Bifidobacterium and the intestinal mucus layer

Bifidobacterium species are integral members of the human gut microbiota and these microbes have significant interactions with the intestinal mucus layer. This review delves into Bifidobacterium-mucus dynamics, shedding light on the multifaceted nature of this relationship. We cover conserved features of Bifidobacterium-mucus interactions, such as mucus adhesion and positive regulation of goblet cell and mucus production, as well as species and strain-specific attributes of mucus degradation. For each interface, we explore the molecular mechanisms underlying these interactions and their potential implications for human health. Notably, we emphasize the ability of Bifidobacterium species to positively influence the mucus layer, shedding light on its potential as a mucin-builder and a therapeutic agent for diseases associated with disrupted mucus barriers. By elucidating the complex interplay between Bifidobacterium and intestinal mucus, we aim to contribute to a deeper understanding of the gut microbiota-host interface and pave the way for novel therapeutic strategies.


Intestinal mucus
The intestine is continually exposed to a multitude of luminal antigens and bacterial components.To protect itself, the intestinal epithelium harbors specialized cells known as goblet cells, which synthesize and secrete mucus.The structure of intestinal mucus is intricately designed to form a protective barrier.The primary structural component of intestinal mucus is a gel-forming glycoprotein called MUC2.MUC2 is a large, heavily glycosylated protein that forms disulfide-bonded dimers.These dimers undergo further polymerization and crosslinking, resulting in the formation of a gel-like network that constitutes the mucus layer.
In addition to the gel-forming MUC2, intestinal mucus contains a diverse array of compounds that contribute to its composition and functionality.Mucus harbors Antimicrobial Peptides (AMPs): small cationic peptides that possess antimicrobial properties.AMPs in the mucus layer help to maintain the balance of microbial populations by inhibiting the growth of pathogenic bacteria and promoting the growth of beneficial commensal bacteria.Immunoglobulin A (IgA) antibodies are also abundant in the mucus layer of the gut.They are produced by specialized immune cells called plasma cells and secreted into the mucus, where they play a crucial role in immune defense by neutralizing pathogens, preventing their adherence to the intestinal epithelium, and promoting their clearance from the gut.In addition to MUC2, goblet cells also secrete trefoil factors, a family of small peptides that contribute to the maintenance of mucosal integrity and repair by promoting epithelial cell migration, enhancing wound healing, and providing protection against injury and inflammation.Other mucus-associated proteins, such as FCGBP, metalloenzyme CLCA1, ZG16, Lypd8, glycosaminoglycans, and chitinases, contribute to the structural organization, hydration, and stability of the mucus layer [1] .These compounds play various roles in shaping the mucus layer and modulating host-microbe interactions within the gut.
The structural organization of intestinal mucus is highly dynamic, exhibiting regional variations along the gastrointestinal tract.In the small intestine, the mucus layer is thinner and less firmly attached to the epithelium, allowing for efficient absorption of nutrients.In contrast, the mucus layer in the colon is thicker and firmly adheres to the epithelial surface, serving as a physical barrier that limits direct contact between luminal contents and the epithelium.In the colon, the mucus layer is stratified, consisting of two distinct regions: the inner mucus layer and the outer mucus layer.The inner mucus layer, also known as the firmly adherent mucus layer, is in direct contact with the intestinal epithelium.It is tightly packed with MUC2, forming a dense and organized matrix that provides a protective barrier against luminal contents.The outer mucus layer, also referred to as the loose mucus layer, is less compact and acts as a reservoir for commensal bacteria and other luminal components.This outer layer is more penetrable and allows for the establishment of symbiotic interactions between the gut microbiota and the host.
One feature of mucus that makes it amenable to microbe interactions is the structure of the mucin proteins.The MUC2 protein is extensively O-glycosylated with branched oligosaccharides [2][3][4][5][6][7] .O-glycans are attached at serine and threonine residues in the MUC2 protein and consist of core structures of α-and β-linked N-acetyl-glucosamine, N-acetyl-galactosamine, and galactose.The core structures are then elongated and generally modified by α-linked fucose, sialic acid, and sulfate residues [4] .Mucin glycoproteins serve as both an adhesion site and nutrient source for the resident gut microbes, providing an array of complex microbehost interactions.

Bifidobacteria and mucus
Among the bacteria found in the gut microbiota, Bifidobacterium species are known to reside within the intestinal mucus layer [8][9][10][11][12][13][14] and exert multiple beneficial effects on the host [15][16][17][18][19][20] .Bifidobacteria are Grampositive anaerobic bacteria from the phylum Actinobacteria that can have a rod or a distinctive bifid (i.e., Y) shape.There are currently 55 recognized species and subspecies of Bifidobacterium [21][22][23]  Bifidobacteria are predominant in the healthy breast-fed infant gut due to the presence of human milk oligosaccharides (HMOs), which these bacteria are adept at utilizing [24][25][26][27] .Studies have suggested that Bifidobacterium species make up ~80% of a breast-fed infant gut microbiota [28][29][30][31][32] .The benefits of Bifidobacterium strains are especially pronounced in early life, encompassing epithelial maturation, immune cell activation, and gut-brain-axis crosstalk [33][34][35][36][37][38][39] .Upon the introduction of solid food and weaning, the level of intestinal bifidobacteria continually decreases until adulthood, at which point bifidobacteria are maintained at a relative abundance of about ~10% throughout adult life [31,[40][41][42] .In the elderly, the level of bifidobacteria further diminishes to about 0%-5% relative abundance [42] .This reduction in bifidobacteria levels in the elderly has been linked to age-related alterations in lifestyle and environment.Interestingly, this decline in Bifidobacterium abundance coincides with a simultaneous decrease in the thickness of intestinal mucus and an increase in its permeability [43][44][45] .It remains uncertain whether there is a direct link between decreased Bifidobacterium and decreased mucus, but this interesting observation suggests a relationship.Independent of age, Bifidobacterium species can be found in both the small intestine and colon, although they exhibit a higher abundance in the colon.Several Bifidobacterium species have been observed to interact with intestinal mucus, colonize the mucus layer, consume mucus glycans, and exert strain-specific modulatory effects on the mucus layer.This review covers the existing literature for the following Bifidobacterium-mucus interactions: (1) mucus adhesion; (2) mucin glycan degradation; (3) positive modulation of goblet cell cells; (4) goblet cell retention during inflammation; and (5) suppression of proinflammatory cytokines and production of anti-inflammatory IL-10.
Interestingly, Bifidobacterium animalis subsp.lactis and unclassified Bifidobacterium species were shown to adhere well to mucus isolated from the feces of newborns, 2-month-old infants, 6-month-old infants, and adults (25 to 52 years), but had substantially lower adhesion to mucus derived from the feces of elderly individuals (74 to 93 years) [41] .It was also found that B. animalis subsp.lactis had diminished adhesion to mucus isolated during episodes of diarrhea [50] .These findings point to the integrity of mucus for adhesion.
The binding of Bifidobacterium to intestinal mucus is regulated by diverse adhesins [Figure 1].Bifidobacterium species employ pili, surface adhesion proteins, moonlighting proteins, and other surfaceanchored proteins to adhere to intestinal mucus [Table 2] [73][74][75] .For example, B. bifidum has several known   mucin-binding partners.B. bifidum possesses two sortase-dependent pili that promote bacterial coaggregation and bind to mucus-producing Caco-2 cells [76] .Another study found that B. bifidum produces an extracellular sialidase that mediates adhesion to mucus via a conserved sialidase domain peptide that interacts with mucin carbohydrates [77] .Similar to B. bifidum, B. longum also expresses multiple mucusbinding proteins.B. bifidum and B. longum both have been shown to express extracellular transaldolases that function as an adhesin that is capable of binding mucin [78,79] .A recent study found that B. longum harbors 21 putative adhesion proteins [75] .Using an overexpression system in a heterologous host, it was found that FimM exhibited significant adhesion to mucus-producing LS174T goblet cells, and it was further found that mucin was one of the major adhesion receptors for the FimM protein [75] .Homologs of FimM were also identified in B. bifidum, B. gallinarum, and 23 other B. longum strains by sequence similarity analysis.Another study found that B. longum harbors a protein with high homology to type 2 glycoproteinbinding fimbriae that may mediate mucus adhesion [80] .B. longum additionally produces the moonlighting proteins EF-Tu and enolase, which indirectly promote adhesion to mucus-producing Caco-2 cells through interactions with host plasminogen [81] .Likewise, enolase plays the role of an adhesion factor in B. lactis Bl07 [82] , and GroEL is another moonlighting protein that has been indicated as an adhesion factor for B. animalis subsp.lactis [83] .
As another example of the various adhesins employed by Bifidobacterium species, B. longum was found to possess a 26-amino-acid peptide called Blap-1 that mediates adhesion to HT-29 cells.Interestingly, genomic analysis revealed that Blap-1 was an identical match to a site in a large extracellular transmembrane protein encoded by the BL0155 open reading frame of B. longum NCC2705 [84] .Additionally, B. longum possesses an endo-α-N-acetylgalactosaminidase that contains binding sites specific to the protein core of mucin glycoproteins [85] .Furthermore, the genome of B. longum subsp.infantis encodes several family 1 of solute binding proteins (F1SBPs), and these proteins were shown to bind and transport mucin oligosaccharides [86,87] .In addition to B. bifidum and B. longum, B. breve has type IVb pilus-type proteins that facilitate colonization in the host gut [82,83,88] .Interestingly, it has also been shown that B. longum produces extracellular vesicles that export mucin-binding cytoplasmic proteins, and these proteins promote the adhesion of B. longum to mucus [89] .It has also been recently shown that the polyamine Spermidine significantly increased the adhesion of B. bifidum Bb12 to mucus isolated from healthy infants [90] , suggesting that secreted factors could also influence the adherence of Bifidobacterium to mucus.Together, these studies indicate that although multiple Bifidobacterium species can bind to mucus, the mechanisms of adhesion appear to be diverse, even among strains of the same species.
The structure of mucus likely dictates the consequences of mucus binding for Bifidobacterium species.In the small intestine, the mucus is loose and not attached to the epithelium.As a result, mucus adhesion likely does not promote persistent colonization of the small intestine.In contrast, in the colon, the mucus is highly organized and adhesion to colonic mucus most likely allows Bifidobacterium species to persist and colonize the colon.The adhesion of Bifidobacterium to colonic mucus is also thought to increase the transit time of the bacteria in the gut, thereby maximizing its beneficial properties [91,92] .It has also been shown that colonization of the mucus layer by Bifidobacterium species positively regulates goblet cells.These interactions are all viewed as beneficial for the host.As a result of these positive attributes, the ability to adhere to human intestinal mucus is a commonly employed criterion for the selection of probiotic organisms [75,93,94] .
The binding of Bifidobacterium to intestinal mucus extends beyond a mere physical attachment; it serves as a gateway for host-microbe crosstalk.By positioning themselves within the mucus, Bifidobacterium strains gain proximity to host cells, enabling the effective delivery of health-promoting molecules, metabolites, and signaling compounds [18,95,96] .Furthermore, the presence of Bifidobacterium within the mucus layer influences the spatial organization and composition of the gut microbiota, thereby impacting the overall microbial ecosystem.In several studies, the ability to bind to the mucus layer allowed Bifidobacterium species to create a niche and exclude pathogens [54,56,57,62,64,92,97] .One study found that a probiotic containing Bifidobacterium could inhibit pathogenic colonization of Escherichia coli, and this protective effect was dependent on MUC2 expression by Caco-2 cells [98] .This data suggests that mucus adhesion is critical for excluding pathogens.In addition to excluding pathogens, Bifidobacterium species likely have synergistic interactions with other commensal microbes in the mucus layer.Bifidobacterium has been shown to cross-fed commensal Eubacterium rectale [99] , E. hallii [100,101] , and Faecalibacterium prausnitzii [102] .In each of these scenarios, Bifidobacterium-commensal co-cultures generated elevated levels of butyrate, a beneficial short-chain fatty acid, compared to the mono-cultures.The literature clearly indicates that Bifidobacterium species readily bind to mucus, and this mucus adhesion likely sets the stage for a range of beneficial effects on both the host and the gut microbial community.
Mucin degradation is considered to be a normal process of intestinal mucus turn-over [116] and begins within the first few months of life [117,118] .Infants are commonly colonized with mucin-degrading B. bifidum, B. longum subsp.infantis, and B. breve [29,30,118,119] , as well as Akkermansia muciniphila and Bacteroides species [116] .Interestingly, breast-fed babies that are dominated by Bifidobacterium species exhibit a delay in the mucin degradation profile as compared with babies fed with formula milk [118] .Consistent with this notion, Karav et al. found that supplementation of B. longum subsp.infantis EVC001 to healthy breast-fed infants significantly reduced the proportion of free colonic mucin-derived O-glycans in the total glycan pool to 1.87% compared to 37.68% in the control infants who did not receive supplemented B. longum [120] .The level of freed mucin-derived O-glycans was negatively correlated with populations of Bifidobacteriaceae, indicating that mucus degradation was not occurring at the same level in B. longum supplemented infants [120] .Along the same lines, genes involved in mucus-degrading pathways, particularly in carbohydrate metabolism, in Bifidobacterium species were found to be expressed to a greater degree in formula-fed infants than in breast-fed infants [121] .It has been speculated that HMOs, which are similar to mucus in some of the glycan structures [121,122] , or other mucin-like glycoproteins present in breast milk, may compete with intestinal mucus as a substrate [118] .
In addition to being found in infants, mucus-degrading Bifidobacterium species are present in adults and have been linked to the suppression of detrimental mucus degradation.One example of excessive mucus degradation that may be prevented by Bifidobacteria is in the context of a Westernized diet, a diet characterized by low fiber but high fat and sugar.It has been demonstrated in mice harboring defined microbial communities that consuming a Westernized diet leads to an expansion of mucin-degrading bacteria such as Akkermansia muciniphila and Bacteroides caccae, and this shift enables the bacterial community to target the mucus layer for digestion in lieu of dietary fibers [123] .In a model with complex native gut microbiota, mice fed a Westernized diet similarly exhibited an expansion of Akkermansia and a corresponding decrease in Bifidobacterium species [124] and increased susceptibility to pathogens and inflammation.In this setting, the addition of B. longum NCC 2705 or the prebiotic inulin resulted in elevated levels of endogenous Bifidobacterium species, reduced mucus degradation, and restored the mucus barrier.In a similar vein, B. bifidum G9-1 was shown to protect against mucus degradation by A. muciniphila following small intestine injury caused by a proton pump inhibitor and aspirin [125] .Another study found that the administration of B. pseudolongum Patronus increased mucosal thickness in rats and decreased the levels of A. muciniphila [126] .These data suggest that mucus degradation by Bifidobacterium species is not detrimental to the host and that Bifidobacterium species keep mucus degradation in check.

Modulation of mucus by Bifidobacteria in homeostasis
Although some bifidobacteria have mucolytic properties, they generally have an overall positive net effect in regulating intestinal mucus.Several studies have found that Bifidobacterium species elevate mucus levels in vitro and in vivo [Table 3 and Figure 2].In vitro, B. infantis, B. breve, B. longum and a probiotic cocktail containing these microbes and others (VSL#3) was found to stimulate mucus secretion in human mucusproducing LS174T cells [127] .The probiotic cocktail was also found to increase MUC2 expression and secretion in rat colonic loops [127] .In another study, B. dentium was reported to increase MUC2 in human mucus-producing T84 cells [18] .Short-chain fatty acids (SCFA) have been demonstrated to increase MUC2 expression [128] , and Bifidobacterium species are known to produce high levels of SCFA acetate.The application of acetate was likewise able to increase MUC2 gene and protein levels in T84 cells [18] .In vivo, B. dentium was found to colonize germ-free mice, elevate intestinal acetate levels, and increase MUC2 at the gene and protein levels [18] .An elevated number of goblet cells and goblet cell-specific genes were observed in B. dentium mono-associated mice, as well as increased mucin glycosylation [18] .In this model, it was speculated that B. dentium-generated gamma-aminobutyric acid (GABA) was able to activate autophagy and calcium signaling to stimulate the release of mucus from goblet cells and bolster the mucus barrier [18] .In addition to B. dentium, B. bifidum and B. longum colonize germ-free mice and increase intestinal mucin glycoproteins [129,130] .These studies using mono-associated gnotobiotic animals provide very powerful evidence that B. dentium, B. bifidum and B. longum can modulate goblet cell function and increase mucus production.In mice with complex gut microbiota, B. breve supplementation led to 3,996 upregulated and 465 downregulated genes in supplemented neonatal mice relative to the untreated group [35] .Upregulated genes in the neonatal mice encoded multiple mucus layer-associated proteins such as MUC2.These data suggest that B. breve in early life modulates goblet cells.In adult mice, administration of a probiotic cocktail  containing B. breve also increased the number of goblet cells per crypt and increased the production of mucus compared with controls [131] .Collectively, these data indicate that Bifidobacterium strains influence goblet cell function and mucus production.

Modulation of mucus by Bifidobacteria in inflammation and infectious diseases
There is a wide array of data that demonstrate the substantial benefits of Bifidobacterium in the context of disease.Colitis is one of the most frequently investigated intestinal diseases, and a variety of Bifidobacterium species have exhibited the ability to alleviate major complications of colitis.In general, Bifidobacterium species have been shown to (1) limit inflammation-associated goblet cell and mucus depletion and MUC2 and (2) reduce pro-inflammatory cytokines [Figure 3, Tables 4 and 5].Colitis-inducing compounds are known to activate ER stress [132][133][134][135] , and ER stress has been linked to intestinal inflammation in multiple animal models [136][137][138][139][140] .Goblet cells are particularly sensitive to ER stress since producing and folding MUC2 is a complex process [141,142] .It has been speculated that modulation of goblet cell ER stress by Bifidobacterium species may represent a key pathway by which bifidobacteria promote intestinal health.In mucus-producing Caco-2 cells, the application of live B. breve YIT 12272 and B. adolescentis YIT 4011T alleviated tunicamycin-induced ER stress [143] .In another study using mucusproducing T84 cells, it was shown that B. dentium ATCC 27678-secreted metabolites could also suppress tunicamycin-or thapsigarin-induced ER stress [96] .Analysis of the B. dentium metabolites revealed that this strain generated substantial levels of γ-glutamylcysteine, a compound that can be converted into the powerful antioxidant glutathione and suppress oxidative and ER stress [132,134,[144][145][146][147][148] .B. dentium metabolites harboring γ-glutamylcysteine and application of commmerically available γ-glutamylcysteine both elevated glutathione, suppressed inflammatory NF-κB activation, reduced IL-8 secretion, and attenuated the induction of the unfolded protein response (UPR) genes GRP78, CHOP, and sXBP1 in T84 cells and TNBStreated mice [96] .These data suggest that Bifidobacterium species can reduce goblet cell ER stress.When goblet cells undergo ER stress, they are unable to adequately synthesize and secrete MUC2, leading to a reduction in goblet cell number and a thinning of the intestinal mucus layer.Several animal models have shown that goblet cell ER stress or loss of mucus leads to intestinal inflammation (Winnie, MUC2 -/-, AGR2 -/-, glycan deficiency, etc.) [149][150][151][152][153][154] .These animal model phenotypes closely resemble the intestinal issues observed in inflammatory bowel disease (IBD) patients, particularly in ulcerative colitis patients [139,[155][156][157][158] .
Several studies have found that Bifidobacterium species can limit the reduction of goblet cells and improve the mucus barrier in the setting of chemically induced intestinal inflammation [  [167][168][169][170][171][172][173] .A probiotic mixture containing B. infantis was also shown to enhance the mucus barrier in DSS-treated mice [174] .B. infantis and B. breve were likewise found to limit the reduction of goblet cells in DSS models [175][176][177] , and B. lactis was found to improve goblet cell counts in a zebrafish model of intestinal inflammation [178] .Along the same lines, B. dentium was also shown to increase MUC2, limit goblet cell reduction, and improve the mucus layer in a TNBS-induced model of colitis [96] .B. infantis and B. longum were also found to improve goblet cell numbers in TNBS-induced colitis [177,179] , while B. animalis subsp.lactis restored goblet cell populations in dinitrobenzene sulfonicacid (DNBS)-challenged mice [180] .These studies indicate that Bifidobacterium species can reduce goblet cell loss and mucus depletion in the setting of TNBS and DNBS-induced colitis.
Bifidobacterium species also have positive roles in modulating mucus in other inflammatory models.For example, B. bifidum, B. infantis, and B. lactis increased MUC2 in the small intestine during LPS-induced injury [181] .In a rat model of necrotizing enterocolitis (NEC), B. bifidum was shown to increase mucin and TFF3 expression and decrease the disease severity [182] .B. longum EVC001 and B. infantis BB-02 also decreased NEC occurrence in animals [183,184] .Even more promising is a double-blind, randomized, controlled study of very-low-birth-weight preterm infants, in which a combination of B. breve strain Yakult and L. casei strain Shirota completely prevented the occurrence of NEC in the intervention group, whereas 3.5% of the cases developed NEC in control without probiotics [185] .The mechanism by which Bifidobacterium confers its benefits in NEC is not fully understood but may be similar to colitis involving the mucus layer, intestinal permeability, and inflammation.
Rotavirus gastroenteritis is another disease where Bifidobacterium species have been shown to beneficially modulate the mucus layer.B. bifidum G9-1 was shown to increase MUC2, normalize mucin-positive goblet cells in the small intestine, and reduce the incidence, diarrheal scores, and intestinal damage in the supplemented group with rotavirus compared to the control group with rotavirus alone [186] .B. infantis PCM has also been shown to maintain goblet cells and reduce epithelial damage in the small intestine of mice infected with the pathogen Cronobacter sakazakii [187] .These data demonstrate that goblet cells and mucin production are also beneficially influenced by bifidobacteria in the small and large intestines in multiple inflammatory models.
Pro-inflammatory cytokines have been shown to negatively regulate goblet cells, while anti-inflammatory compounds such as IL-10 are known to alleviate ER stress and enhance goblet cell function.Another pathway by which Bifidobacterium species positively modulate goblet cells is through the modulation of intestinal cytokines [  [171,177,179] .These strains additionally led to rises in the anti-inflammatory cytokine IL-10 [171,177,179] .Similarly, B. dentium reduced serum IFN-γ, IL-1α, IL-1β, IL-12, and TNF-α with a concomitant increase in IL-10 in comparison to the TNBS control mice [96] .Another study using DSS revealed that B. breve and B. longum lowered both systemic and colonic levels of TNF-α, IL-1β, and IL-6 [172] .These studies suggest that in addition to directly modulating goblet cells through metabolites and suppression of ER stress, Bifidobacterium strains may be indirectly modulating goblet cell function via immune regulation.

OVERALL EFFECTS OF BIFIDOBACTERIUM-MUCUS INTERACTIONS ON THE HOST
The literature suggests that the intestinal mucus layer plays a crucial role in the interaction of Bifidobacterium species with the host.It appears that the majority of Bifidobacterium species bind to intestinal mucus and establish a unique niche that affords them an advantageous position for their beneficial activities.Within the mucus layer, some Bifidobacterium species can degrade mucus, while others must rely on other nutrient sources.In the mucus layer, Bifidobacterium species likely perform the following functions: (1) exclude pathogens; (2) cross-fed commensal bacteria; (3) limit excessive mucus degradation; (4) secrete compounds such as acetate, which elevate MUC2 expression and increase mucus production; (5) reduce goblet cell ER stress; (6) limit inflammation-and infection-driven goblet cell loss; (7)  suppress pro-inflammatory cytokines; and (8) increase anti-inflammatory pro-goblet cell IL-10.
The literature points to the capacity for Bifidobacterium species to beneficially modulate goblet cell number and function, thereby regulating the mucus layer and intestinal barrier function.This modulation of the goblet cells by Bifidobacterium is likely even more important during the setting of infection and inflammation.Through these interactions, Bifidobacterium species facilitate a dynamic interplay that contributes to gut homeostasis and overall host health.

LIMITATIONS AND GAPS IN THE FIELD
While these findings are compelling, there are still several gaps in knowledge.First, it is unclear which Bifidobacterium strains are the most effective at positively regulating goblet cell function.Very few studies have performed head-to-head comparisons of different Bifidobacterium strains and studies vary in terms of mouse strain (C57B6/J, BALBc, Swiss Webster, etc.), colonization status (mono-association, gnobotioic with defined communities, conventional, etc.), and challenge (TNBS, DSS, DNBS, LPS, infection etc.).These variables make it difficult to tease out the nuances between strains and effects.Second, the metabolites that drive goblet cell-specific attributes of Bifidobacterium are not well characterized.It is well documented that Bifidobacterium species can generate acetate and this SCFA can elevate MUC2 levels, but it is likely that other metabolites also stimulate MUC2.In addition to modulating MUC2 levels, Bifidobacterium species can influence goblet cells in other ways, such as suppressing ER stress, promoting autophagy, and stimulating mucus expulsion.Likewise, it is not clear how bifidobacteria members regulate IL-10 production, which could indirectly affect goblet cell homeostasis.These pathways need to be explored with multiple Bifidobacterium strains.
The advent of intestinal organoids is a promising new technology to address Bifidobacterium-goblet cell interactions.This model maintains segment specificity, is not immortalized, and is not cancer-derived.Importantly, intestinal organoids harbor MUC2-positive goblet cells and have been previously used to examine bacterial-host interactions, including Bifidobacterium [95,[188][189][190] .We anticipate that many future studies will employ this model to define the mechanisms by which Bifidobacterium species regulate goblet cells and interact with intestinal mucus.
Although there are still large gaps in the field, the wealth of literature allows us to make some key observations on conserved bifidobacteria functions, such as mucus binding, suppression of inflammationdriven goblet cell depletion, and elevation of MUC2.Understanding the interaction between Bifidobacterium and the intestinal mucus layer is imperative for unraveling the mechanisms underlying their beneficial effects.With this knowledge, there is immense potential for developing targeted therapeutic interventions.

Figure 1 .
Figure 1.Schematic outlining examples of the various mechanisms by which Bifidobacterium adhere to mucus.(A) Extracellular vesicles released by Bifidobacterium can bind to mucus, and in turn, this binding can inhibit pathogen colonization; (B) Bifidobacterium possess a wide array of pili and fimbriae, including FimM and its homologs, type 2 fimbriae, and type IVb pili, which bind to mucus; (C) Other proteins such as F1SBPs (family-1 binding proteins), BL0155 (a type of ABC transport transmembrane protein), GroEL (a heat shock protein), and EF-Tu (Elongation Factor Tu) are involved in mucus binding; (D) Endo-α-N-acetylgalactosaminidase, transaldolase, sialidase, and enolase are enzymes that facilitate mucus adhesion.

Figure 2 .
Figure 2. Representative diagram of Bifidobacterium-goblet cell interactions.(A) Bifidobacterium species can generate acetate, which can elevate MUC2 expression and protein; (B) Bifidobacterium species can also generate varying levels of GABA, which can activate autophagy-driven expulsion of mucus.Through these mechanisms, Bifidobacteria are speculated to positively regulate goblet cells.GABA: B. dentium-generated gamma-aminobutyric acid.

Figure 3 .
Figure 3. Diagram outlining the major beneficial effects, especially in improving goblet cell function and in reducing inflammation, per Bifidobacterium species in disease models.GABA: B. dentium-generated gamma-aminobutyric acid; NEC: necrotizing enterocolitis; SCFAs: short-chain fatty acids.