Bacteria Penetrate the Inner Mucus Layer before Inflammation in the Dextran Sulfate Colitis Model

Background

Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described.

Principal Findings

When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells.

Conclusion

DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.     

Spatial Homogeneity of Bacterial Communities Associated with the Surface Mucus Layer of the Reef-Building Coral Acropora palmata

Coral surface mucus layer (SML) microbiota are critical components of the coral holobiont and play important roles in nutrient cycling and defense against pathogens. We sequenced 16S rRNA amplicons to examine the structure of the SML microbiome within and between colonies of the threatened Caribbean reef-building coral Acropora palmata in the Florida Keys. Samples were taken from three spatially distinct colony regions—uppermost (high irradiance), underside (low irradiance), and the colony base—representing microhabitats that vary in irradiance and water flow. Phylogenetic diversity (PD) values of coral SML bacteria communities were greater than surrounding seawater and lower than adjacent sediment. Bacterial diversity and community composition was consistent among the three microhabitats. Cyanobacteria, Bacteroidetes, Alphaproteobacteria, and Proteobacteria, respectively were the most abundant phyla represented in the samples. This is the first time spatial variability of the surface mucus layer of A. palmata has been studied. Homogeneity in the microbiome of A. palmata contrasts with SML heterogeneity found in other Caribbean corals. These findings suggest that, during non-stressful conditions, host regulation of SML microbiota may override diverse physiochemical influences induced by the topographical complexity of A. palmata. Documenting the spatial distribution of SML microbes is essential to understanding the functional roles these microorganisms play in coral health and adaptability to environmental perturbations.     

Altered Mucus Glycosylation in Core 1 O-Glycan-Deficient Mice Affects Microbiota Composition and Intestinal Architecture

A functional mucus layer is a key requirement for gastrointestinal health as it serves as a barrier against bacterial invasion and subsequent inflammation. Recent findings suggest that mucus composition may pose an important selection pressure on the gut microbiota and that altered mucus thickness or properties such as glycosylation lead to intestinal inflammation dependent on bacteria. Here we used TM-IEC C1galt ^-/- mice, which carry an inducible deficiency of core 1-derived O-glycans in intestinal epithelial cells, to investigate the effects of mucus glycosylation on susceptibility to intestinal inflammation, gut microbial ecology and host physiology. We found that TM-IEC C1galt ^-/- mice did not develop spontaneous colitis, but they were more susceptible to dextran sodium sulphate-induced colitis. Furthermore, loss of core 1-derived O-glycans induced inverse shifts in the abundance of the phyla Bacteroidetes and Firmicutes. We also found that mucus glycosylation impacts intestinal architecture as TM-IEC C1galt^-/- mice had an elongated gastrointestinal tract with deeper ileal crypts, a small increase in the number of proliferative epithelial cells and thicker circular muscle layers in both the ileum and colon. Alterations in the length of the gastrointestinal tract were partly dependent on the microbiota. Thus, the mucus layer plays a role in the regulation of gut microbiota composition, balancing intestinal inflammation, and affects gut architecture.     

Secretory Antibodies Do Not Affect the Composition of the Bacterial Microbiota in the Terminal Ileum of 10-Week-Old Mice

Terminal restriction fragment length polymorphism (T-RFLP) analysis was conducted on the 16S rRNA genes of the bacterial communities colonizing the epithelial surfaces of the terminal ilea of open conventionally housed mice in an institutional small-animal facility. Polymeric-immunoglobulin-receptor-deficient (pIgR^−/−) mice that were unable to secrete antibodies across mucosal surfaces were cohoused with normal and otherwise genetically identical wild-type (C57BL/6) mice for 4 weeks. If secretory antibodies played a role in modeling the gastrointestinal microbiota, C57BL/6 mice would have had a more distinct and uniform microbiota than their pIgR^−/− cage mates. The T-RFLP profiles of the bacterial communities were compared by using Sorensen's pairwise similarity coefficient, a newly developed weighted pairwise similarity coefficient, and on the basis of Shannon's and Simpson's diversity indices. No systematic differences were observed between the dominant components of the mucosa-associated bacterial communities of the terminal ileal walls of the two types of mice, indicating that secretory antibodies do not control the composition of this microbiota. Similar analyses of experiments conducted at two different times, between which the bacterial community composition of the mouse colony in the small-animal facility appeared to have changed, showed that differences could have been detected, had they existed.     

An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ～40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.     

Mucus Properties and Goblet Cell Quantification in Mouse,Rat and Human Ileal Peyer's Patches

Peyer''s patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.     

Importance and regulation of the colonic mucus barrier in a mouse model of colitis

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.     

Mice Overexpressing β-1,4-Galactosyltransferase I Are Resistant to TNF-Induced Inflammation and DSS-Induced Colitis

Glycosylation is an essential post-translational modification, which determines the function of proteins and important processes such as inflammation. β-1,4-galactosyltransferase I (βGalT1) is a key enzyme involved in the addition of galactose moieties to glycoproteins. Intestinal mucins are glycoproteins that protect the gut barrier against invading pathogens and determine the composition of the intestinal microbiota. Proper glycosylation of mucus is important in this regard. By using ubiquitously expressing βGalT1 transgenic mice, we found that this enzyme led to strong galactosylation of mucus proteins, isolated from the gut of mice. This galactosylation was associated with a drastic change in composition of gut microbiota, as TG mice had a significantly higher Firmicutes to Bacteroidetes ratio. TG mice were strongly protected against TNF-induced systemic inflammation and lethality. Moreover, βGalT1 transgenic mice were protected in a model of DSS-induced colitis, at the level of clinical score, loss of body weight, colon length and gut permeability. These studies put βGalT1 forward as an essential protective player in exacerbated intestinal inflammation. Optimal galactosylation of N-glycans of mucus proteins, determining the bacterial composition of the gut, is a likely mechanism of this function.     

Differences in the gastrointestinal microbiota of specific pathogen free mice: an often unknown variable in biomedical research

Large differences were found in the numbers of facultatively anaerobic Gram-negative bacteria in the gastrointestinal tract of mice from 3 major specific pathogen free (SPF) units in Australia. The species isolated also differed between mouse colonies. In one unit the presence of Enterobacter cloacae was found to dramatically influence the survival of mice following total body irradiation. This finding conforms with previous studies which have shown the influence of variation in gastrointestinal microbiota on the immune system and on susceptibility to infection. Given that the presence or absence of Enterobacteriaceae in the intestines of mice under investigation may influence experimental results, researchers using SPF rodents are encouraged to determine the baseline loading of these bacteria in their animals. Where results of immunological or irradiation studies from different colonies are likely to be compared, the enterobacterial status of the colony being used should be reported.     

Host adaptive immunity alters gut microbiota

It has long been recognized that the mammalian gut microbiota has a role in the development and activation of the host immune system. Much less is known on how host immunity regulates the gut microbiota. Here we investigated the role of adaptive immunity on the mouse distal gut microbial composition by sequencing 16 S rRNA genes from microbiota of immunodeficient Rag1^−/− mice, versus wild-type mice, under the same housing environment. To detect possible interactions among immunological status, age and variability from anatomical sites, we analyzed samples from the cecum, colon, colonic mucus and feces before and after weaning. High-throughput sequencing showed that Firmicutes, Bacteroidetes and Verrucomicrobia dominated mouse gut bacterial communities. Rag1⁻ mice had a distinct microbiota that was phylogenetically different from wild-type mice. In particular, the bacterium Akkermansia muciniphila was highly enriched in Rag1^−/− mice compared with the wild type. This enrichment was suppressed when Rag1^−/− mice received bone marrows from wild-type mice. The microbial community diversity increased with age, albeit the magnitude depended on Rag1 status. In addition, Rag1^−/− mice had a higher gain in microbiota richness and evenness with increase in age compared with wild-type mice, possibly due to the lack of pressure from the adaptive immune system. Our results suggest that adaptive immunity has a pervasive role in regulating gut microbiota''s composition and diversity.     

Physiological recycling of endogenous nitrate by oral bacteria regulates gastric mucus thickness

BackgroundInorganic nitrate from exogenous and endogenous sources is accumulated in saliva, reduced to nitrite by oral bacteria and further converted to nitric oxide (NO) and other bioactive nitrogen oxides in the acidic gastric lumen. To further explore the role of oral microbiota in this process we examined the gastric mucus layer in germ free (GF) and conventional mice given different doses of nitrate and nitrite.MethodsMice were given either nitrate (100 mg/kg/d) or nitrite (0.55–11 mg/kg/d) in the drinking water for 7 days, with the lowest nitrite dose resembling the levels provided by swallowing of fasting saliva. The gastric mucus layer was measured in vivo.ResultsGF animals were almost devoid of the firmly adherent mucus layer compared to conventional mice. Dietary nitrate increased the mucus thickness in conventional animals but had no effect in GF mice. In contrast, nitrite at all doses, restored the mucus thickness in GF mice to the same levels as in conventional animals. The nitrite-mediated increase in gastric mucus thickness was not inhibited by the soluble guanylyl cyclase inhibitor ODQ. Mice treated with antibiotics had significantly thinner mucus than controls. Additional studies on mucin gene expression demonstrated down regulation of Muc5ac and Muc6 in germ free mice after nitrite treatment.ConclusionOral bacteria remotely modulate gastric mucus generation via bioactivation of salivary nitrate. In the absence of a dietary nitrate intake, salivary nitrate originates mainly from NO synthase. Thus, oxidized NO from the endothelium and elsewhere is recycled to regulate gastric mucus homeostasis.     

AGR2, an Endoplasmic Reticulum Protein,Is Secreted into the Gastrointestinal Mucus

The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2^−/− mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.     

Secretory antibodies do not affect the composition of the bacterial microbiota in the terminal ileum of 10-week-old mice

Terminal restriction fragment length polymorphism (T-RFLP) analysis was conducted on the 16S rRNA genes of the bacterial communities colonizing the epithelial surfaces of the terminal ilea of open conventionally housed mice in an institutional small-animal facility. Polymeric-immunoglobulin-receptor-deficient (pIgR(-/-)) mice that were unable to secrete antibodies across mucosal surfaces were cohoused with normal and otherwise genetically identical wild-type (C57BL/6) mice for 4 weeks. If secretory antibodies played a role in modeling the gastrointestinal microbiota, C57BL/6 mice would have had a more distinct and uniform microbiota than their pIgR(-/-) cage mates. The T-RFLP profiles of the bacterial communities were compared by using Sorensen's pairwise similarity coefficient, a newly developed weighted pairwise similarity coefficient, and on the basis of Shannon's and Simpson's diversity indices. No systematic differences were observed between the dominant components of the mucosa-associated bacterial communities of the terminal ileal walls of the two types of mice, indicating that secretory antibodies do not control the composition of this microbiota. Similar analyses of experiments conducted at two different times, between which the bacterial community composition of the mouse colony in the small-animal facility appeared to have changed, showed that differences could have been detected, had they existed.     

Fast renewal of the distal colonic mucus layers by the surface goblet cells as measured by in vivo labeling of mucin glycoproteins

The enormous bacterial load and mechanical forces in colon create a special requirement for protection of the epithelium. In the distal colon, this problem is largely solved by separation of the bacteria from the epithelium by a firmly attached inner mucus layer. In addition, an outer mucus layer entraps bacteria to be cleared by distal transport. The mucus layers contain a network of Muc2 mucins as the main structural component. Here, the renewal rate of the inner protective mucus layer was studied as well as the production and secretion of Muc2 mucin in the distal colon. This was performed by intraperitoneal injection of N-azidoacetyl-galactosamine (GalNAz) that was in vivo incorporated during biosynthesis of O-glycosylated glycoproteins. The only gel-forming mucin produced in the colon is the Muc2 mucin and as it carries numerous O-glycans, the granulae of the goblet cells producing Muc2 mucin were intensely stained. The GalNAz-labeled glycoproteins were first observed in the Golgi apparatus of most cells. Goblet cells in the luminal surface epithelium had the fastest biosynthesis of Muc2 and secreted material already three hours after labeling. This secreted GalNAz-labeled Muc2 mucin formed the inner mucus layer. The goblet cells along the crypt epithelium accumulated labeled mucin vesicles for a longer period and secretion of labeled Muc2 mucin was first observed after 6 to 8 h. This study reveals a fast turnover (1 h) of the inner mucus layer in the distal colon mediated by goblet cells of the luminal surface epithelium.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeostasis of the colon to prevent spontaneous development of murine inflammatory bowel disease

Fine-tuning of host cell responses to commensal bacteria plays a crucial role in maintaining homeostasis of the gut. Here, we show that tumor necrosis factor receptor-associated factor (Traf)2(-/-) mice spontaneously developed severe colitis and succumbed within 3 weeks after birth. Histological analysis revealed that apoptosis of colonic epithelial cells was enhanced, and B cells diffusely infiltrated into the submucosal layer of the colon of Traf2(-/-) mice. Expression of proinflammatory cytokines, including Tnfa, Il17a, and Ifng, was up-regulated, whereas expression of antimicrobial peptides was down-regulated in the colon of Traf2(-/-) mice. Moreover, a number of IL-17-producing helper T cells were increased in the colonic lamina propria of Traf2(-/-) mice. These cellular alterations resulted in drastic changes in the colonic microbiota of Traf2(-/-) mice compared with Traf2(+/+) mice. Treatment of Traf2(-/-) mice with antibiotics ameliorated colitis along with down-regulation of proinflammatory cytokines and prolonged survival, suggesting that the altered colonic microbiota might contribute to exacerbation of colitis. Finally, deletion of Tnfr1, but not Il17a, dramatically ameliorated colitis in Traf2(-/-) mice by preventing apoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, and restoration of wild-type commensal bacteria. Together, TRAF2 plays a crucial role in controlling homeostasis of the colon.     

Changes in the microbial communities associated with Gorgonia ventalina during aspergillosis infection

The surface mucopolysaccharide layer (SML) secreted by corals is a rich environment where bacteria live and proliferate, with population levels often being several orders of magnitude higher than in the surrounding waters (at least for culturable microbes). Some studies have suggested that these communities play an important role in energy and nutrient flux in marine environments. We hypothesize that the microbial community structure of the SML also plays a role in protection against disease. This hypothesis is based on studies that have shown differences in the bacterial composition of the mucus of healthy and diseased corals. In this study we tested the differences in the microbial communities living in association with the SML of healthy and diseased Gorgonia ventalina by comparing their metabolic profiles using Biolog EcoPlates. Overall, metabolic profiles of the coral surface microbiota were significantly different to those in the water column based on stepwise canonical discriminant analyses (CDAs). Furthermore, differences between communities living in healthy and diseased corals were also found. Changes were observed between affected and unaffected areas of the same colony, although these changes were not as obvious as between individual healthy and diseased colonies. Results suggest that the microbial communities living in the SML of G. ventalina are affected by the presence of aspergillosis, even if the area is not in direct contact with the infection. This suggests the possibility of changes in the composition of the SML throughout the colony as a response to aspergillosis infection.     

Population Regulation in Magellanic Penguins: What Determines Changes in Colony Size?

Seabirds are often studied at individual colonies, but the confounding effects of emigration and mortality processes in open populations may lead to inappropriate conclusions on the mechanisms underlying population changes. Magellanic penguin (Spheniscus magellanicus) colonies of variable population sizes are distributed along the Argentine coastline. In recent decades, several population and distributional changes have occurred, with some colonies declining and others newly established or increasing. We integrated data of eight colonies scattered along ∼ 600 km in Northern Patagonia (from 41°26´S, 65°01´W to 45°11´S, 66°30´W, Rio Negro and Chubut provinces) and conducted analysis in terms of their growth rates, production of young and of the dependence of those vital rates on colony age, size, and location. We contrasted population trends estimated from abundance data with those derived from population modeling to understand if observed growth rates were attainable under closed population scenarios. Population trends were inversely related to colony size, suggesting a density dependent growth pattern. All colonies located in the north—which were established during the last decades—increased at high rates, with the smallest, recently established colonies growing at the fastest rate. In central-southern Chubut, where colonies are the oldest, the largest breeding aggregations declined, but smaller colonies remained relatively stable. Results provided strong evidence that dispersal played a major role in driving local trends. Breeding success was higher in northern colonies, likely mediated by favorable oceanographic conditions. However, mean foraging distance and body condition of chicks at fledging were influenced by colony size. Recruitment of penguins in the northern area may have been triggered by a combination of density dependence, likely exacerbated by less favorable oceanographic conditions in the southern sector. Our results reaffirm the idea that individual colony trends do not provide confident indicators of population health, highlighting the need to redefine the scale for the study of population changes.     

Initial gut microbiota structure affects sensitivity to DSS-induced colitis in a mouse model

The dextran sulfate sodium (DSS)-induced colitis model is a widely applied mouse model, but controversial results have been obtained from experiments using the same mouse strain under the same conditions. Because the gut microbiota play an important role in DSS-induced colitis, it is essential to evaluate the influence of the initial gut microbiota in this model. Here, we identified significant variations in the initial gut microbiota of different batches of mice and found that the initial intestinal microbiota had a profound influence on DSS-induced colitis. We performed three independent trials using the same C57BL/6J mouse model with DSS treatment and used high-throughput 16S rRNA gene sequencing to analyze the gut microbiota. We found that the structure and composition of the gut microbiota in mice with severe colitis, as compared with mice with milder colon damage, had unique features, such as an increase in Akkermansia bacteria and a decrease in Barnesiella spp. Moreover, these varied gut bacteria in the different trials also showed different responses to DSS treatment. Our work suggests that, in studies using mouse models, the gut microbiota must be considered when examining mechanisms of diseases, to ensure that comparable results are obtained.     

An adherent mucus layer attenuates the genotoxic effect of colibactin

The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase‐island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide‐nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA‐DSBs. Removal of the adherent mucus layer restored the occurrence of DNA‐DSBs.