database created from the UniProtKB database. Search parameters and quantitative analysis was performed as reported (Latour et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) partner repository with the dataset identifier PXD069622\u003c/p\u003e\u003cp\u003eIngenuity pathway analysis (IPA) software (QIAGEN) was used for the functional interpretation of differential expression results obtained from the proteomic analyses. The pathways related to Diseases and Functions were generated.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis\u003c/div\u003e\u003cp\u003eProteins were extracted from isolated GECs as reported (Latour et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and concentrations were determined using the BCA Protein Assay (Pierce). Western blots were performed using 10 \u0026micro;g protein per lane using a rabbit polyclonal anti-DHPS antibody (Ab; Abcam, Cat#ab202133; 1:5000), a rabbit polyclonal anti-EIF5A\u003csup\u003eHyp\u003c/sup\u003e Ab (Millipore, Cat#ABS1064-I; 1:8000), or a mouse monoclonal anti-b-actin Ab (MilliporeSigma, Cat#A5316; 1:10000). The Peroxidase AffiniPure\u0026reg; Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Jackson ImmunoResearch, Cat#111-035-003; 1:5000) or the goat anti-mouse IgG, HRP-labeled (Jackson ImmunoResearch, Cat#115-035-003; 1:5000) were the used as secondary Abs.\u003c/p\u003e\n\u003ch3\u003eImmunostaining\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence was performed on human gastric biopsies and murine gastric tissues. Sections were deparaffinized and incubated at room temperature with 3% hydrogen peroxide in phosphate-buffered saline to block endogenous peroxidase. Tissues were then blocked for 1 h in Protein Block, Serum-Free (Dako, Cat#X0909). Slides were sequentially incubated with a rabbit polyclonal anti-DHPS Ab (Proteintech, Cat#11184-1-AP; 1:1000) or a rabbit anti-EIF5A\u003csup\u003eHyp\u003c/sup\u003e Ab (MilliporeSigma Cat#ABS1064; 1:2000) overnight at 4\u0026deg;C and with a donkey anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor\u0026trade; Plus 488 (ThermoFisher Scientific, Cat#A32790; 1:700) 45 min at room temperature. Slides were mounted with VECTASHIELD HardSet\u0026trade; Antifade Mounting Medium with DAPI (Vector Laboratories, Cat#H-1500-10) and visualized using a Nikon E800 microscope and a SPOT Imaging CMOS camera.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003ePrism 10.6.0 (GraphPad Inc.) was used for figure design and statistical analysis. All the data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Data that were not normally distributed according to the D\u0026rsquo;Agostino \u0026amp; Pearson normality test were log transformed. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test was used to determine significant differences between two groups, whereas a one-way ANOVA followed by a Tukey\u0026rsquo;s test or Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s test was used for multiple groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIncreased levels of DHPS and EIF5A\u003c/b\u003e\u003csup\u003e\u003cb\u003eHyp\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eH. pylori\u003c/b\u003e\u003cb\u003e-infected patients\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing immunostaining, we evidenced that that the level of DHPS and hypusinated EIF5A were increased overall in the gastric mucosa of endoscopic biopsies from patients with \u003cem\u003eH. pylori\u003c/em\u003e gastritis compared to individuals without infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of importance, the staining was abundant in GECs in \u003cem\u003eH. pylori\u003c/em\u003e-infected patients but also present in the immune infiltrates, as we reported (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDeletion of epithelial hypusination reduces the inflammatory response to\u003c/b\u003e \u003cb\u003eH. pylori\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of hypusination in epithelial cells during \u003cem\u003eH. pylori\u003c/em\u003e infection we utilized a genetic approach by generating C57BL/6 animals with a gastric epithelial-specific knockout of \u003cem\u003eDhps\u003c/em\u003e. First, we verified by immunoblots and densitometry that \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice exhibited reduced expression of DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e levels in the gastric epithelium compared to \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThen, we infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice with \u003cem\u003eH. pylori\u003c/em\u003e PMSS1 for 8 weeks. We confirmed by immunofluorescence that DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e were less expressed in GECs from naive \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Upon infection, the levels of DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e were increased in GECs and immune infiltrates of \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice compared to uninfected animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA); there was markedly less staining in the GECs of \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice, whereas immune cells were still positive for DHPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAll 30 \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice were colonized and only 1 of the 21 \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice was not colonized and was thus removed from the analysis. We observed no difference in gastric bacterial burden between both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). An inflammatory infiltrate and mild foveolar hyperplasia were mainly observed at the antrocorporal transitional mucosa of \u003cem\u003eH. pylori\u003c/em\u003e-infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These parameters were less observed in the stomach of infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Using a comprehensive score, we found increased inflammation in both genotypes compared to uninfected animals, but also significantly less inflammation in the gastric tissue of infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice compared to the infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/fl\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003cb\u003eProteome of\u003c/b\u003e \u003cb\u003eH. pylori\u003c/b\u003e\u003cb\u003e-infected animals with\u003c/b\u003e \u003cb\u003eDhps\u003c/b\u003e \u003cb\u003edeficiency\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the role of the hypusination pathway during \u003cem\u003eH. pylori\u003c/em\u003e-mediated inflammation, we performed TMT proteomics on isolated GECs from uninfected and infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\u003cp\u003eFirst, we analyzed the proteome in GECs from na\u0026iuml;ve mice. There were 79 proteins significantly induced by the specific \u003cem\u003eDhps\u003c/em\u003e deletion in epithelial cells (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These proteins were signaling molecules, such as cyclin-dependent kinase 1 (CDK1) or Src substrate cortactin (SRC8), the superoxide dismutase SODM, and numerous heterogeneous nuclear ribonucleoproteins (ROA1/A2/A3/AA), which are RNA-binding proteins playing critical roles in multiple cellular processes such as DNA repair and regulation of gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We also found 183 proteins downregulated in GECs from \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice. Among them we found numerous ribosomal proteins (e.g., RM24, RS4X, RT33), as expected, different cytochromes P450 (e.g. CP3AB, CP2E1, CP1A2), and one glutathione-S-transferase (MGST1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The changes in the level of these proteins in GECs were overall associated with a lessening of the pathways associated with organismal injury, cancer, and gastrointestinal diseases in \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e); pathways associated with cell movement were mainly induced in \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe identified 110 and 174 proteins significantly induced by \u003cem\u003eH. pylori\u003c/em\u003e infection in the GECs from the stomach tissues from \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice, respectively. Among them, 37 were in common to both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These included mediators of adaptive immunity (IGHA, HB2A/2I, HG2A, and TGTP2), and regulators of the innate immune response, such as DOXA2, I23O1, and STAT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However overall, the level of expression of these proteins in \u003cem\u003eH. pylori\u003c/em\u003e-infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice was lower compared to infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003eanimals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, there were 170 proteins significantly downregulated in GECs from \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice with infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas only 61 proteins were less expressed in infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e GECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA); only 18 proteins were similar in both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The proteins downregulated by \u003cem\u003eH. pylori\u003c/em\u003e infection in \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003eanimals included numerous cytochromes, e.g., CP2F, CP2E1, CP3AB, CP2DA, and CP240, which are known to be downregulated during infection and inflammation, TFF1, the stabilizer of the mucous gel overlying the gastrointestinal mucosa that provides a physical barrier against bacteria, and the marker of M2/Mreg macrophages, ARGI1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e); most of these effectors were less altered in infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen we analyzed the functional clusters corresponding to the proteins differentially expressed between infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e versus infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e mice (see Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), we evidenced that numerous pathways related to infections were significantly upregulated, whereas the biological processes related to inflammation were mainly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDHPS is the rate-limiting enzyme for the synthesis of hypusine on EIF5A, thus controlling its activation and the translation of specific proteins. In this report, we found that DHPS was induced in GECs of mice infected by the gastric pathogen \u003cem\u003eH. pylori\u003c/em\u003e; consequently, the level of EIF5A\u003csup\u003eHyp\u003c/sup\u003e was also increased in the gastric epithelium. Interestingly, when we specifically knocked-down DHPS in intestinal epithelial cells, we found less \u003cem\u003eH. pylori\u003c/em\u003e-induced gastritis in the stomach, suggesting that hypusination in GECs supports inflammation. Our proteomic investigation in isolated GECs from the mice confirmed that the reduction of hypusination was associated with reduced expression of proteins involved in pathophysiological processes in both na\u0026iuml;ve and infected mice. Lastly, the increased level of hypusination in GECs from patients with \u003cem\u003eH. pylori\u003c/em\u003e gastritis underlines the clinical relevance of our findings and highlights DHPS as a potential target to reduce the development of the diseases associated with \u003cem\u003eH. pylori\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003eThe homozygous deletion of the \u003cem\u003eDhps\u003c/em\u003e gene results in embryonic lethality (Nishimura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and biallelic variants in the \u003cem\u003eDHPS\u003c/em\u003e gene in humans have been linked to a neurodevelopmental disorder (Ganapathi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), evidencing that DHPS activity is globally essential for embryogenesis and homeostasis, and therefore constitutively expressed. Notably, this gene can be induced, as we reported increased DHPS levels in macrophages infected with pathogenic bacteria, including \u003cem\u003eH. pylori\u003c/em\u003e (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), in human colonic epithelial cells (CECs) infected \u003cem\u003ein vitro\u003c/em\u003e with enteropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e, and in the colon of mice infected with \u003cem\u003eCitrobacter rodentium\u003c/em\u003e, a bacterial pathogen of the colon that induces colitis in mice (Gobert et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, the expression of DHPS is also increased in adipose tissue macrophages of obese mice and in bone marrow-derived macrophages from C57BL/6J mice stimulated toward an M1 phenotype with LPS\u0026thinsp;+\u0026thinsp;IFN-g (Anderson-Baucum et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, we found increased expression of DHPS in GECs from \u003cem\u003eH. pylori\u003c/em\u003e-infected humans and mice; this was associated with an enhanced level of EIF5A\u003csup\u003eHyp\u003c/sup\u003e, as expected.\u003c/p\u003e\u003cp\u003eWe previously reported that the specific deletion of DHPS in myeloid cells using a Lyz2-Cre driver yields an increased colonization of the colon by the rodent pathogen \u003cem\u003eC. rodentium\u003c/em\u003e and of the stomach by \u003cem\u003eH. pylori\u003c/em\u003e (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This observation led us to propose that hypusination supports macrophage activity, which was consistent with the loss of expression of innate proteins with antimicrobial functions in infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eDmye\u003c/em\u003e\u003c/sup\u003e mice (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, we have reported that mice with knock-down of \u003cem\u003eDhps\u003c/em\u003e in intestinal epithelial cells also exhibit increased \u003cem\u003eC. rodentium\u003c/em\u003e burden in the colon (Gobert et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), although the hypusine-dependent proteome of macrophages differed from that of CECs in infected mice (Gobert et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gobert et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Herein, we found that \u003cem\u003eDhps\u003c/em\u003e deletion in the stomach has no impact on gastric colonization by \u003cem\u003eH. pylori\u003c/em\u003e, demonstrating that hypusination in GECs does not play a major role in the antimicrobial effect of the gastric mucosa and the cell-specificity of DHPS activity. Moreover, we found reduced inflammation and histological damage in \u003cem\u003eH. pylori\u003c/em\u003e-infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆epi\u003c/em\u003e\u003c/sup\u003e mice. In contrast, mice with specific \u003cem\u003eDhps\u003c/em\u003e deletion in intestinal epithelial cells exhibited spontaneous colitis and inflammation of the small intestine, increased susceptibility to dextran sulfate sodium-induced and \u003cem\u003eC. rodentium\u003c/em\u003e-mediated colitis, and exacerbated tumorigenesis in response to the carcinogen azoxymethane (Gobert et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Gobert et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) compared to \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e animals. In this context, we propose that the role of hypusination in epithelial cells throughout the gastrointestinal tract is clearly organ specific, being protective in the colon and deleterious in the stomach. The reason behind this discrepancy is likely related to the nature of the transcriptomes of GECs and CECs that are drastically different due to their distinct physiological functions and microenvironments; therefore, the proteins regulated by hypusination in these organs are different, as we have observed in our previous report (Gobert et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and in the present study.\u003c/p\u003e\u003cp\u003eDHPS activity is also controlled by the availability of its substrate spermidine (Gobert et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The concentration of this polyamine is regulated by the enzyme spermine oxidase (SMOX) in the gastrointestinal tract (Gobert et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, we reported that deletion of \u003cem\u003eSmox\u003c/em\u003e in C57BL6 mice and in cancer-prone transgenic FVB/N mice overexpressing the human gastrin gene reduces the development of gastritis and gastric carcinoma, respectively (McNamara et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sierra et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), demonstrating that SMOX activity mediates \u003cem\u003eH. pylori\u003c/em\u003e pathogenesis. Although we attributed the deleterious effects of SMOX on the synthesis of the monocarbonyl electrophile acrolein in the stomach (McNamara et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), it is also possible that DHPS activity can be enhanced by the generation of spermidine by SMOX. Thus, collectively these data indicate that the spermidine/hypusine pathway is a critical mediator of \u003cem\u003eH. pylori\u003c/em\u003e pathogenesis.\u003c/p\u003e\u003cp\u003eThe development of precancerous lesions in \u003cem\u003eH. pylori\u003c/em\u003e-infected patients often occurs in the context of chronic gastritis (Correa \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Piazuelo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, eradication of \u003cem\u003eH. pylori\u003c/em\u003e does not necessarily reduce cancer risk once precancerous lesions are present (Ma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mera et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Therefore, the inhibition of hypusination in the stomach might represent a therapeutic approach to dampen gastritis, but also a preventive strategy to reduce the risk of gastric cancer development. Further, it has been reported that hypusination supports the growth and proliferation of various established cancer cell lines (Bandino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Fang et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), including from the gastrointestinal tract (Coni et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this context, the study of the role of the spermidine/hypusine pathway on gastric cancer cells is warranted and is underway in our laboratory.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors disclose no conflicts.\u003c/p\u003e\n\u003ch2\u003eGrant Support\u003c/h2\u003e\n\u003cp\u003eThis study was funded by NIH grants R01DK128200, P01CA116087, and P01CA028842 (KTW); Veterans Affairs Merit Review grants I01CX002171 and I01CX002473 (KTW) and I01BX004366 (LAC); Department of Defense Peer Reviewed Cancer Research Program Impact Award W81XWH-21-1-0617 (KTW); a gift from CURE for IBD (KTW); the Thomas F Frist Sr. Endowment (KTW); and the Vanderbilt Center for Mucosal Inflammation and Cancer (KTW). KMM was supported by T32CA009592 and F31CA278330. The Tissue Morphology Core (MBP) of the Vanderbilt Digestive Disease Research Center is supported by P30DK058404, and the proteomics studies were supported by the Proteomics and Metabolomics Core of P01CA116087 and the Mass Spectrometry Cores of P30DK058404 and the Vanderbilt Ingram Cancer Center supported by NIH grant P30CA068485.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, A.P.G., K.M.M., and K.T.W.; Formal Analysis, K.L.R.; Investigation, K.M.M., C.V.H., M.A., D.P.B., A.G.D., P.P., R.N.T., K.S.C., L.A.C., M.B.P., and K.T.W.; Writing \u0026ndash; Original Draft, A.P.G. and K.M.M.; Writing \u0026ndash; Review \u0026amp; Editing, A.P.G. and K.T.W.; Visualization, A.P.G. and K.M.M.; Supervision, A.P.G. and K.T.W.; Funding Acquisition, A.P.G. and K.T.W.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2025) partner repository with the dataset identifier PXD069622.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbruzzese A, Park MH, Folk JE (1986) Deoxyhypusine hydroxylase from rat testis. 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Gastroenterology 160 (4):1106-1117\u003c/li\u003e\n\u003cli\u003ePuleston DJ, Baixauli F, Sanin DE, Edwards-Hicks J, Villa M, Kabat AM, Kaminski MM, Stanckzak M, Weiss HJ, Grzes KM, Piletic K, Field CS, Corrado M, Haessler F, Wang C, Musa Y, Schimmelpfennig L, Flachsmann L, Mittler G, Yosef N, Kuchroo VK, Buescher JM, Balabanov S, Pearce EJ, Green DR, Pearce EL (2021) Polyamine metabolism is a central determinant of helper t cell lineage fidelity. Cell 184 (16):4186-4202\u003c/li\u003e\n\u003cli\u003eSchuller AP, Wu CC, Dever TE, Buskirk AR, Green R (2017) EIF5A functions globally in translation elongation and termination. Mol Cell 66 (2):194-205\u003c/li\u003e\n\u003cli\u003eSierra JC, Piazuelo MB, Luis PB, Barry DP, Allaman MM, Asim M, Sebrell TA, Finley JL, Rose KL, Hill S, Holshouser SL, Casero RA, Cleveland JL, Woster PM, Schey KL, Bimczok D, Schneider C, Gobert AP, Wilson KT (2020) Spermine oxidase mediates \u003cem\u003eHelicobacter pylori-\u003c/em\u003einduced gastric inflammation, DNA damage, and carcinogenic signaling. Oncogene 39 (22):4465-4474 \u003c/li\u003e\n\u003cli\u003eXu A, Jao DL, Chen KY (2004) Identification of mRNA that binds to eukaryotic initiation factor 5a by affinity co-purification and differential display. Biochem J 384 (Pt 3):585-590 \u003c/li\u003e\n\u003cli\u003eZhao G, Zhao X, Liu Z, Wang B, Dong P, Watari H, Pfeffer LM, Tigyi G, Zhang W, Yue J (2025) Knockout or inhibition of dhps suppresses ovarian tumor growth and metastasis by attenuating the tgfbeta pathway. Sci Rep 15 (1):917 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"amino-acids","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amac","sideBox":"Learn more about [Amino Acids](http://link.springer.com/journal/726)","snPcode":"726","submissionUrl":"https://submission.nature.com/new-submission/726/3","title":"Amino Acids","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Infection, Gastritis, Polyamines, Hypusine, Proteome","lastPublishedDoi":"10.21203/rs.3.rs-7906468/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7906468/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypusine is a unique amino acid synthesized on the eukaryotic initiation factor 5A (EIF5A) from the polyamine spermidine by deoxyhypusine synthase (DHPS). Hypusination of EIF5A plays a key role in translation. Here, we examined the contribution of the epithelial hypusination pathway to gastric inflammation induced by \u003cem\u003eHelicobacter pylori\u003c/em\u003e. Immunohistochemical analyses revealed increased expression of DHPS and hypusinated EIF5A (EIF5A\u003csup\u003eHyp\u003c/sup\u003e) in the gastric mucosa of patients with \u003cem\u003eH. pylori\u003c/em\u003e gastritis compared to uninfected individuals, notably within gastric epithelial cells (GECs) and immune infiltrates. Then, we created a mouse model with epithelial-specific deletion of Dhps (\u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e) and confirmed the reduction of DHPS and EIF5A\u003csup\u003eHyp\u003c/sup\u003e in GECs. \u003cem\u003eH. pylori\u003c/em\u003e-infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e mice exhibited an attenuation of gastric histologic inflammation scores compared with infected \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e controls, without alteration in bacterial colonization levels. Quantitative proteomics of isolated GECs showed that \u003cem\u003eDhps\u003c/em\u003e deletion altered the expression of proteins involved in organismal injury, cancer, and gastrointestinal diseases in na\u0026iuml;ve mice. Upon \u003cem\u003eH. pylori\u003c/em\u003e infection, inflammatory and immune response proteins, including signaling factors and immunoglobulin mediators, were less induced in \u003cem\u003eDhps\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔepi\u003c/em\u003e\u003c/sup\u003e GECs, and pathways linked to tissue injury and inflammation were selectively downregulated. Together, these findings demonstrate that epithelial hypusination supports \u003cem\u003eH. pylori\u003c/em\u003e-driven gastric inflammation without affecting bacterial persistence. Targeting DHPS-dependent EIF5A hypusination may thus represent a novel therapeutic strategy to limit \u003cem\u003eH. pylori\u003c/em\u003e-associated mucosal injury and disease progression.\u003c/p\u003e","manuscriptTitle":"Epithelial Hypusination Regulates Helicobacter pylori-induced Gastric Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 13:13:07","doi":"10.21203/rs.3.rs-7906468/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-10T20:53:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T17:12:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T02:30:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339682435488476120541698726505995957579","date":"2025-11-20T11:16:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-20T00:27:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100235940781787953763691339166659898251","date":"2025-11-19T18:47:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25603147354816673993983400971213509883","date":"2025-11-10T20:48:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T20:44:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-21T19:25:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-21T07:26:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Amino Acids","date":"2025-10-20T13:52:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amino-acids","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amac","sideBox":"Learn more about [Amino Acids](http://link.springer.com/journal/726)","snPcode":"726","submissionUrl":"https://submission.nature.com/new-submission/726/3","title":"Amino Acids","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"77e2835b-ea79-4e1d-95ce-419b93e76e67","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:05:13+00:00","versionOfRecord":{"articleIdentity":"rs-7906468","link":"https://doi.org/10.1007/s00726-026-03496-3","journal":{"identity":"amino-acids","isVorOnly":false,"title":"Amino Acids"},"publishedOn":"2026-02-02 15:58:19","publishedOnDateReadable":"February 2nd, 2026"},"versionCreatedAt":"2025-11-05 13:13:07","video":"","vorDoi":"10.1007/s00726-026-03496-3","vorDoiUrl":"https://doi.org/10.1007/s00726-026-03496-3","workflowStages":[]},"version":"v1","identity":"rs-7906468","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7906468","identity":"rs-7906468","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}