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Three-dimensional structure of the gut microbial community is preserved by embedding in methacrylate resin Projections along the xz- and yz-planes show that the methacrylate-embedded section is of consistent thickness throughout, whereas the microbial community collapses onto the slide or is partially washed away when using cryosectioning, paraffin, and polyester embedding. From a stack of 35 images at half-micron intervals forming a z-series for each section, the projections of the stack along the xz-plane (top) and yz-plane (bottom) are shown. The positions of the xz- and yz-images in the xy-plane are shown by the magenta lines in Fig 3. Nominal thicknesses of the sections differ because of limitations of the cryotome we employed (minimum section thickness 12.5 μm) and the maximum recommended thickness for methacrylate sections (5 μm) while the thickness of the paraffin and polyester sections was kept similar to that of the cryosection (15 μm). Scale bar = 20 μm.
Source publication
Preservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embe...
Citations
... Mucin and gastric epithelial cell nuclei staining was accomplished via sectioning of OCT-preserved mouse colonic tissue. Select tissue slices were fixed and dehydrated 84 . Postfixation, all slides were incubated with blocking buffer (2% fetal bovine serum, 1% bovine serum albumin, 0.2% Triton X-100 and 0.05% Tween 20) for 1 h at room temperature and washed twice with 1× PBS. ...
Oral administration of biologic drugs is challenging because of the degradative activity of the upper gastrointestinal tract. Strategies that use engineered microbes to produce biologics in the lower gastrointestinal tract are limited by competition with resident commensal bacteria. Here we demonstrate the engineering of bacteriophage (phage) that infect resident commensals to express heterologous proteins released during cell lysis. Working with the virulent T4 phage, which targets resident, nonpathogenic Escherichia coli, we first identify T4-specific promoters with maximal protein expression and minimal impact on T4 phage titers. We engineer T4 phage to express a serine protease inhibitor of a pro-inflammatory enzyme with increased activity in ulcerative colitis and observe reduced enzyme activity in a mouse model of colitis. We also apply the approach to reduce weight gain and inflammation in mouse models of diet-induced obesity. This work highlights an application of virulent phages in the mammalian gut as engineerable vectors to release therapeutics from resident gut bacteria.
... Notably, PEG chains with MWs of 2 and 5 kDa contributed to the most efficient infiltration of EcN in the mucus at an observation duration of 24 h (Fig. 3a). For detailed visualization of the penetration of EPP, 1 × 10 5 c.f.u.s of bacteria were transferred to a glass-bottom dish pre-spread with a fluorescent mucus layer in which mucin was specifically marked with Alexa Fluor 488-labelled wheat germ agglutinin (AF488-WGA) 31 . LSCM images showed that both uncoated EcN and EP mainly stayed at the top of the mucus layer at 15 min after incubation (Fig. 3b). ...
The breakdown of the gut’s mucosal barrier that prevents the infiltration of microorganisms, inflammatory cytokines and toxins into bodily tissues can lead to inflammatory bowel disease and to metabolic and autoimmune diseases. Here we show that the intestinal mucosal barrier can be reinforced via the oral administration of commensal bacteria coated with poly(ethylene glycol) (PEG) to facilitate their penetration into mucus. In mice with intestinal homoeostatic imbalance, mucus-penetrating PEGylated bacteria preferentially localized in mucus at the lower gastrointestinal tract, inhibited the invasion of pathogenic bacteria, maintained homoeostasis of the gut microbiota, stimulated the secretion of mucus and the expression of tight junctions, and prevented the mice from developing colitis and diabetes. Orally delivered PEGylated bacteria may help prevent and treat gastrointestinal disorders.
... Utilizing SEM with a modified sample preparation technique, this research provided the first insight into the ultrastructure of bovine ileal and rectal mucus. While effective mucus preservation and visualization techniques have been explored extensively for histological and immunostaining evaluations [29][30][31][32], comparative studies for ultrastructural evaluations with SEM remain sparse [16]. The modified GA/PFA/AB-fixation technique explored in this study effectively preserved and visualized the fine net-like structure of mucus in our bovine model, which aligns with the structures seen in human and porcine intestinal mucus [16]. ...
Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.
... Additionally, fixation of tissue sections by conventional cross-linking agents causes complete mucous layer compression, limiting our under standing of its architecture and microbial composition (14). However, tissue fixation with Carnoy's fixative or paraformaldehyde has allowed for better preservation of mucous structure (15,16) and microscopic observation of bacterial species localized to this region of the mucosa. C. difficile has also been found to associate with the loose outer mucous layer of the cecum and colon in a murine model of infection (17). ...
Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others. Clostridioides difficile is an anaerobic bacterium, which colonizes the mammalian gastrointestinal (GI) tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. Although C. difficile toxicity stems from secreted toxins, colonization is a prerequisite for C. difficile disease. While C. difficile is known to associate with the mucous layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well understood. To understand the molecular mechanisms by which C. difficile interacts with mucins, we used ex vivo mucosal surfaces to test the ability of C. difficile to bind to mucins from different mammalian tissues. We found significant differences in C. difficile adhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed defects in adhesion by mutants deficient in flagella but not type IV pili. These results imply that interactions between host mucins and C. difficile flagella facilitate the initial host attachment of C. difficile to host cells and secreted mucus.
Importance
Clostridioides difficile is one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by which C. difficile colonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.
... Staining of the cellular mucus model: Cell samples were fixed using freshly prepared 4% (v/v) paraformaldehyde (Electron microscopy science, Hatfield, UK) and permeabilized with 0.2% Triton-X 100 solution (Carl Roth, Karlsruhe, Germany). Paraformaldehyde as a crosslinking fixative has been used, as it has been described before to successfully preserve mucus structure (Meziu et al., 2021;Hasegawa et al., 2017;Esther et al., 2019). Subsequently, samples were blocked with 5% (w/v) bovine serum albumin (Carl Roth, Karlsruhe, Germany). ...
Human respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.
... Improvement may also come in the form of better sample preparation. For intestinal samples, mucus-preserving fixative can be used to avoid detaching of the microbes and skewing of the spatial distribution and ideally to obtain a more comprehensive overview of the microbes (Hasegawa et al. 2017). However, the 10x ST platform used in this study requires freshly frozen samples for RNA yield and is thus not readily compatible with this protocol. ...
We developed an analysis pipeline that can extract microbial sequences from spatial transcriptomic (ST) data and assign taxonomic labels, generating a spatial microbial abundance matrix in addition to the default host expression matrix, enabling simultaneous analysis of host expression and microbial distribution. We called the pipeline spatial metatranscriptome (SMT) and applied it on both human and murine intestinal sections and validated the spatial microbial abundance information with alternative assays. Biological insights were gained from these novel data that showed host–microbe interaction at various spatial scales. Finally, we tested experimental modification that can increase microbial capture while preserving host spatial expression quality and, by use of positive controls, quantitatively showed the capture efficiency and recall of our methods. This proof-of-concept work shows the feasibility of SMT analysis and paves the way for further experimental optimization and application.
... Furthermore, we did note that ITLN1 expression, in both wild-type and Tg Vil1-Itln1 mice, was associated with increased A. muciniphila infiltration of the stratified mucus layer relative to Itln1 −/− mice (Fig. S4 F). To better assess microbial communities morphologically under SPF conditions, tissues were fixed in methacrylate, which preserves the three-dimensional structure of the intestinal microbiota Hasegawa et al., 2017). As expected, the inner mucus layer, defined as the wheat germ agglutinin (WGA) stratified layer between the bacterial biomass and the epithelial border, of Tg Vil1-Itln1 mice was thinner than that of wild-type littermates in methacrylate-fixed tissues (Fig. S4, G-H). ...
... Having validated methacrylate fixation, we conducted proximity analyses to quantify if the ITLN1-associated reduction in mucus thickness affected bacterial penetration of the inner mucus layer under SPF conditions. As previously reported, the bulk of the bacteria (Eubacteria in Fig. S4 K) displayed a repulsive relationship toward the mucus edge close to the epithelium Hasegawa et al., 2017;Johansson et al., 2010;Vaishnava et al., 2011). However, species-specific proximity measurements revealed that A. muciniphila localized significantly closer to the epithelial surface (<3 μm) in Tg Vil1-Itln1 mice ( Fig. S4 K) compared to their wild-type littermates. ...
... To quantify the spatial distribution of A. muciniphila regarding the mucus layer, we used methacrylate-fixed tissues to preserve better the three-dimensional structure of the intestinal microbial communities Hasegawa et al., 2017). ImageJ was used to binarize Eubacteria and A. muciniphila channels using auto local thresholding with the Bernsen method (Nichele et al., 2020) as well as size (0.1-1 µm 2 ) and circularity (0.6-1.0) filters. ...
Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host–microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell–mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.
... melanogaster (Akhtar et al., 2021), vaginal biofilms (Hardy et al., 2015), gastrointestinal tract (Hasegawa et al., 2017), and nematodes (Sinnathamby et al., 2018). In some cases, however, FISH has been unsuccessful when used in microbiome studies due to crossreactivity (Huys et al., 2008;Swidsinski et al., 2017) and requires confirmation of probe specificity prior to use but can be useful for visualizing localization of bacteria within an organism when successfully applied. ...
Parasitic nematodes, including the large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarid, Parascaris spp., was the first ascarid parasite to develop wide-spread anthelmintic drug resistance, with other species slowly following suit. There are no new classes of anthelmintics currently in development, and a solution to the ever-increasing prevalence of resistance is desperately needed. The microbiome has been shown to be an important factor in the fitness and health of many organisms and changes to microbiome composition have been associated with a plethora of diseases. The microbiome is also important to the health of parasitic nematodes, and the endosymbiotic bacterium Wolbachia, whose presence is essential for the viability of filarial nematodes, has been exploited for treatment of filariasis in humans by using both broad-range and, more recently, specific anti-Wolbachial antimicrobial treatments. Despite this success, parasite microbiomes are understudied. The overarching goal of this dissertation was to characterize the microbiome of Parascaris spp. by identifying a common core microbiota, by comparing microbiota diversity metrics for the whole worm at different life stages and in individual organs in male and female parasites, and by assessing the female gonad microbiota in greater detail.
... Additionally, fixation of tissue sections by conventional crosslinking agents causes complete mucus layer compression, limiting our understanding of its architecture and microbial composition (Johansson et al., 2011). However, tissue ation of mucus structure (Hasegawa et al., 2017;Swidsinski et al., 2005) and microscopic observation of bacterial species localized to this region of the mucosa. Recently, C. ...
Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacterium which causes gastrointestinal disease and is a leading cause of nosocomial infections. Although infection typically occurs following antibiotic therapy, in recent years there has been an increase in infections which are not preceded by antibiotic use. Additionally, community-associated infections and rates of disease recurrence have increased. While it is understood that a healthy gastrointestinal microbiota provides protection against infection, the molecular mechanisms which underly C. difficile's ability to colonize and persist in the gut are mostly unknown. Building on work from others that suggests C. difficile associates with the outer mucus layer during infection, we utilized an in vitro mucus layer model to probe attachment and colonization mechanisms. Using gene-interruption mutants of the major subunits of type IV pili and flagella, two extracellular appendages implicated in adhesion, we observed that the presence of flagella facilitates initial mucus attachment. Adhesion was variable across multiple strains of C. difficile and was also dependent on the source of mucin derivation. When mucin glycans were modified by mucin-degrading bacteria, C. difficile attachment decreased in our model. Collectively, our findings suggest that adherence to mucin likely influences C. difficile's ability to colonize and may offer novel strategies for future therapeutics once the underlying mechanisms are better understood. Advisor: Kurt H. Piepenbrink
... 10 Several studies that interrogated the gastrointestinal (GI) mucus layer report that traditional aqueous chloroform, and 10% glacial acetic acid is capable of preserving mucus integrity, allowing for the measurement of mucus thickness in human and animal guts. [11][12][13] While, others suggest instant freezing of tissues in liquid nitrogen or optimal cutting temperature (OCT) compound followed by cryosectioning and post-fixation in formalin, paraformaldehyde (PFA), or sucrose solution to achieve optimal mucus preservation. 14 Animals were euthanized with CO 2 according to BRMS guidelines. ...
Objectives
The epithelial associated mucus layer of vocal fold (VF) mucosa, plays an essential role in protecting and lubricating the tissue, as well as promoting normal voice quality. Serving as a habitat for laryngeal microbiota involved in the regulation of host immunity, VF mucus contributes to laryngeal health and disease. However, its unstable structure renders its' investigation challenging. We aim to establish a reproducible histological protocol to recover the natural appearance of the VF mucus layer for investigation.
Methods
Using a murine model, we compared the suitability of multiple fixation methods—methacarn, formalin, and cryopreservation followed by post‐fixation with formalin, paraformaldehyde (PFA), acetone, and two staining methods—Alcian Blue (pH 2.5)/Periodic Acid Schiff (AB/PAS) or PAS. Fixation and staining outcomes were evaluated based on the preservation of tissue morphology and mucus layer integrity. Mucin proteins, Muc1 and Muc4, were stained to validate the presence of mucus layer overlaying the VF mucosa.
Results
Methacarn fixation followed by PAS staining was capable of preserving and displaying the smooth and continuous mucus layer, ensuring the determination of mucus thickness and mucin staining.
Conclusions
Our study if the first to establish a histological protocol for the visualization of the in situ VF mucus layer whereby facilitating the study of VF mucus biology including VF surface hydration, ion/nutrients transports, biomechanical properties that maintains normal voice quality as well as VF pathophysiology and host‐microbe interactions in the larynx.
Level of Evidence
N/A.
