Obesity is a complex condition with genetic and microbial factors playing a significant role. The gut microbiota, a vast community of microorganisms, influences various physiological processes, including appetite regulation. While traditional probiotics like *Lactobacillus* and *Bifidobacterium* have shown limited efficacy in obesity management, research has identified *E. coli* heat shock protein ClpB as a mimetic of the anorexigenic α-melanocyte stimulating hormone (α-MSH). This discovery led to the hypothesis that commensal bacteria producing ClpB, such as *Hafnia alvei*, could serve as anti-obesity probiotics. This study aimed to validate *H. alvei* HA4597, a food-grade strain, as a potential probiotic for appetite and body weight control in two mouse models of obesity: genetically obese (*ob/ob*) mice and diet-induced obese (HFD) mice. The study also aimed to investigate the role of ClpB in the observed anti-obesity effects and assess the relevance of enterobacterial ClpB gene abundance in human obesity using in silico analysis of the MetaHIT database.

The literature review extensively covered the multifactorial etiology of obesity, highlighting the role of both host genetics and the gut microbiome. Studies demonstrating the transfer of obesity phenotypes with the transfer of gut microbiota in experimental animals were cited, establishing the causal link between gut microbiota composition and obesity. The limited effectiveness of traditional probiotic strains in obesity treatment was also reviewed, motivating the search for novel probiotics with potentially superior mechanisms of action. The pivotal discovery of *E. coli* ClpB's α-MSH mimetic properties and its influence on food intake and body weight in lean mice laid the groundwork for this study, emphasizing the potential of ClpB-producing commensal bacteria as therapeutic agents.

The study used two mouse models of obesity: leptin-deficient *ob/ob* mice and high-fat diet (HFD)-induced obese mice. To assess the role of ClpB, *ob/ob* mice were treated with either native *E. coli* K12, ClpB-deficient *E. coli* K12, or a control. Separately, *ob/ob* and HFD mice received daily oral gavages of *H. alvei* HA4597 or a control (LB medium) for 18 and 46 days, respectively. Body weight, food intake, body composition (fat and lean mass), and plasma ClpB levels were measured. Proteomic analysis of *H. alvei* HA4597 was performed to confirm the presence of α-MSH-like epitopes in ClpB. The expression of hypothalamic neuropeptides (AgRP, NPY, POMC) and phosphorylated hormone-sensitive lipase (pHSL) levels in fat tissue were also assessed. In silico analysis of the MetaHIT database was conducted to examine the relationship between enterobacterial ClpB gene richness and BMI in a cohort of 569 healthy individuals.

Administration of native *E. coli* K12, but not the ClpB-deficient mutant, reduced body weight gain, fat mass, and daily food intake in *ob/ob* mice. *H. alvei* HA4597 treatment significantly reduced body weight gain and fat mass in both *ob/ob* and HFD-fed obese mice. In *ob/ob* mice, *H. alvei* also significantly decreased food intake. Proteomic analysis confirmed the presence of α-MSH-like epitopes in *H. alvei* HA4597 ClpB. *H. alvei*-treated *ob/ob* mice showed elevated fat tissue levels of phosphorylated hormone-sensitive lipase (pHSL), suggesting increased lipolysis. In silico analysis revealed significantly lower enterobacterial ClpB gene richness in obese compared to non-obese humans, with a negative correlation between ClpB gene abundance and BMI in *Enterobacter*, *Klebsiella*, and *Hafnia* genera.

The findings strongly support the hypothesis that *H. alvei* HA4597 can effectively reduce food intake, body weight gain, and fat mass in obese mice. The reduced food intake in *ob/ob* mice suggests a central effect on appetite regulation, possibly mediated by the α-MSH-mimetic activity of ClpB. The observed increase in pHSL levels suggests enhanced lipolysis as a contributing factor to fat mass reduction. The lower abundance of the enterobacterial ClpB gene in obese humans further strengthens the rationale for exploring *H. alvei* as a therapeutic probiotic. The study's findings open up new avenues for developing novel probiotics targeting obesity.

This preclinical study demonstrates that *H. alvei* HA4597 effectively reduces food intake, body weight, and fat mass gain in mouse models of obesity, potentially through the α-MSH-mimetic action of its ClpB protein. The correlation between reduced ClpB gene abundance and obesity in humans further supports its potential as a probiotic for obesity management. Future research should focus on human clinical trials to confirm these findings and elucidate the precise mechanisms of action.

The study utilized mouse models, which may not perfectly replicate the complexity of human obesity. The long-term effects and safety of *H. alvei* HA4597 administration require further investigation in larger-scale studies. While the in silico analysis suggests a correlation between ClpB gene abundance and obesity, further research is needed to determine the causal relationship.