Blankenberger, Amanda

Background: Exogenous and endogenous polyamines are critical for gastrointestinal (GI) barrier integrity, of which putrescine, spermidine and spermine are most well studied. Our recent work shows n-acetylputrescine, a putrescine derivative, correlates with increased intestinal permeability and loss of microbial diversity. Despite this, bacterial mechanisms of n-acetylputrescine production or direct effects on barrier integrity are unknown. Aim: Determine the role of microbiota in shaping n-acetylputrescine abundance and the effect on barrier integrity. Methods: N-acetylputrescine effects on barrier were assessed both in vitro using Caco-2 and ex vivo in Ussing using colonic strips from conventional mice. Germ-free (GF) mice were monocolonized with wild-type E. coli (SK929) or E.coli SK930 (2-weeks, n=4mice/group), strains given by Dr. Shin Kurihara. E. coli SK930 is deficient in SpeC, SpeF and SpeB, genes generating putrescine, required for n-acetylputrescine production. Untargeted metabolomics (Metabolon) was performed on fecal pellets. Targeted isotope tracing with [13C6, 15N4]-arginine delivered via enema was done to track bacterial production of n-acetylputrescine by Fusobacterium varium, a known producer, in vivo with isotopolog labeling measured by LC/HRMS. Results: In vitro n-acetylputrescine (100mM) addition to Caco-2 monolayers caused significant barrier disruption compared to controls (%Baseline TER, 13.7±6.7 vs 102.8±9.4, 3h postexposure, n=3 p=0.0002), not seen at lower (10mM) concentration. N-acetylputrescine caused increased flux of 300Da fluorescein (12.6±2.0 vs 6.0±0.2 mg/mL, n=3 p=0.004) and 4kDa FITC-Dextran flux on Ussing compared to untreated controls (999±94 vs 562±121 ng/hr/cm2, n=3 p= 0.01). Mutant E. coli SK930 monocolonized mice had similar fecal n-acetylputrescine to GF animals. In contrast, wild type E. coli SK929 monocolonization led to significantly greater n-acetylputrescine (5-fold, FDR< 0.001) compared to SK930, indicating a bulk of n-acetylputrescine production is driven by bacterial mechanisms. Pathway enrichment analysis predicted decreased spermidine and spermine biosynthesis in SK930 monocolonization, indicative of impaired polyamine metabolism, compared to SK929 (FDR< 0.01). Targeted metabolomics on fecal pellets showed a shift in the production of isotopolog [13C1, 15N0]-N-acetylputrescine to increased isotopolog [13C4, 15N2]-N-acetylputrescine concentration after monocolonization with F. varium compared to GF mice (5.9±5.0 vs. 2.1±3.6 pg/mg stool, n=3mice/group p=0.05) suggesting microbiota driven shifts in n-acetylputrescine metabolism. Conclusion: Luminal n-acetylputrescine production and profile is driven by bacteria in the GI tract. Concentration dependent effects of n-acetylputrescine suggest insufficient regulation by intestinal microbiota can lead to accumulation of n-acetylputrescine and the intestinal barrier disruption seen in human studies. Ongoing studies aim to identify bacterial taxa critical for n-acetylputrescine suppression, the mechanisms involved, and impact on GI health. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Polyamines produced by host cells and intestinal microbiota and are critical to overall health. Studies have shown putrescine, a polyamine, can strengthen, or in some instances disrupt, gastrointestinal (GI) barrier integrity, yet the regulation of polyamine concentrations in the GI tract is unknown. Host colonocytes and microbiota both metabolize polyamines, and in GI diseases like irritable bowel syndrome (IBS) where intestinal microbiota is significantly altered, polyamine metabolism may be affected. Aim: Determine regulation of luminal polyamine concentration and its effects on intestinal barrier integrity. Hypothesis: Luminal polyamine concentration is suppressed by commensal intestinal microbiota and increasing concentration of putrescine results in intestinal barrier disruption. Methods: Microbiota effects on luminal polyamine concentrations were assessed by humanizing germ-free (GF) mice with a single oral gavage of commensal human microbiota. Polyamine effect on barrier integrity was assessed in vitro using Caco-2 cells and ex vivo on Ussing using mouse colonic tissue with 300Da fluorescein and 4kDa FITC-Dextran tracers. Changes in transepithelial resistance (TER) and rate of tracer flux was recorded. Untargeted metabolomics with shotgun metagenomics was completed on human fecal samples and sigmoid colonic biopsies were collected for bulk RNAseq. Bray-Curtis distance was determined at the species level using the PERMANOVA method and used to test relationships between beta-diversity and fecal polyamine concentration. Results: Humanization with human commensal microbiota (1.2% C. aerofaciens) lead to a 20-fold increase in cecal putrescine and a 20-fold decrease in fecal putrescine compared to GF controls. In vitro addition of putrescine (100mM) caused increased fluorescein flux (3.50±0.24 vs 1.44±0.04 mg/mL) and a rapid loss of transepithelial resistance compared to controls (% of Baseline TER, 36.6±8.2 vs 102.8±5.4, 3h postexposure). Ex vivo addition of putrescine caused a loss of TER (57% vs 89% of baseline by 3h) and an increased rate of 4kDa FITC-Dextran flux compared to untreated controls (1236±131 vs 562±70 ng/hr/cm2). Transcriptomic profiles show decreased expression of ornithine decarboxylase antienzyme (OAZ1, FDR=0.018) in IBS which inhibits the rate limiting enzyme for putrescine production. Increased lactulose excretion (2-24h), suggestive of increased distal colonic permeability, correlated with N-acetylputrescine concentration (p=0.07). N-acetylputrescine also correlated positively with increased stool frequency (p=0.009) and looser consistency (p=0.013). C. aerofaciens, a polyamine consumer, negatively correlated with n-acetylputrescine (FDR=0.02) suggesting a loss of specific taxa may lead to insuffcient polyamine metabolism. Conclusion: Polyamines, namely putrescine, disrupts intestinal barrier integrity, correlate with in vivo permeability, and are decreased in the colon by commensal microbiota. These observations identify putrescine as a negative effector of barrier function and suggest a novel role for the commensal microbiome in regulating barrier integrity through polyamine metabolism. Further work investigating host and microbiota mechanisms of polyamine metabolism, signaling and the specific taxa involved is needed to better understand the effects polyamines have on GI physiology. NIH DK 127998 (MG), support from Mayo Clinic Center for Individualized Medicine (CIM), and Mayo Clinic Center for Cell Signalling in Gastroenterology (C-Sig) P30DK084567. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.