**1.3 Mitochondria cross-talking with intestinal microbiota maintaining intestinal barrier integrity**

Maintenance of TJ integrity is an energy-dependent process, and it is not surprising that disruption of the barrier by toxins, pathogens, or noxious stimuli can be initiated by damaged mitochondria [39, 54, 55].

Mitochondria in animals, as well as chloroplasts in plant cells, are old- primitive bacteria that have lost the ability to live a "*free*" life by entering into a complex system of cooperation, the eukaryotic cell, and leaving some fundamental functions to the nucleus and other cellular organelles. The fact that mitochondria are ancestral bacteria makes them particularly sensitive to metabolic "motifs" produced by other bacteria. New research shows bidirectional communication exists between the gut microbiota and mitochondria [56, 57].

Certain insults, such as NSAID exposure, are known to disrupt the structure and function of the mitochondria, and at least transiently, increase gut permeability [58–60]. Additionally, it has been reported that some patients with Crohn's disease develop immune reactivity against components of their gut microbiome [61]. Consistent with these reports, Nazli et al. [44] demonstrated that treating a cell monolayer with dinitrophenol (an oxidative phosphorylation uncoupler) resulted in cellular internalization of a non-invasive strain of *Escherichia coli*. From this, the authors hypothesized that under metabolic stress resulting from mitochondrial dysfunction, the enteric epithelium loses its ability to distinguish between commensals and pathogens, and as a result, begins internalizing commensal organisms, which can lead to an exacerbated intestinal inflammatory response [44]. Studies do suggest that both mitochondrial dysfunction [62] and increased gut permeability [63] affect the overall competence of the intestinal epithelial barrier, but the stimuli that initiate either process are not known. Nonetheless, these studies reinforce the implication of epithelial mitochondrial dysfunction as a predisposing factor for an increase in gut epithelial permeability and a loss of gut barrier function, resulting in intestinal inflammation. The intestinal lumen and epithelium are continuously exposed to noxious stimuli, such as ingested nutrients, local microbes or infections, gastric acid production, and periods of ischemia/reperfusion that have the potential to stimulate the generation of oxygen and nitrogen radicals [64–66]. Additionally, the infiltration of leukocytes, monocytes, and neutrophils during inflammation can further enhance intestinal ROS production through both respiratory burst enzymes and prostaglandin and leukotriene metabolism [67]. Several studies have demonstrated increased ROS/ RNS levels within the intestinal epithelium of animals and patients with spontaneous and experimentally induced IBD [68–70].

Typical gut bacterial families found in healthy dogs and cats include Bacteroidaceae, Clostridiaceae, Prevotellaceae, Eubacteriaceae, Ruminococcaceae, Bifidobacteriaceae, Lactobacillaceae, Enterobacteriaceae, Saccharomycetaceae, and Methanobacteriaceae [71].

The gut microbiota are key to host metabolism as they aid in the digestion and absorption of food, neutralize drugs and carcinogens, synthesize choline [72], secondary bile acids [73, 74], folate, vitamin K2 and short chain fatty acids (SCFA). Additionally, the gut microbiota protects the host against pathogenic infection, stimulating and maturing the immune system [75] and epithelial cells [76] and regulating oxidative stress [77].

Bacterial metabolites, including short-chain fatty acids (SCFAs) and hydrogen sulfide (H2S), serve as messengers to enteric/colonic epithelial and immune cells, impacting their metabolism, epigenetic modifications, and gene expression. SCFAs are currently the most studied bacterial metabolites and are beneficial to intestinal and colon homeostasis. The three major SCFAs, acetate, propionate, and butyrate, are produced in the colon by bacterial fermentation of carbohydrates and are an important source of energy for colon epithelial cells. SCFAs are ligands for free fatty acid receptors 2 and 3, which modulate glucose metabolism and mitochondrial fatty acid β-oxidation (FAO). Additionally, SCFAs regulate PGC1α, a transcriptional coactivator that is a central inducer of mitochondrial biogenesis in cells [78]. These responses to SCFAs result, at the organelle level, in increased glucose uptake, FAO, oxidative phosphorylation, and mitochondrial biogenesis. In terms of intestinal homeostasis, these responses to SCFAs in colon epithelial cells facilitate the development of a tolerant mucosal immune system, promote epithelial barrier integrity, promote "physiologic hypoxia", and suppress colitis [7]. In addition, steady-state inflammasome machinery activation in the colon is mediated by SCFAs, which produces basal IL-18 levels, regulates the microbiome composition, and dampens overt inflammatory responses.

Butyrate, a by-product of the microbial fermentation of SCFAs, is one of the key molecules of mitochondria/gut microbiota cross-talk; butyrate may influence mitochondrial-endoplasmic reticulum (ER) contact signaling pathways. A body of recent evidence reveals that the microbiome impacts the host by communicating with its intracellular relatives, the mitochondria. This perspective mode of chemical communication between bacteria and mitochondria may help us understand complex and dynamic environment-microbiome-host interactions regarding their vital impacts on health and diseases. Communications between bacteria and mitochondrial are mediated by chemical signals from intestinal bacteria. In one case, a cluster of bacterial metabolites including betaine, methionine, and homocysteine initiate a signaling cascade that triggers the nuclear receptor 5A nuclear receptor and activates hedgehog signaling to regulate mitochondrial fission-fusion balance in intestinal cells [79]. This bacteria-mitochondria communication ultimately regulates fat storage homeostasis in the host [80]. Additionally, a slime polysaccharide named *colanic acid*, a major biofilm component of *E. coli*, secreted from intestinal bacteria, after entering the host cytoplasm via endocytosis, increases the fragmentation of intestinal mitochondria in a dependent fashion to the Dynamin Related protein-1 (Drp-1), a cytosolic guanosine triphosphate (GTPase) protein-key player of mitochondria fission, as well as enhances Mitochondrial Unfolded-Protein Response (UPRmt) in response to mitochondrial stress. These signaling effects of bacterial colanic acid on mitochondrial dynamics and UPRmt consequently lead to lifespan extension and protection against age-associated pathologies, like germline tumor progression and toxic amyloid-beta accumulation, in the host [81]. Besides SCFA, secondary bile acids produced by the gut microbiota also play an important role in regulating mitochondrial energy metabolism. Anaerobic bacteria of the genera *Bacteroides, Eubacterium*, and *Clostridium hiranonis* degrade 5–10% of the primary bile acids, forming secondary bile acids [71, 82, 83] Secondary bile acids interact with mitochondria by
