Microbial adhesins to gastrointestinal mucus

The gastrointestinal tract (GIT) is lined by a layer of mucus formed by mucin glycoproteins. This layer constitutes a physical and chemical barrier between the intestinal contents and the underlying epithelia. In addition to this protective role, mucins harbor glycan-rich domains that provide preferential binding sites for pathogens and commensal bacteria. Although mucus-microbial interactions in the GIT play a crucial role in determining the outcome of relationships of both commensal and pathogens with the host, the adhesins and ligands involved in the interaction are poorly delineated. This review focuses on the current knowledge of microbial adhesins to gastrointestinal mucus and mucus components.     

The intestinal epithelium as guardian of gut barrier integrity

A single layer of epithelial cells separates the intestinal lumen from the underlying sterile tissue. It is exposed to a multitude of nutrients and a large number of commensal bacteria. Although the presence of commensal bacteria significantly contributes to nutrient digestion, vitamin synthesis and tissue maturation, their high number represents a permanent challenge to the integrity of the epithelial surface keeping the local immune system constantly on alert. In addition, the intestinal mucosa is challenged by a variety of enteropathogenic microorganisms. In both circumstances, the epithelium actively contributes to maintaining host–microbial homeostasis and antimicrobial host defence. It deploys a variety of mechanisms to restrict the presence of commensal bacteria to the intestinal lumen and to prevent translocation of commensal and pathogenic microorganisms to the underlying tissue. Enteropathogenic microorganisms in turn have learnt to evade the host's immune system and circumvent the antimicrobial host response. In the present article, we review recent advances that illustrate the intense and intimate host‐microbial interaction at the epithelial level and improve our understanding of the mechanisms that maintain the integrity of the intestinal epithelial barrier.     

Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis

Toll-like receptors (TLRs) play a crucial role in host defense against microbial infection. The microbial ligands recognized by TLRs are not unique to pathogens, however, and are produced by both pathogenic and commensal microorganisms. It is thought that an inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia. Here, we show that commensal bacteria are recognized by TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, we find that activation of TLRs by commensal microflora is critical for the protection against gut injury and associated mortality. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury-and provide a new perspective on the evolution of host-microbial interactions.     

The human commensal Bacteroides fragilis binds intestinal mucin

The mammalian gastrointestinal tract harbors a vast microbial ecosystem, known as the microbiota, which benefits host biology. Bacteroides fragilis is an important anaerobic gut commensal of humans that prevents and cures intestinal inflammation. We wished to elucidate aspects of gut colonization employed by B. fragilis. Fluorescence in situ hybridization was performed on colonic tissue sections from B. fragilis and Escherichia coli dual-colonized gnotobiotic mice. Epifluorescence imaging reveals that both E. coli and B. fragilis are found in the lumen of the colon, but only B. fragilis is found in the mucosal layer. This observation suggests that physical association with intestinal mucus could be a possible mechanism of gut colonization by B. fragilis. We investigated this potential interaction using an in vitro mucus binding assay and show here that B. fragilis binds to murine colonic mucus. We further demonstrate that B. fragilis specifically and quantitatively binds to highly purified mucins (the major constituent in intestinal mucus) using flow cytometry analysis of fluorescently labeled purified murine and porcine mucins. These results suggest that interactions between B. fragilis and intestinal mucin may play a critical role during host-bacterial symbiosis.     

Incorporating a mucosal environment in a dynamic gut model results in a more representative colonization by lactobacilli

To avoid detrimental interactions with intestinal microbes, the human epithelium is covered with a protective mucus layer that traps host defence molecules. Microbial properties such as adhesion to mucus further result in a unique mucosal microbiota with a great potential to interact with the host. As mucosal microbes are difficult to study in vivo, we incorporated mucin‐covered microcosms in a dynamic in vitro gut model, the simulator of the human intestinal microbial ecosystem (SHIME). We assessed the importance of the mucosal environment in this M‐SHIME (mucosal‐SHIME) for the colonization of lactobacilli, a group for which the mucus binding domain was recently discovered. Whereas the two dominant resident Lactobacilli, Lactobacillus mucosae and Pediococcus acidilactici, were both present in the lumen, L. mucosae was strongly enriched in mucus. As a possible explanation, the gene encoding a mucus binding (mub) protein was detected by PCR in L. mucosae. Also the strongly adherent Lactobacillus rhamnosus GG (LGG) specifically colonized mucus upon inoculation. Short‐term assays confirmed the strong mucin‐binding of both L. mucosae and LGG compared with P. acidilactici. The mucosal environment also increased long‐term colonization of L. mucosae and enhanced its stability upon antibiotic treatment (tetracycline, amoxicillin and ciprofloxacin). Incorporating a mucosal environment thus allowed colonization of specific microbes such as L. mucosae and LGG, in correspondence with the in vivo situation. This may lead to more in vivo‐like microbial communities in such dynamic, long‐term in vitro simulations and allow the study of the unique mucosal microbiota in health and disease.     

How Microbial Community Composition Regulates Coral Disease Development

Reef coral cover is in rapid decline worldwide, in part due to bleaching (expulsion of photosynthetic symbionts) and outbreaks of infectious disease. One important factor associated with bleaching and in disease transmission is a shift in the composition of the microbial community in the mucus layer surrounding the coral: the resident microbial community—which is critical to the healthy functioning of the coral holobiont—is replaced by pathogenic microbes, often species of Vibrio. In this paper we develop computational models for microbial community dynamics in the mucus layer in order to understand how the surface microbial community responds to changes in environmental conditions, and under what circumstances it becomes vulnerable to overgrowth by pathogens. Some of our model''s assumptions and parameter values are based on Vibrio spp. as a model system for other established and emerging coral pathogens. We find that the pattern of interactions in the surface microbial community facilitates the existence of alternate stable states, one dominated by antibiotic-producing beneficial microbes and the other pathogen-dominated. A shift to pathogen dominance under transient stressful conditions, such as a brief warming spell, may persist long after environmental conditions have returned to normal. This prediction is consistent with experimental findings that antibiotic properties of Acropora palmata mucus did not return to normal long after temperatures had fallen. Long-term loss of antibiotic activity eliminates a critical component in coral defense against disease, giving pathogens an extended opportunity to infect and spread within the host, elevating the risk of coral bleaching, disease, and mortality.     

Luminal host-defense mechanisms against invasive amebiasis

Most humans infected with the virulent protozoan parasite Entamoeba histolytica do not develop invasive disease. Available evidence indicates that beneficial bacteria and the mucus gel layer in the colon lumen protect the host mucosa. Glycosidases produced by some normal colonic bacteria and luminal proteases degrade the key adherence lectin on E. histolytica trophozoites and decrease their adherence to epithelial cells. The mucus gel layer prevents those trophozoites that escape the hydrolases from reaching the epithelial cells. Trophozoite mucosal invasion is triggered only when both protective mechanisms are lost, as might occur during an unrelated pathogenic enteric bacterial infection. A newly developed gnotobiotic model of intestinal amebiasis should enable testing of this hypothesis and provide clues to help design practical studies in humans.     

肠道菌群功能与成瘾机制的研究进展

烟酒成瘾、药物滥用和停药反应的增多,对社会和家庭造成巨大的经济损失,同时也产生了一系列的健康问题,其中神经系统是成瘾的关键。近年来,越来越多的资料证明脑-肠轴的联系,人们发现肠道微生物的扰动对神经系统的调节有至关重要的作用。综述了烟酒和药物的成瘾机制,脑-肠轴影响宿主的代谢和对神经功能的调节,益生元和益生菌的摄入能引起肠道菌群的改变。深入分析脑-肠轴和肠道菌群代谢,通过益生菌,益生元改变菌群结构治疗成瘾成为今后研究的重点方向。     

Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer

Recent reports have suggested the involvement of gut microbiota in the progression of colorectal cancer (CRC). We utilized pyrosequencing based analysis of 16S rRNA genes to determine the overall structure of microbiota in patients with colorectal cancer and healthy controls; we investigated microbiota of the intestinal lumen, the cancerous tissue and matched noncancerous normal tissue. Moreover, we investigated the mucosa-adherent microbial composition using rectal swab samples because the structure of the tissue-adherent bacterial community is potentially altered following bowel cleansing. Our findings indicated that the microbial structure of the intestinal lumen and cancerous tissue differed significantly. Phylotypes that enhance energy harvest from diets or perform metabolic exchange with the host were more abundant in the lumen. There were more abundant Firmicutes and less abundant Bacteroidetes and Proteobacteria in lumen. The overall microbial structures of cancerous tissue and noncancerous tissue were similar; however the tumor microbiota exhibited lower diversity. The structures of the intestinal lumen microbiota and mucosa-adherent microbiota were different in CRC patients compared to matched microbiota in healthy individuals. Lactobacillales was enriched in cancerous tissue, whereas Faecalibacterium was reduced. In the mucosa-adherent microbiota, Bifidobacterium, Faecalibacterium, and Blautia were reduced in CRC patients, whereas Fusobacterium, Porphyromonas, Peptostreptococcus, and Mogibacterium were enriched. In the lumen, predominant phylotypes related to metabolic disorders or metabolic exchange with the host, Erysipelotrichaceae, Prevotellaceae, and Coriobacteriaceae were increased in cancer patients. Coupled with previous reports, these results suggest that the intestinal microbiota is associated with CRC risk and that intestinal lumen microflora potentially influence CRC risk via cometabolism or metabolic exchange with the host. However, mucosa-associated microbiota potentially affects CRC risk primarily through direct interaction with the host.     

Coral mucus-associated bacteria: a possible first line of defense

Interactions among microorganisms found in coral mucus can be either symbiotic or competitive. It has been hypothesized that microbial communities found on the surface of coral play a role in coral holobiont defense, possibly through production of antimicrobial substances. Selected microorganisms isolated from the mucus layer of a number of coral species were grown using agar-plating techniques. Screening for antimicrobial substances was performed using overlay and drop techniques, employing several indicator microorganisms. Between 25% and 70% of cultivable mucus-associated bacteria from scleractinian corals demonstrated bioactivity. Higher percentages of activity were evident in mucus-associated cultivable bacteria from massive and solitary corals, as compared with bacteria from branching or soft corals. Isolates related to the genera Vibrio and Pseudoalteromonas demonstrated high activity against both Gram-positive and Gram-negative bacteria. Gram-positive bacteria ( Bacillus, Planomicrobium ) demonstrated lower levels of activity, primarily against other Gram-positive bacteria. In some cases, inhibitory effects were confined to the cell fraction, suggesting the involvement of a cell-bound molecule, sensitive to temperature and most likely proteinaceous in nature. These results demonstrate the existence of microorganisms with antimicrobial activity on the coral surface, possibly acting as a first line of defense to protect the coral host against pathogens.     

Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation

The body's surfaces form the interface with the external environment, protecting the host. These epithelial barriers are also colonized by a controlled diversity of microorganisms, disturbances of which can give rise to disease. Specialized intraepithelial lymphocytes (IELs), which reside at these sites, are important as a first line of defense as well as in epithelial barrier organization and wound repair. We show here that the aryl hydrocarbon receptor (AhR) is a crucial regulator in maintaining IEL numbers in both the skin and the intestine. In the intestine, AhR deficiency or the lack of AhR ligands compromises the maintenance of IELs and the control of the microbial load and composition, resulting in heightened immune activation and increased vulnerability to epithelial damage. AhR activity can be regulated by dietary components, such as those present in cruciferous vegetables, providing a mechanistic link between dietary compounds, the intestinal immune system, and the microbiota.     

益生菌与肠黏膜互作的分子机制研究进展

益生菌是一类定植于动物肠道,可辅助动物消化功能,维护肠道菌群平衡并可影响肠道免疫系统,有益于动物健康的重要调节性菌群。该类菌群与动物肠上皮细胞间互作的分子机制包括菌体表面分子如磷脂壁酸（phosphatidicacid,LTA）、表面层蛋白（Slayerprotein）等与宿主的粘附相关蛋白分子结合,通过占位效应抑制有害菌群在肠道内的定植;益生菌还可刺激肠道细胞分泌B防御素2、细菌素和有机酸等可抑制甚至杀灭有害菌群;在益生菌作用下,肠道上皮细胞可增强粘液糖蛋白、紧密连接蛋白occludin和ZO-1等分子的表达,加厚并加固肠道黏膜屏障;益生菌相关抗原可通过与抗原递呈细胞表面模式识别受体（TLRs等）分子结合,激活递呈细胞,启动各免疫细胞的交互作用,调节肠道免疫状态。     

Lung protease/anti-protease network and modulation of mucus production and surfactant activity

Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local ‘alarm’ anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs.     

肠道微生物功能宏基因组学与代谢组学关联分析方法研究进展

近年来基于高通量基因测序的微生物组学研究极大加深了人们对微生物与健康和疾病关系的认识。然而基因测序方法不能直接测定微生物的功能活性,难以鉴定微生物中的关键功能分子,单独使用无法回答肠道微生物何种成员通过何种方式影响宿主等关键科学问题。单一组学研究弊端尽显,多组学联用势在必行。肠道微生物代谢组学以微生物群落所有小分子代谢物为研究对象,可发现肠道微生物随宿主病理生理变化的关键代谢物,为微生物组-宿主互作机制研究提供线索,成为微生物组学研究的重要补充。肠道微生物功能基因组学与代谢组学关联分析在宿主生理、疾病病理、药物药理等方面取得众多进展,展现良好应用前景。然而目前肠道微生物功能基因组学与代谢组学关联分析存在方法滥用、相关性结论与生物学知识相悖等突出问题。为帮助正确应用肠道微生物功能宏基因组学与代谢组学关联分析,本文综述了各种多组学数据整合分析方法的原理、优缺点与适用范围,并给出了应用建议。     

Physiological basis for novel drug therapies used to treat the inflammatory bowel diseases I. Pathophysiological basis and prospects for probiotic therapy in inflammatory bowel disease

Mechanisms underlying the conditioning influence of the intestinal flora on mucosal homeostasis, including development and function of immune responses, are attracting increasing scientific scrutiny. The intestinal flora is a positive asset to host defense, but some of its components may, in genetically susceptible hosts, become a risk factor for development of inflammatory bowel disease (IBD). It follows that strategies to enhance assets or offset microbial liabilities represent a therapeutic option; therein lies the rationale for manipulation of the flora in IBD. In addition, the diversity of regulatory signalling among the flora and host epithelum, lymphoid tissue, and neuromuscular apparatus is an untapped reservoir from which novel therapeutics may be mined. Moreover, the capacity to engineer food-grade or commensal bacteria to deliver therapeutic molecules to the intestinal mucosa promises to extend the scope of microbial manipulation for the benefit of mankind.     

Translationally Controlled Tumor Protein,a Dual Functional Protein Involved in the Immune Response of the Silkworm,Bombyx mori

Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori.     

Current and future uses of real-time polymerase chain reaction and microarrays in the study of intestinal microbiota, and probiotic use and effectiveness

Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in adequate amounts. In addition to human health benefits, probiotics can improve various aspects of growth and performance in livestock and poultry, as well as control undesirable microorganisms in food animals. Studies indicate that probiotics can prevent or treat certain conditions, including atopic disease in infants, food allergy, infection after surgery, acute diarrhea, and symptoms associated with irritable bowel syndrome. Understanding the complete mechanism, effectiveness, and potential use of probiotics is limited by the availability and sensitivity of current methods (i.e., culturing techniques). In recent years, real-time polymerase chain reaction (PCR) and microarrays have become prominent and promising methods to examine quantitative changes of specific members of the microbial community and the influence of probiotics on the structure and function of human and animal intestinal ecosystems. Culture-independent studies have established that only a fraction of organisms present in feces are cultivable, therefore, results obtained by cultivation are limited. Conversely, in-depth knowledge of microbial genomes has enabled real-time PCR and microarrays to be more sensitive and has resulted in precise methods for comprehensive analysis of the complex gut microbiota. Additionally, these technologies can assess the influence of intestinal microorganisms on host metabolism, nutrient status, and disease. This paper reviews method technologies and applications of real-time PCR and microarray assays as they relate to the effect and use of probiotics on the intestinal microbiota and gastrointestinal disease.     

油藏微生物群落研究的方法学

油藏微生物群落的解析和认知是开发和应用微生物采油技术的基础。利用各种提高油藏微生物可培养性的方法和非培养技术解析不同油藏微生物的群落结构、功能和多样性,对定向调控油藏微生物群落、开发和应用有效微生物驱油技术具有重要的指导意义。通过调查新近发展的提高微生物可培养性的方法和措施以及不依赖于培养的分子微生物生态学技术,总结了油藏微生物群落研究方法学的最新进展。提高微生物可培养性的方法和措施主要通过模拟微生物的生存环境,减少富营养的毒害作用、添加信号分子维持微生物细胞间的作用和提供新型电子供体和受体等手段采用稀释法、高通量培养法等方法得以实现;不依赖于培养的分子微生物生态学技术主要包括荧光原位杂交、末端限制性片断长度多态性分析、变性梯度凝胶电泳和构建克隆文库等技术。这些方法学的进展为更有效的获得各种油藏微生物资源、调控油藏微生物群落以提高石油采收率提供理论指导。     

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.