Commensal *Hafnia alvei* strain reduces food intake and fat mass in obese mice—a new potential probiotic for appetite and body weight management

Obesity arises from multifactorial causes involving both the human genome and the gut microbiota metagenome. The gut microbiota composition is dynamic, influenced by diet, age, sex, geography and medications, and alterations have been linked causally to obesity as shown by phenotype transfer in animal models. Conversely, healthy microbiota can regulate physiological processes including feeding behavior. Traditional probiotics (Lactobacillus, Bifidobacterium) show limited efficacy in obesity, indicating a need for new probiotics based on mechanistic insights into host–microbe interactions. A key finding is the identification of E. coli heat shock protein ClpB as a molecular mimic of the anorexigenic α-MSH. Prior work showed that oral gavage of lean mice with native, but not ClpB-deficient, E. coli reduced food intake and body weight, suggesting ClpB’s role in anorexigenic effects. Given the central role of melanocortin signaling (α-MSH) in energy balance, the hypothesis is that commensals producing ClpB with α-MSH-like epitopes could modulate appetite and weight. This study aims to validate Hafnia alvei HA4597, a food-grade commensal producing ClpB with α-MSH-like motif, as a candidate probiotic in genetic (ob/ob) and diet-induced (HFD) obesity models and to assess the relevance of enterobacterial ClpB gene abundance to human obesity via metagenomic analysis.

Prior literature establishes that gut microbiota composition is altered in obesity and can causally transfer obesity phenotypes to germ-free or antibiotic-treated animals. Diet, age, sex, geography, and antibiotics rapidly modulate microbiota composition. Traditional probiotics from Lactobacillus and Bifidobacterium have shown limited effectiveness for obesity management. Mechanistic insights into host–microbe signaling suggest novel targets: E. coli ClpB is an antigenic mimic of α-MSH, implicating bacterial factors in appetite regulation via the melanocortin system. Deficiency in melanocortin signaling leads to hyperphagia and obesity in rodents and humans, underscoring the relevance of α-MSH mimetics. These data motivate exploration of commensals expressing ClpB as next-generation probiotics for appetite and weight control.

Animal studies: Male B6.V-Lep ob/ob (n=75) and male C57BL/6 (n=70) mice (6 weeks old) were housed under controlled conditions with ad libitum access to standard chow or high-fat diet (HFD; 45% fat, 35% carbohydrate, 20% protein) for 19 weeks to induce obesity. Food intake (per cage) and individual body weight were recorded daily. For feeding behavior with E. coli strains, ob/ob mice were singly housed in BioDAQ cages. E. coli experiment: ob/ob mice were randomized into three groups (n=8 each): native E. coli K12, E. coli K12 ΔClpB mutant, or vehicle (LB medium). Bacteria or vehicle were given by daily intragastric gavage; effects on body weight and detailed meal patterns were assessed over ~20 days. H. alvei experiment: ob/ob and HFD-induced obese mice were randomized into H. alvei HA4597 or control (LB medium) groups (ob/ob n=12 per group; HFD n=22 per group). Randomization was by cage. Once daily at 18:00, mice received intragastric gavage (10 mL/kg) of H. alvei HA4597 cultured in LB or LB alone. Doses: ob/ob, 3×10^8 CFU/day; HFD, 4×10^7 CFU/day. Duration: ob/ob, 18 days; HFD, 46 days. Body composition was measured pre- and post-treatment (MiniSpec LF50). Terminal procedures included anesthesia, blood collection, and dissection of hypothalamus, epididymal fat, and colonic fecal content for storage at −80°C. Proteomics: H. alvei HA4597 proteins were analyzed for α-MSH-like epitopes by western blot with anti-α-MSH antibodies and by immunoprecipitation followed by LC-MS/MS to identify proteins bound by α-MSH antibodies; comparative analyses with E. coli K12 were performed. ClpB assays: Plasma ClpB levels were quantified using a validated sandwich immunoassay with anti-E. coli K12 ClpB antibodies and alkaline phosphatase detection. Fecal ClpB DNA: Total DNA from colonic contents was extracted and PCR-amplified using ClpB and 16S rRNA primers. Adipose lipolysis marker: Phosphorylated hormone-sensitive lipase (pHSL) in epididymal fat was assessed by western blot. Hypothalamic neuropeptides: RT-qPCR quantified AgRP, NPY, and POMC mRNA in hypothalami. In silico metagenomics: The human gut Integrated Gene Catalog (IGC; 9.88 million genes) from 1267 individuals was screened; analysis focused on 569 healthy individuals (384 European, 185 Chinese) with BMI data. Blastp (e-value ≤1E−5, ≥40% identity over ≥90% length) with E. coli K12 ClpB sequence (NP_417083.1) queried ClpB homologs; abundances from IGC tables were used to assess presence/abundance and correlations with BMI, including genus-level analyses for Enterobacter, Klebsiella, and Hafnia. Statistics: Normality by Kolmogorov–Smirnov; group comparisons via ANOVA with Tukey or Kruskal–Wallis with Dunn; pairwise via two-sided t-test or Mann–Whitney; longitudinal data via two-way RM-ANOVA with Bonferroni; significance at p<0.05; results as mean±SEM.

- In ob/ob mice, native E. coli K12 reduced body weight gain within 4 days and throughout the ~20-day treatment, whereas the ClpB-deficient mutant had no significant effect. Fat mass decreased in the native E. coli group versus control (t-test p<0.05). - Feeding behavior: native E. coli K12 reduced total daily food intake versus both control and ΔClpB groups (ANOVA p<0.0001), driven by fewer meals per day (Dunn’s p<0.001) without changing meal size; the ΔClpB strain showed a weaker anorexigenic effect (reduced meal number vs controls, t-test p<0.01). - Proteomics confirmed that H. alvei HA4597 produces ClpB containing an α-MSH-like epitope; ClpB was enriched ~10-fold more in H. alvei HA4597 than in E. coli K12 in α-MSH antibody immunoprecipitates (ANOVA p<0.0001). - H. alvei HA4597 gavage was well tolerated. In ob/ob mice, body weight gain was significantly lower from day 9, culminating in a 50.1% reduction at treatment end (day 18). Body composition showed reduced fat mass gain (−38.3%) and reduced lean mass loss (−126.8% relative decrease in loss) versus controls. Cumulative food intake decreased, evident from day 9, reaching −20.8% at the end (p<0.001). - In HFD-induced obese mice, H. alvei HA4597 reduced body weight gain during treatment, with the largest difference at day 33 (0.13±0.41 vs 2.53±0.43 g; −94.7% difference) and a 38.1% lower gain at the end (2.3±0.4 vs 3.8±0.41 g; t-test p<0.05). Fat mass gain decreased by 51.9%; the reduction in lean mass change (−72.4%) did not reach significance. No significant cumulative food intake difference was observed in HFD mice. - Adipose tissue from H. alvei-treated ob/ob mice showed elevated phosphorylated hormone-sensitive lipase (pHSL) levels (p<0.01), consistent with enhanced lipolysis. - Human metagenomic analysis showed lower richness of enterobacterial ClpB genes in obese vs non-obese individuals (p<0.0001) and negative correlations with BMI within Enterobacter, Klebsiella, and Hafnia genera, supporting translational relevance.

Findings support the hypothesis that bacterial ClpB, a molecular mimic of α-MSH, contributes to appetite and weight regulation via melanocortin-linked mechanisms. The absence of significant effects with ClpB-deficient E. coli, contrasted with robust anorexigenic and anti-adiposity effects of native E. coli and H. alvei HA4597, indicates ClpB’s involvement in the observed phenotypes. H. alvei HA4597’s ClpB carries an α-MSH-like epitope and is abundantly recovered by α-MSH antibodies, consistent with functional mimicry. In ob/ob mice, reductions in food intake and body weight gain alongside increased adipose pHSL suggest both central appetite modulation and peripheral enhancement of lipolysis. In HFD mice, body weight and fat mass reductions without significant changes in cumulative food intake suggest additional metabolic effects beyond hypophagia. The in silico human data—lower enterobacterial ClpB gene abundance in obese individuals and negative BMI correlations—provide ecological support linking ClpB-expressing commensals to leanness, reinforcing the rationale for probiotic strategies using H. alvei HA4597 for appetite and weight management.

Hafnia alvei HA4597, a commensal bacterium producing ClpB with an α-MSH-like epitope, reduces food intake (in hyperphagic ob/ob mice), body weight gain, and fat mass in both genetic and diet-induced mouse models of obesity, and increases adipose pHSL levels. Native but not ClpB-deficient E. coli similarly reduces weight gain and meal frequency, implicating ClpB in the effect. Metagenomic analyses show decreased enterobacterial ClpB gene richness in obese humans with negative correlations to BMI in relevant genera. Together, these findings provide a strong preclinical rationale for developing H. alvei HA4597 as a probiotic for appetite and body weight management in overweight and obesity. Future work should include controlled clinical trials to assess efficacy and safety in humans, mechanistic studies to delineate central versus peripheral pathways, and dose–response and formulation optimization.