Food Science and Human Wellness

Volume 12, Issue 5, September 2023, Pages 1526-1537

Yang Liu , Qing Liu , Chengcheng Zhang , Jianxin Zhao , Hao Zhang , Wei Chen , Qixiao Zhai

Abstract
Akkermansia muciniphila, one of the most promising next-generation probiotics, was reported to exhibit beneficial modulatory effects on the gut barrier. However, the strain-specific and underlying regulatory mechanisms of this species on gut barrier function were not well studied. Therefore, this study evaluated the protective effect of A. muciniphila strains on the intestinal barrier and investigated the mode of action and material basis of this modulatory effect. We first confirmed the strain-specific effects of A. muciniphila on intestinal barrier regulation and found that this phenomenon may be explained by the different abilities of strains to affect tight junction protein expression in enterocytes. Comparative genomic analysis proved that the ability of A. muciniphila to regulate the intestinal barrier was exerted in part by the functional genes (such as COG0438, COG0463, and COG2244) related to the synthesis of cellular surface proteins. The role of these surface proteins in intestinal barrier regulation was further verified by strain-comparative experiments in animal and cell models and surface protein removal trials. This study confirmed the different effects of A. muciniphila strains on gut barrier modulation and provided molecular and genetic targets for the screening of A. muciniphila strains with superior protection against gut barrier dysfunction.

Keywords
Akkermansia muciniphilaIntestinal barrierStrain specificityComparative genomicsBacterial surface components

1. Introduction
The integrity of the gut barrier is crucial for the maintenance of the health of the host [1]. Pathogens and harmful substances pass through the lumen and translocate to the bloodstream by increasing intestinal permeability induced by gut barrier impairment [2]. Recent surveys have shown that intestinal barrier dysfunction is closely associated with several diseases, including enteropathy [3], neurodegeneration [4], and metabolic disorders [5]. Based on several gene-knockout mice studies, spontaneous intestinal inflammation is caused by intestinal barrier dysfunction [6], [7], [8]. Notably, a number of studies suggest that impaired gut barrier was associated with the progression of inflammatory bowel disease (IBD) [9], [10]. Despite the obscurity of the causal relationship, intestinal barrier dysfunction is considered to exacerbate the progression of IBD, and maintenance of gut barrier function has been reported to inhibit proinflammatory cascades and physiological oxidative stress, which will ultimately contribute to maintaining intestinal homeostasis and tolerance in host-intestinal microbiota interactions [11]. A recent review summarized that dietary factors such as phenolic compounds (including resveratrol, curcumin, and quercetin) could improve the integrity of the gut barrier of animals with intestinal inflammation, which may be explained by the reduction of the release of proinflammatory factors, the enhancement of the expression of tight proteins, and an increase in the antioxidant activity of enterocytes [12]. Moreover, the treatment of the gastrointestinal tract area with drugs, such as prucalopride [13] and ghrelin [14], significantly reduces inflammation in the colons of mice by improving gut barrier function. Therefore, it is necessary to find an effective treatment for the recovery of barrier integrity loss.

Akkermansia muciniphila has been widely recognized as one of the most promising next-generation probiotics. The abundance of this bacterium is inversely associated with a series of dysfunctions including appendicitis [15], hypertension [16], autism [17], and IBD [18]. Chelakkot et al. [19] demonstrated that supplementation of the extracellular vesicles of A. muciniphila markedly reduced weight and improved glucose tolerance in obese mice. Lower insulin resistance and adipose tissue inflammation were also observed in diet-induced diabetic mice after the treatment with A. muciniphila [20]. One of the key mechanisms for the probiotic function of A. muciniphila is based on its regulation of the host intestinal barrier, thereby inhibiting the entry of intestinal bacteria-borne endotoxin into the blood and relieving chronic inflammation in vivo [21]. Several animal studies have indicated that A. muciniphila has beneficial effects on the secretion of antimicrobial peptides and mucin in intestinal epithelial cells [22], [23]. Kang et al. [24] reported that oral administration of A. muciniphila significantly reversed the inflammatory infiltration of colon tissue and the alterations in the expression tight junctions in dextran sodium sulphate (DSS)-induced mice. Based on a clinical trial, treatment with pasteurized A. muciniphila enhanced the gut barrier function of volunteers, which showed a reduced endotoxin level in plasma and an alleviated overweight or insulin resistance [25].

However, several studies have shown strain-specific effects on intestinal barrier regulation of intestinal microorganisms. Escherichia coli Nissle 1917 supplementation is effective against inflammatory factors secreted by immune cells in multiple sclerosis mice and further repairs intestinal permeability disorders [26]. Conversely, adherent-invasive E. coli strains have been reported to disrupt epithelial mitochondrial networks and increase gut permeability [27]. Toxigenic Bacteroides fragilis impairs gut barrier integrity and induces colon tumorigenesis by activating nuclear factor-kappa B (NF-κB) signaling in colonic epithelial cells [28], while non-toxigenic B. fragilis administration reduces intestinal permeability and alleviates bacteria-driven chronic colitis and tumors [29]. Limited by the isolation and incubation methods necessary for A. muciniphila, the strain-specific effect of this bacterium on intestinal functions has not received much attention. Zhai et al. [30] demonstrated that compared to the A. muciniphila ATCC-BAA835 strain, oral administration of A. muciniphila 139 strain showed a more significant reduction in proinflammatory cytokines expression, including tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, thus reversing disease symptoms in colitic mice. Moreover, it is noteworthy that although A. muciniphila could accelerate intestinal stem cell-mediated epithelial development, the AK32 strain, not the ATCC-BAA835 strain, exhibited better probiotic effects [22]. Thus, we hypothesized that different A. muciniphila strains may possess strain-specific effects on the integrity of the gut barrier.

The aim of this study was, therefore, to evaluate the effects of six A. muciniphila strains on gut barrier dysfunction, with a focus on mouse and Caco-2 cell line models. Furthermore, we sought to gain insight into the possible material basis for the action of A. muciniphila through comparative genomic analysis and the isolation and verification of specific bacterial components.

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